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HYDROCARBONS Here insert the full name and address of the Candidate. ?Strike out whichever of these is not required. APPLICATION FORM FOR MEMBERSHIP I , ........................................................................................................................................................................................................ ................................................................................................................................................................................................................... being ........................... t Member ...y ears of age, and desirous of becoming a (under 25) of THE FARADAY SOCIETY, do hereby apply for Membership. I agree to be bound by the Rules of the Society. I enclose the Annual Subscription and Entrance Fee.(Members i 3 , Entrance Fee &I, Juniors &I IOS., no entrance fee.) *If the Candidate My qualifications are * ..................................................................................................................................................... is a Member of other Societies stated here. this should be ...... ...................................................................................................................................................................................................... .................................................................................................................................................................................................................. Signature of Candidate ............................................................................................................ I, the undersigned Member, from my personal knowledge, do hereby propose the above Candidate for election.Witness my hand this day of ............................................................ 19 .................................... ........................................................................................................................ Proposer This space for the use of the Secretary :- Application received on Application accepted by Council on ...................................................................................................... I To THE SECRETARY, The Faraday Society, Gray's Inn, 6 Gray's Inn Square, London, W.C.1.PRINTED IN GREAT BRITAIN AT THE UNIVERSITY PRESS, ABERDEENHYDROCARBONS Here insert the full name and address of the Candidate. ?Strike out whichever of these is not required. APPLICATION FORM FOR MEMBERSHIP I , ........................................................................................................................................................................................................ ................................................................................................................................................................................................................... being ........................... t Member ...y ears of age, and desirous of becoming a (under 25) of THE FARADAY SOCIETY, do hereby apply for Membership.I agree to be bound by the Rules of the Society. I enclose the Annual Subscription and Entrance Fee. (Members i 3 , Entrance Fee &I, Juniors &I IOS., no entrance fee.) *If the Candidate My qualifications are * ..................................................................................................................................................... is a Member of other Societies stated here. this should be ...... ...................................................................................................................................................................................................... .................................................................................................................................................................................................................. Signature of Candidate ............................................................................................................ I, the undersigned Member, from my personal knowledge, do hereby propose the above Candidate for election. Witness my hand this day of ............................................................ 19 .................................... ........................................................................................................................ Proposer This space for the use of the Secretary :- Application received on Application accepted by Council on ...................................................................................................... I To THE SECRETARY, The Faraday Society, Gray's Inn, 6 Gray's Inn Square, London, W.C.1. PRINTED IN GREAT BRITAIN AT THE UNIVERSITY PRESS, ABERDEEN

ISSN:0366-9033    

DOI:10.1039/DF94906FX001

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

Lipo-Protein Discussion BIRMINGHAM A u g u s t 2gth-31st, 194.9Lipo-Protein Discussion BIRMINGHAM A u g u s t 2gth-31st, 194.9NUMERICAL LIST FOR PHOTOGRAPH I. Astbury, W. T. 2. McFarlane, A. S. 3. Chargaff, E. 4. 5. Boscott, R. J. 6. Dervichian, D. G. 7. Jellinek, H. H. G. 8. Thomas, Garfield. 30. Macheboeuf, M. 31. Woolf, L. I. 32. Gaade, W. 33. O’Connor, J. M. 34. Lawrence, A. S. C. 35. Butler, J. A. V. 36. Robinson, C. 37. Tompkins, F. C. 38. Bircumshaw, L. L. 39. Beale, R. N. 40. Conway, B. E. 41. Claude, A. 42. Murray Luck, J. M. 43. Lasnitzki, A. 44. Booij, H. L. 45. Frazer, A. C. 46. Booij, Mrs. H. L. 72. Teale, J. W. F. 73. Kornitzer, B. 74. Meiklejohn, M. 75. Czeczowiczka, N. 76. Faber, hl. 77. Faber, J. G. 93. Goddard, E. D. 78. Carter, S. R. 94. Pethica, B.A. 79. Harris, R. C. J. 95. Parsons, T. R. 80. Hoffmann-Ostenhof, 0. 96. Hamer, W. E. 81. Haurowitz, F. 97. Stewart, H. C. 82. Pangborn, M. C. 98. Cockbairn, E. G. 83. Roe, E. gg. Armstrong, WT. McD. 84. Small, R. 1%‘. H. 85. Carr, J. G. 86. Iball, J. 87. Elford, W. J. 88. Herschdoerfer, S. M. 89. Bavin, E. If. go. Mansour, T. 91. Joujovsky, N. I. 92. Liberman, A. 100. Finean, J. B. 101. Brown, W. D. 102. Niemierko, W. 103. Li, C. H. 104. Dustin, J. P. 105. Neuzil, E. 106. Tayeau, F. 107. Meduski, -. 108. Sagrott, P. E. 109. Pearcy, R. 110. Smith, R. H. I I I . Bradish, C. J. 112. Ratcliffe, P. W. 113. Squire, J. R. 114. O’Connor, D. A. 115. Elkes, J. 116. McCarthy, E. F. 117. Claesson, S. 118. Garrod Thomas, G. 119. Godfrey, D. J. 120. Beznak, A. 121. Popjak, G. 122. Pankhurst, K. 123. Hurst, H. 124. Hanson, S. W. F. 125. Lovern, J. A. 126. Moore, D. H. 127. Wassink, E. C. 128. Frey-Wyssling, A. 129. Sammons, H. G. 130. Matalon, R. 131. Schulman, J. H. 132. Blokker, P. C. 133. Wajda, S. H. 134. Rowland, P. R. 135. Ehrensvard, G. 136. Raison, M. 137. Baker, L. C. 138. Baker, R. G. 139. Wajda, I. 140. Mounfield, J. D.

ISSN:0366-9033    

DOI:10.1039/DF949060X003

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

NUMERICAL LIST FOR PHOTOGRAPH I. Astbury, W. T. 2. McFarlane, A. S. 3. Chargaff, E. 4. 5. Boscott, R. J. 6. Dervichian, D. G. 7. Jellinek, H. H. G. 8. Thomas, Garfield. 30. Macheboeuf, M. 31. Woolf, L. I. 32. Gaade, W. 33. O’Connor, J. M. 34. Lawrence, A. S. C. 35. Butler, J. A. V. 36. Robinson, C. 37. Tompkins, F. C. 38. Bircumshaw, L. L. 39. Beale, R. N. 40. Conway, B. E. 41. Claude, A. 42. Murray Luck, J. M. 43. Lasnitzki, A. 44. Booij, H. L. 45. Frazer, A. C. 46. Booij, Mrs. H. L. 72. Teale, J. W. F. 73. Kornitzer, B. 74. Meiklejohn, M. 75. Czeczowiczka, N. 76. Faber, hl. 77. Faber, J. G. 93. Goddard, E. D. 78. Carter, S. R. 94. Pethica, B. A. 79. Harris, R. C. J. 95. Parsons, T. R. 80. Hoffmann-Ostenhof, 0. 96. Hamer, W. E. 81. Haurowitz, F. 97. Stewart, H. C. 82. Pangborn, M.C. 98. Cockbairn, E. G. 83. Roe, E. gg. Armstrong, WT. McD. 84. Small, R. 1%‘. H. 85. Carr, J. G. 86. Iball, J. 87. Elford, W. J. 88. Herschdoerfer, S. M. 89. Bavin, E. If. go. Mansour, T. 91. Joujovsky, N. I. 92. Liberman, A. 100. Finean, J. B. 101. Brown, W. D. 102. Niemierko, W. 103. Li, C. H. 104. Dustin, J. P. 105. Neuzil, E. 106. Tayeau, F. 107. Meduski, -. 108. Sagrott, P. E. 109. Pearcy, R. 110. Smith, R. H. I I I . Bradish, C. J. 112. Ratcliffe, P. W. 113. Squire, J. R. 114. O’Connor, D. A. 115. Elkes, J. 116. McCarthy, E. F. 117. Claesson, S. 118. Garrod Thomas, G. 119. Godfrey, D. J. 120. Beznak, A. 121. Popjak, G. 122. Pankhurst, K. 123. Hurst, H. 124. Hanson, S. W. F. 125. Lovern, J. A. 126. Moore, D. H. 127. Wassink, E. C. 128. Frey-Wyssling, A.129. Sammons, H. G. 130. Matalon, R. 131. Schulman, J. H. 132. Blokker, P. C. 133. Wajda, S. H. 134. Rowland, P. R. 135. Ehrensvard, G. 136. Raison, M. 137. Baker, L. C. 138. Baker, R. G. 139. Wajda, I. 140. Mounfield, J. D.NUMERICAL LIST FOR PHOTOGRAPH I. Astbury, W. T. 2. McFarlane, A. S. 3. Chargaff, E. 4. 5. Boscott, R. J. 6. Dervichian, D. G. 7. Jellinek, H. H. G. 8. Thomas, Garfield. 30. Macheboeuf, M. 31. Woolf, L. I. 32. Gaade, W. 33. O’Connor, J. M. 34. Lawrence, A. S. C. 35. Butler, J. A. V. 36. Robinson, C. 37. Tompkins, F. C. 38. Bircumshaw, L. L. 39. Beale, R. N. 40. Conway, B. E. 41. Claude, A. 42. Murray Luck, J. M. 43. Lasnitzki, A. 44. Booij, H. L. 45. Frazer, A. C. 46. Booij, Mrs. H. L. 72. Teale, J. W. F. 73. Kornitzer, B. 74. Meiklejohn, M. 75.Czeczowiczka, N. 76. Faber, hl. 77. Faber, J. G. 93. Goddard, E. D. 78. Carter, S. R. 94. Pethica, B. A. 79. Harris, R. C. J. 95. Parsons, T. R. 80. Hoffmann-Ostenhof, 0. 96. Hamer, W. E. 81. Haurowitz, F. 97. Stewart, H. C. 82. Pangborn, M. C. 98. Cockbairn, E. G. 83. Roe, E. gg. Armstrong, WT. McD. 84. Small, R. 1%‘. H. 85. Carr, J. G. 86. Iball, J. 87. Elford, W. J. 88. Herschdoerfer, S. M. 89. Bavin, E. If. go. Mansour, T. 91. Joujovsky, N. I. 92. Liberman, A. 100. Finean, J. B. 101. Brown, W. D. 102. Niemierko, W. 103. Li, C. H. 104. Dustin, J. P. 105. Neuzil, E. 106. Tayeau, F. 107. Meduski, -. 108. Sagrott, P. E. 109. Pearcy, R. 110. Smith, R. H. I I I . Bradish, C. J. 112. Ratcliffe, P. W. 113. Squire, J. R. 114. O’Connor, D. A. 115. Elkes, J. 116. McCarthy, E. F. 117. Claesson, S. 118. Garrod Thomas, G. 119. Godfrey, D. J. 120. Beznak, A. 121. Popjak, G. 122. Pankhurst, K. 123. Hurst, H. 124. Hanson, S. W. F. 125. Lovern, J. A. 126. Moore, D. H. 127. Wassink, E. C. 128. Frey-Wyssling, A. 129. Sammons, H. G. 130. Matalon, R. 131. Schulman, J. H. 132. Blokker, P. C. 133. Wajda, S. H. 134. Rowland, P. R. 135. Ehrensvard, G. 136. Raison, M. 137. Baker, L. C. 138. Baker, R. G. 139. Wajda, I. 140. Mounfield, J. D.

ISSN:0366-9033    

DOI:10.1039/DF94906BX006

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

STRUCTURAL ASPECT OF LIPO-PROTEIN ASSOCIATION BY D. G. DERVICHIAN Received 2nd June 1949 From a detailed analysis of all the available work done on the association between proteins and different colloidal electrolytes general and consistent conclusions could be derived concerning the simultaneous influence of pH salt concentration and proportions of the two constituents ; the important point being that these different factors intervene in a similar and complementary way. From this and other considerations it is concluded that the interaction is purely ionic. The necessity of bringing in van der Waals’ forces in some cases results from the binding together of the molecules in the colloidal electrolyte micelle. The nature of the lipid-lipid associations is discussed from the point of view of mixed micelles.If their ionic behaviour is considered natural lipo-proteins are similar to the artificial associations of proteins with ionic colloids If the extraction of lipids is considered natural lipo-proteins show the characteristics of the artificial lipid-lipid associations. A tentative structure is proposed for the natural lipo-proteins in solution. Lipids would form separate mixed micelles in which the non-ionic are solubilized by the ionic. It is further postulated that a purely ionic interaction takes place between these mixed lipidic micelles and the protein particles as well as with all other small ions present. The natural lipo-proteins i.e. those encountered in living organisms contain ionic lipids (phospholipids fatty acids) and non-ionic lipids (esters of cholesterol cholesterol glycerides).First will be examined the protein-ionic lipid association. No doubt it i s the protein-lecithin interaction which is the most instructive from the point of view of the natural lipo-proteins. But if one examines and compares simply the experimental facts very satisfactory correlations are found in the behaviour of lecithin alone and of the different associations protein- lecithin protein-fatty acids protein-synthetic detergents and in a general way protein-colloidal electrolytes such as gum arabic nucleic acid dyes. I t appears that non-ionic lipids cannot associate by them- selves with proteins while they can associate with the other ionic lipids. The study of those lipid-lipid associations coming next will introduce some structural considerations which can explain some of the specific behaviour of the natural lipo-proteins.Finally the behaviour and properties of natural lipo-proteins will be examined in the light of the conclusions of the preceding studies. Protein-Ionic Lipid Interaction The precipitation of protein-lipid systems gives certain information on the interaction of these two classes of substances. In certain cases instead of ordinary precipitation or flocculation a fluid phase separates which is called coacervate.l Dervichian Research 1949 2 210. 7 LIPO-PROTEIN ASSOCIATION 8 (a) Mutual Influence of pH and Proportion on Precipitation. -Concerning zones of precipitation at certain proportions of the con- stituents and the influence of pH on these proportions the same peculiarities are found either in the protein-lecithin interaction 2-6 or in the interaction of proteins with other ionic lipids,5 detergents *-14 colloidal electrolytes such as gum arabic,l59 l6 or nucleic acid,16 l7 and dyes.1s-20 The behaviour of anionic detergents on the one hand and cationic detergents on the other are symmetric about the isoelectric point of the protein on which they act.(b) Influence of Electrolytes.-Even with lecithin alone the precipitation by an acid is inhibited by salts.3 The presence of neutral salts also inhibits the precipitation of lecithin by protein^.^ More exactly to precipitate lecithin by protein the acidity of the medium must be higher the more neutral salt is present.2 This property is also found in interactions with other colloidal electrolyte^.^ With anionic detergents the amount of detergent necessary to produce complete precipitation increases with the amount of added electrolyte.1° The same phenomenon is observed with cationic detergents in basic medium.ll3 l4 Above the isoelectric point precipitation by an anionic detergent is only possible in the presence of a considerable quantity of salt.In addition the ratio detergent/protein in the precipitate is nearly ten times its value in acid medium in the absence of salt.21 Finally coacervation of gelatin by gum arabic is strongly influenced by the presence of salts.15 For example 0.35 % KC1 is sufficient to suppress the separation.One sees that pH and saline concentration play an identical role and this in accord with the fact that with proteins alone the addition of salts causes an increase in the pH of the solution if the initial pH is less than that of the isoelectric point and a decrease in pH if the initial pH is greater than that of the isoelectric point.22 I t should be pointed out also that the protein and the other colloidal electrolyte each carries its gegenions which explains the fact that an excess of one or the other influences the precipitation in the same manner as ions of added salt or OH- or Hf ion (pH). a Mayer and Terroine Compt. rend. SOC. Biol. 1907 62 398. Handovsky and Wagner Biochem. Z. 1911 31 32. Przylecki and Hober Biochem.Z. 1936 288 303. 4 Parsons Biochem. J . 1928 22 800. 8 Bull and Neurath J . Biol. Chern. 1937 XIS 163. 6 Feinschmidt Biochem. Z. 1912 38 244. 7 Matsumura Kolloid-Z. 1923 32 171. * Putnam and Neurath J . Amer. Chem. Soc. 1944 66 692. lo Pankhurst and Smith Trans. Faraday SOC. 1945 41 630. l1 Schmidt 2. Physiol. Chem. 1943 277 117. l2 Elkes and Finean in Surface Chemistry (Butterworth London 1g4g) p. 281. l3 Dervichian and Magnant Bull. SOC. Chim. biol. 1947 29 655. l4 Polonovski and Macheboeuf Ann. Inst. Pasteur 1948 74 196 l5 Bungenberg de Jong and Dekher Kolloid. Beih 1935 43 143 ; 1936 43 213. l6 Dervichian and Magnant Bull. SOC. Chim. biol. 1947 29 660-666. l7 Bungenberg de Jong and Hoskam Proc. Ned. Akad. Wetensch. 1942 45 387. Mathews Amer.J . Physiol. 1898 I 445 lo Chapman Greenberg and Schmidt J . Bzol. Chem. 1927 72 707. 20 Rawling and Schmidt ibid. 1929 82 709. 21 Pankhurst and Smith Trans. Faraday SOC. 1944 40 565. 22 Schmidt The Chemistry of Amino Acids and Protezns (Charles C . Thomas 1945) p. 1209. D. G. DERVICHIAN 9 (c) Interaction in Solution. - The separation of proteins by other colloidal electrolytes can occur only in a pH region such that the net charge of the protein is opposite to that of the precipitating agent There are nevertheless interactions between the two con- stituents in regions where the net charges are of the same sign and where the constituents remain in solution. These interactions have been demonstrated mostly by electrophoresis studies.23 24 Well-defined boundaries appear which correspond to the migration of the associated protein and detergent.These interactions have also been demon- strated by measurements of rotation of plane polarized light surface tension and pH.25 More recently Pankhurst 26 has added evidence based on viscosity measurements. Interactions between acid or basic dyes and proteins outside the pH zone of precipitation has been demonstrated by measurement of the shift of absorption bands and determination of the quantity of dye held by the protein in dialysis eq~ilibrium.~' ( d ) Nature of Protein-Ionic Lipid Interaction.-Mayer and Terroine established that in an electric field the charge (positive) of the lecithin-albumin complex was opposite to that of lecithin alone.It was Putnam and Neurath9 who were the first to realize the con- nection between the maximum quantity of detergent bound and the total acid-binding capacity of the protein. They concluded that pre- cipitation was produced by electrostatic forces between the ionized groups of the two constituents. In studying the electrophoresis of small drops of gelatin-gum arabic coacervate Bungenberg de Jong and Dekker l5 found they were drawn to the positive or negative pole according to the relative amount of gum arabic. For each pH the change of sign occurs in the neighbourhood of the maximum separation. It appears then as if coacervation were connected to the mutual neutral- ization of the charges of the two colloids. In what form is the lipid bound to the protein in the precipitate? The method of titration used by Parson might give us some informa- tion on this point.The lecithin present in the precipitate was titrated by dissolving in it a lipo-soluble dye (but not water-soluble). This dye remained in the associated lipid. There is strong evidence that lipo- soluble dyes (like other lipo-soluble substances) can only be dissolved between the paraffinic chains of lecithin or detergent when these mole- cules are associated in m i ~ e l l e s . ~ - ~ ~ The work of Parsons tends to prove that in the protein-lecithin precipitate the lecithin is associated in the form of lamellar micelles. The fact that this micellar structure is necessary is shown by the observation that proteins cannot be pre- cipitated by detergents (anionic or cationic) having less than 10 carbon atoms.Besides it is well known that at ordinary temperature micelle structure exists only in solutions of ionic lipids having more than 8 carbon atoms in their chain. To satisfy all these conditions i.e. ionic interaction between lipids and proteins necessity of micellar state for the lipid and the appearance 23 Lundgren Etam and O'ConneII J . Biol. Chem. 1943 149 183. 24 Putnam and Neurath ibid. 1945 159 195. 25 Desreux and Fabry Bull. SOC. Chem. biol. 1946 28 478. 26 Pankhurst ibid. 1949 31. Klotz Chem. Rev. 1947 41 373. a8 Dervichian and Magnant Compt. rend. SOC. Biol. 1946 140 95. 28 Kiessig and Philippoff Naturwiss 1939 27 593. 30 Hughes Sawger and Vinograd J . Chem. Physic.1945 13 131. A " LIPO-PROTEIN ASSOCIATION I 0 of paraffinic character in organic solvents,2* 31 one could suggest that in the precipitate the double layer micelles of the lipid stick by their ionic faces to the ionic faces of the protein particles and thus con- stitute alternate layers of lipid and protein.32* 33 The structure proposed here might not seem to differ from that of Pankhurst and Smith.2l In fact it originates from a different point of view. The interaction of the lipid micelle as a whole has to be con- sidered at the moment of precipitation starting from the principle that no lipid molecules can adsorb in a single layer without the double layer lattice being built up and precipitation following. One could as well say that the micelle of the detergent fixes the protein particles by its two external ionic faces (and vice versa) and that the ionic lattice (Hartley) being thus formed similarly to the crystal of fatty substances the whole precipitates.Should the “ dissolved complexes” be considered as formed of complex particles with lipid molecules sticking to the protein molecules as they are in the precipitate? To see the problem clearly one must not lose sight of a very trivial fact ionic combination in solution does not mean aggregation of oppositely charged ions in one particle. Proportions are stoichiometric whether the reaction gives rise to aggregation and precipitation or to a dissociated compound which remains in solution. It is generally admitted that the length of the lipid or detergent chain serves to increase the forces of interaction between the lipid and the protein in a discontinuous way when there are more than eight carbons in the lipid chain.24* 34p 35 Some see here a specific action.Thus two factors would determine the force of the interaction an electrostatic attraction due to oppositely charged ions and van der Waals’ forces. This is also the conclusion arrived at by Klotz 27 con- cerning the bonds between proteins and dyes. The basis of this con- clusion is the difference in behaviour between dyes having one charged group and those having two or three charged groups. However the point should be considered that with amphipatic molecules the tendency to form micelles diminishes with the increasing number of solubilizing groups.I t seems reasonable to think that the necessity of bringing in van der Waals’ forces is not concerned with the bond between the protein and the lipid but with the linkages which hold together the lipid molecules in the lipid rnicelle in the neighbourhood of the protein particle. The interaction between the lipid micelle as a whole and the protein particle is purely ionic. The “ soluble compounds ” are formed in the pH region where the protein and the lipid have the same charge. How can ionic inter- actions take place under such conditions? The answer is given by Schwert Putnam and Briggs 36 who admit that such interactions may take place in a pH range where the two components have a net charge of the same sign provided there exist ionized groups on the protein opposite in sign to that of the net charge.Klotz2’ reached the same conclusion concerning the interaction between dyes and proteins. 3l Pankhurst in Surface Chemisfry (Butterworth London 1949)~ p. 109. 32 Elkes Frazer Schulman and Stewart Proc. Roy. Soc. A 1945 184 102. 93 Palmer Schmitt and Chargaff J . Cell. Camp. Physiol. 1941 18 43. 34 McMeekin Federation Proc. 1942 I 125. 35 Boyer Ballou and Luck J . Biol. Chem. 1947 167 407. a6 Schwert Putnam and Briggs Arch. Biochem. 1944 4 371. D. G. DERVICHIAN But how is it that the bond is purely ionic when electrophoresis shows sharp boundaries indicating migration of the two associated constituents with no dissociation ? Such migration toward the same electrode of an anion and a cation have been found by Hartley Collie and Samis 37 with a small ion like Br- associated with a cationic detergent in cetylpyridinium bromide.The bromine ion mobility is so much reduced that it becomes actually negative. More bromine is carried in the reverse direction attached to the micelles than travels un- attached in the normal direction. The adherence of gegenions to the micelle is due to the attraction of the free gegenions into a dense atmosphere around the micelle.= Thus emerge the particular con- ditions which exist at the surface of the micelles of the colloidal electro- lytes due to the high concentration of packed charges. However 39 I 1 in protein-detergent association conditions are even more favourable than those of cetylpyridinium bromide because the electrophoresis is carried out at a pH where both substances have net charges of the same sign and both have micelle structure.One might think that the interaction is localized a t the positive ions of the protein (e.g. protein-anionic lipid interaction at an alkaline pH) only single molecules of the lipid being able to approach and not compact micelles because of the repulsion of the negative protein ions. This is where van der Waals’ forces acting between paraffinic chains in the lipid micelle must come in. This means that it requires more energy to keep paraffin chains separated in the presence of water than t o draw some of the anionic groups of the detergent near to the anionic groups of the protein.Besides if groups of the same sign always repelled one another soaps would never crystallize nor show para- crystalline phases in water. In the lamellar structure of soap the COO- groups are face to face in what Hartley calls an ionic lattice. No doubt this ionic lattice is stabilized by the presence of small Na+ ions. In the present case the small cations of saIt present in the system or even the amino groups of the protein themselves would play the same role. Whatever is the mechanism we can at least say that it is sufficient to have a few points of attraction between the positive groups of the protein and the negative groups of the lipid for the whole lipid micelle to be held in the neighbourhood of the protein molecule. If the solu- tion is acid with respect to the isoelectric point of the protein the ionic interactions become overwhelmingly important and there is precipita- tion.The same reasoning can be applied mutatis mutandis to cationic lipid-protein interaction. In coacervation of gelatin or haemoglobin by gum arabic excess of one constituent causes both constituents to pass into solution. It cannot be seen how the acid groups of gum arabic could be fixed individually to a greater or smaller number of the amino groups of the protein nor could be understood how a second layer of gum arabic could be formed making the surface soluble. Such assumptions have been made to explain the same phenomenon with The influence of the quantity of electrolyte present either on the pH of optimum precipitation or on the proportion of detergent 37 Hartley Collie and Samis Trans.Faraday Soc. 1936 32 795. 38 Hartley Aqueous SoEutions of Parafin-chain Salts (Hermann et Cie Pans 7936). 39 Lundgren Textile Res. J. 1945 15 335- I 2 LIPO-PROTE IN ASSOCIATION precipitated shows that the whole phenomenon is a matter of neutraliza- tion of charge in a given volume element. This explanation is similar to that given in the case of ordinary electrolytes. This spatial neutral- ization in which small and large ions take part causes either precipita- tion or changes in velocity of migration in an electric field. We saw that the presence of salt inhibits precipitation. This effect of small ions may be duplicated by the lipid ions. An excess of detergent (carrying its gegenions) produces the same inhibitory effect by per- turbing the ionic atmosphere.This appears as if the acid binding capacity of the protein were modified (see (a) and (b) above). In the experiments of Pankhurst and Smithz1 a t pH above the isoelectric pH the quantity of detergent fixed is ten times the acid binding capacity. These authors (as has Steinhardt *O) suggest that this quantity corresponds to fixation by the amide groups following shifting of their dissociation constants. This point of view can be reconciled with that presented here if instead of fixation we consider the effect of compensation of charge in each small volume element. Other models have been proposed in which the bond is made by the paraffinic end of the detergent linking itself to the paraffinic groups of the protein.Such models have been proposed by Macheboeuf and S a n d ~ r ~ l by Dervichian,42 and by Palmer.43 The accumulated proofs direct and indirect of the ionic nature of the phenomena seems now to exclude such a possibility. In the model here proposed there would not be fixation of the deter- gent to the protein. Fixation results in precipitation just as with ordinary ionic compounds. In solution what we call a compound or a complex is dissociated but the charge can be modified by the presence of H+ or OH- ions (action of pH) or by other small ions of salts present. A reason for this effect to be so apparent is that the ions of the detergent and the protein are strongly concentrated at the surface of their re- spective micelles.The proximity of the charges introduced by the protein by the fact that they are opposite to that of the lipid could tend to ionize the detergent groups. Thus some ionic lipids insoluble in water are made soluble as soaps are in the presence of alkaline metal ions. Lipid -Lipid Associations A whole series of associations in the bulk can be obtained in definite proportions of lipids in contact with water.44 Generally speaking i t is the structure of the constituents which comes into play and not a particular substance (e.g. a molecule of a long-chain aliphatic com- pound associated with a steroid molecule). Consequently in such an association a molecule may replace another of a similar type. A systematic study of a great number of pairs of lipids the one soluble and the other insoluble have allowed detection of a gradation going from simple swelling to complete d i s ~ e r s i o n .~ ~ In complete 40 Steinhardt Atan. Rev. Biochem. 1945 1 4 ~ 145. 41 Macheboeuf and Sandor Bull. SOC. Chim. biol. 1932 1 4 ~ 1168. 42 Dervichian J . Chim. physique 1941 38 59 ; J . Chem. Physics 1943 I I 219. 43 Palmer J . Physic. Chem. 1944 48 12. 44 Dervichian and Magnant Bull. SOC. Chim. biol. 1946 28 419; Comfit. vend. SOC. Biol. 1946 140 94. 46 Dervichian Trans. Faraday SOC. 1946 42B 180. D. G. DERVICHIAN Natural Lipo-Proteins I 3 dispersion the two constituents are associated in mixed miceiles. Substances insoluble like cholesteryl oleate can thus be dispersed in ~ a t e r .4 ~ The manner of association of molecules of lipids is known. Myelinic figures formed by such associations have a clearly stratified structure 4 7 s @ composed of double lipid layers separated by layers of several mole- cules of water. In each lipid layer the different types of molecule are oriented side by side their polar groups directed toward the water and their paraffinic chains facing the paraffin chains of the adjoining layer likewise oriented toward the water. The same double layer structure is preserved in other forms of swelling and one is led to suppose that it is preserved also in the separate micelles when completely dispersed. The association cannot be explained on purely chemical grounds but from a crystallographic point of view.The principal point is that associ- ation cannot be realized except in the presence of water and that water itself must be regarded as one of the structural constituents. Thus is explained the failure of the authors who have tried to isolate complexes in the absence of water.49 A general examination of lipo-proteins shows a striking parallelism to that of protein-ionic lipid and lipid-lipid associations. We are speaking here only of lipids masked in serum or the cells and not those which are found in the form of microscopic or ultramicroscopic particles (for details see for example50). (a) Ionic Behaviour.-The lipo-proteins of serum precipitate a t a pH lower than the isoelectric pH and go into solution again when the pH is raised to 7,51 exactly like anionic lipid-protein associations.The fact that natural lipo-proteins are not dissociated in the elec- trical field 5 2 9 53 does not as thought by Cohen and Chargaff,53 plead against the assumption of a simple saline bond between proteins and phospholipids. I t was shown that not only in protein-detergent mixtures but even in cetylpyridinium bromide both constituents or ions migrate together. (b) Extraction of Lipids.-From the point of view of extraction natural lipo-proteins show predominantly the characteristics of lipid- lipid associations. I t is not a matter of solubility accident contrary to the idea of Cha~-gaff,~* “ that substances as disparate as cephalin and cholesterol” are extracted jointly. This type of pair gives the best lipid-lipid association.Extraction by ether can only be accomplished after dehydration by cold alcohol for example (Hardy and Gardiner method). This is 46 Valette and Cavier Bull. SOC. Chim. biol. 1938 20 1256. 48 Browaes and Dervichian Compt. rend. SOC. Biol. 1946 140 136. 49Partington J . Chem. SOC. 1911 99 313 318. 47 Nageotte Compt. rend. 1927 185 1021. 60Elkes Frazer and Stewart J . Physiol. 1939 95 68. Frazer Trans. Faraduy SOC. 1941 37 125. 51 Macheboeuf Bull. SOC. Chim. biol. 1929 1 I 268 and 485. Macheboeuf Delsal Lepine and Giuntini Ann. Inst. Pasteur 1943 69 321. 63 Cohen and Chargaff Biochem. J. 1940 136 243. 54 Chargaff Advances in Protein Chemistry (1944) I I 8. LIPO-PROTEIN ASSOCIATION I4 identical with the behaviour of lipids alone or lipid-lipid associations which cannot be extracted by ether as long as they remain dispersed in water in the form of micelles.The lowering of the temperature reconstitutes the ionic lattice45 (Krafft point) even in the presence of water and makes association impossible (e.g. myelinic forms do not develop). Extraction by ether becomes then possible. This explains the effectiveness of the method of M ~ F a r l a n e . ~ ~ The presence of alcohol destroys the micelle structure 56 and prevents lipid association. This explains why the extraction of purely lipidic associations by ether is not possible a t ordinary temperature except in the presence of alcohol,57 58 and allows us to understand why the addition of alcohol to serum helps the extraction of lipids by ether.41 Macheboeuf and Tayeau 59 showed that the addition of soap to serum permits extraction by ether.In addition i t can be seen in the eurves of these authors that the maximum of extraction is realized for a soap/protein ratio of 0.4 which is exactly that found as binding capacity of proteins towards detergent^.^ There is therefore sub- stitution from the point of view of ionic interactions we have seen that fatty acids themselves associate with proteins. The quantity of extracted lipids then diminishes with increasing proportion of soap. This might be due to the association of the excess of soap with the separated lipids keeping them in solution. The action of heparin on the extraction60 is similar to that of soap.(c) Lipid-Lipid Association.-The fact that cholesterol and phospholipids are liberated simultaneously by the action of soap seems t o indicate that there is substitution of soap micelles for the mixed cholesterol-phospholipid micelles as a whole. In addition the specific extraction of cholesterol alone by substitution of molecules of similar structure (saponin with a steroid structure 61 or sodium dehydro- abietate62) reminds us of the typical specific conditions of the purely lipidic associations and seems to show that cholesterol is directly bound with the phospholipids in mixed micelles. Tayeau61 had already been led to the conclusion that cholesterol is mainly bound to the phosphatides and that phosphatides might form the link between cholesterol and the proteins.White63 in 1908 had already foreseen that cholesterol is normally present in the tissues of animal in a state of “weak combination ” with fatty acids and lecithin forming liquid crystals. The suggestion has been made that a t least a small part of the lipids were bound to the protein by stronger bonds. This conclusion is based on the fact that part of the lipids cannot be extracted by the above-mentioned methods. In fact lipids might be included in the structure of the protein. Nevertheless it should be pointed out that this behaviour is not particular to natural lipo-proteins as it is also found with ionic lipids alone or artificially associated to protein^.^ 64 s6 McFarlane Nature 1942 149 439. s6 Ward Proc. Roy. SOC.A 1940 176 412. s7 Delezenne and Fourneau Bull. SOC. chim. 1914 15 421. Baranger Ann. Physiol. 1937 13 341. 69 Macheboeuf and Tayeau Bull. SOC. Chim. biol. 1941 23 49. b o Chargaff 2% and Cohen J . Biol. Chem. 1940 136 257. 61 Tayeau Bull. SOC. Chim. biol. f944 26 287. 62 Macheboeuf and Rebeyrotte ibzd. 26 475. 6 3 White Medic. Chron. 1908 p. 47. 64Long J . Amer. Chem. SOC. 1908 30 881. D. G. DERVICHIAN I 5 Conclusion and Tentative Structure of Natural Lip0 -Proteins I t is easy to conceive that with as many varied constituents as we find in the cell or in serum associations may come undone and be rebuilt in new compositions .in the course of extraction. Water being the indispensable structural constituent if one tries to " extract " by first dehydrating the system the association may vanish.In other cases the extracted complex may well be an artefact. This means that the lipids and the proteins may well be associated in the aqueous phase of the cell or the plasma differently and with some other constituents than those which come out by precipitation. The solubilization of esters of cholesterol and glycerides their simultaneous occurrence with phospholipids and their mode of ex- traction in particular their extraction by substitution of a molecule of similar form all suggest that non-ionic lipids could be associated with ionic lipids in mixed micelles which can be dispersed in aqueous solution. The identical behaviour of natural lipo-proteins and arti- ficial lipid-protein associations favours an interaction of ionic nature between protein particles and lipid micelles.This interaction can take place as in all ionic interactions in solution between dissociated com- ponents which remain separated The union of these particles gives rise to precipitation. This is not in contradiction to the evidence of associated migration in an electric field or simultaneous sedimentation 5 a in a gravitational field. Association in mixed micelles increase the solubility of lipids. In addition ionization of the protein can because of the interaction increase the ionization of the lipid and therefore increase its solubility and dispersion. The following structure is compatible with all these properties. In solution proteins and associated lipids constitute two sets of separated micelles.Interaction takes place between these two species of micelles and all other small ions present producing neutralization of their charges in every small volume element of the solution. This state would be represented in the isotropic media of the cells or the plasma. The alternating lipid and protein layer structure of the myelinic nerve sheath 6 5 9 formed by the same type of elements but arranged regularly would represent the other extreme case. Thus the lipo-protein solution would be to the paracrystalline nerve sheath what the isotropic soap solution is to the middle-soap or neat-soap anisotropic phases.45 I wish to thank Mr. R. S. Titchen for his help in writing this paper in English. Service de Chimie physique Institut Pasteur Paris 66 Elkes and Finean in Swface Chemistry (Buttenvorths London Ig4g) 65 Palmer and Schmitt Cold Spring Harbor Spnp.Quant. Biol. 1940. 8 97. p. 289. STRUCTURAL ASPECT OF LIPO-PROTEIN ASSOCIATION BY D. G. DERVICHIAN Received 2nd June 1949 From a detailed analysis of all the available work done on the association between proteins and different colloidal electrolytes general and consistent conclusions could be derived concerning the simultaneous influence of pH, salt concentration and proportions of the two constituents ; the important point being that these different factors intervene in a similar and complementary way. From this and other considerations it is concluded that the interaction is purely ionic. The necessity of bringing in van der Waals’ forces in some cases results from the binding together of the molecules in the colloidal electrolyte micelle.The nature of the lipid-lipid associations is discussed from the point of view of mixed micelles. If their ionic behaviour is considered natural lipo-proteins are similar to the artificial associations of proteins with ionic colloids If the extraction of lipids is considered natural lipo-proteins show the characteristics of the artificial lipid-lipid associations. A tentative structure is proposed for the natural lipo-proteins in solution. Lipids would form separate mixed micelles in which the non-ionic are solubilized by the ionic. It is further postulated that a purely ionic interaction takes place between these mixed lipidic micelles and the protein particles as well as with all other small ions present.The natural lipo-proteins i.e. those encountered in living organisms, contain ionic lipids (phospholipids fatty acids) and non-ionic lipids (esters of cholesterol cholesterol glycerides). First will be examined the protein-ionic lipid association. No doubt it i s the protein-lecithin interaction which is the most instructive from the point of view of the natural lipo-proteins. But if one examines and compares simply the experimental facts very satisfactory correlations are found in the behaviour of lecithin alone and of the different associations protein-lecithin protein-fatty acids protein-synthetic detergents and in a general way protein-colloidal electrolytes such as gum arabic nucleic acid dyes.I t appears that non-ionic lipids cannot associate by them-selves with proteins while they can associate with the other ionic lipids. The study of those lipid-lipid associations coming next will introduce some structural considerations which can explain some of the specific behaviour of the natural lipo-proteins. Finally the behaviour and properties of natural lipo-proteins will be examined in the light of the conclusions of the preceding studies. Protein-Ionic Lipid Interaction The precipitation of protein-lipid systems gives certain information on the interaction of these two classes of substances. In certain cases, instead of ordinary precipitation or flocculation a fluid phase separates which is called coacervate.l Dervichian Research 1949 2 210.8 LIPO-PROTEIN ASSOCIATION (a) Mutual Influence of pH and Proportion on Precipitation. -Concerning zones of precipitation at certain proportions of the con-stituents and the influence of pH on these proportions the same peculiarities are found either in the protein-lecithin interaction 2-6 or in the interaction of proteins with other ionic lipids,5 detergents *-14 colloidal electrolytes such as gum arabic,l59 l6 or nucleic acid,16 l7 and dyes.1s-20 The behaviour of anionic detergents on the one hand and cationic detergents on the other are symmetric about the isoelectric point of the protein on which they act. (b) Influence of Electrolytes.-Even with lecithin alone the precipitation by an acid is inhibited by salts.3 The presence of neutral salts also inhibits the precipitation of lecithin by protein^.^ More exactly to precipitate lecithin by protein the acidity of the medium must be higher the more neutral salt is present.2 This property is also found in interactions with other colloidal electrolyte^.^ With anionic detergents the amount of detergent necessary to produce complete precipitation increases with the amount of added electrolyte.1° The same phenomenon is observed with cationic detergents in basic medium.ll3 l4 Above the isoelectric point precipitation by an anionic detergent is only possible in the presence of a considerable quantity of salt.In addition the ratio detergent/protein in the precipitate is nearly ten times its value in acid medium in the absence of salt.21 Finally coacervation of gelatin by gum arabic is strongly influenced by the presence of salts.15 For example 0.35 % KC1 is sufficient to suppress the separation.One sees that pH and saline concentration play an identical role and this in accord with the fact that with proteins alone the addition of salts causes an increase in the pH of the solution if the initial pH is less than that of the isoelectric point and a decrease in pH if the initial pH is greater than that of the isoelectric point.22 I t should be pointed out also that the protein and the other colloidal electrolyte each carries its gegenions which explains the fact that an excess of one or the other influences the precipitation in the same manner as ions of added salt or OH- or Hf ion (pH). a Mayer and Terroine Compt.rend. SOC. Biol. 1907 62 398. Handovsky and Wagner Biochem. Z. 1911 31 32. 4 Parsons Biochem. J . 1928 22 800. Przylecki and Hober Biochem. Z. 1936 288 303. 6 Feinschmidt Biochem. Z. 1912 38 244. 7 Matsumura Kolloid-Z. 1923 32 171. 8 Bull and Neurath J . Biol. Chern. 1937 XIS 163. * Putnam and Neurath J . Amer. Chem. Soc. 1944 66 692. lo Pankhurst and Smith Trans. Faraday SOC. 1945 41 630. l1 Schmidt 2. Physiol. Chem. 1943 277 117. l2 Elkes and Finean in Surface Chemistry (Butterworth London 1g4g) p. 281. l3 Dervichian and Magnant Bull. SOC. Chim. biol. 1947 29 655. l4 Polonovski and Macheboeuf Ann. Inst. Pasteur 1948 74 196 l5 Bungenberg de Jong and Dekher Kolloid. Beih 1935 43 143 ; 1936 43, l6 Dervichian and Magnant Bull. SOC. Chim.biol. 1947 29 660-666. l7 Bungenberg de Jong and Hoskam Proc. Ned. Akad. Wetensch. 1942, Mathews Amer. J . Physiol. 1898 I 445 lo Chapman Greenberg and Schmidt J . Bzol. Chem. 1927 72 707. 20 Rawling and Schmidt ibid. 1929 82 709. 21 Pankhurst and Smith Trans. Faraday SOC. 1944 40 565. 22 Schmidt The Chemistry of Amino Acids and Protezns (Charles C . Thomas, 213. 45 387. 1945) p. 1209 D. G. DERVICHIAN 9 (c) Interaction in Solution. - The separation of proteins by other colloidal electrolytes can occur only in a pH region such that the net charge of the protein is opposite to that of the precipitating agent There are nevertheless interactions between the two con-stituents in regions where the net charges are of the same sign and where the constituents remain in solution.These interactions have been demonstrated mostly by electrophoresis studies.23 24 Well-defined boundaries appear which correspond to the migration of the associated protein and detergent. These interactions have also been demon-strated by measurements of rotation of plane polarized light surface tension and pH.25 More recently Pankhurst 26 has added evidence based on viscosity measurements. Interactions between acid or basic dyes and proteins outside the pH zone of precipitation has been demonstrated by measurement of the shift of absorption bands and determination of the quantity of dye held by the protein in dialysis eq~ilibrium.~' ( d ) Nature of Protein-Ionic Lipid Interaction.-Mayer and Terroine established that in an electric field the charge (positive) of the lecithin-albumin complex was opposite to that of lecithin alone.It was Putnam and Neurath9 who were the first to realize the con-nection between the maximum quantity of detergent bound and the total acid-binding capacity of the protein. They concluded that pre-cipitation was produced by electrostatic forces between the ionized groups of the two constituents. In studying the electrophoresis of small drops of gelatin-gum arabic coacervate Bungenberg de Jong and Dekker l5 found they were drawn to the positive or negative pole according to the relative amount of gum arabic. For each pH the change of sign occurs in the neighbourhood of the maximum separation. It appears then as if coacervation were connected to the mutual neutral-ization of the charges of the two colloids.In what form is the lipid bound to the protein in the precipitate? The method of titration used by Parson might give us some informa-tion on this point. The lecithin present in the precipitate was titrated by dissolving in it a lipo-soluble dye (but not water-soluble). This dye remained in the associated lipid. There is strong evidence that lipo-soluble dyes (like other lipo-soluble substances) can only be dissolved between the paraffinic chains of lecithin or detergent when these mole-cules are associated in m i ~ e l l e s . ~ - ~ ~ The work of Parsons tends to prove that in the protein-lecithin precipitate the lecithin is associated in the form of lamellar micelles. The fact that this micellar structure is necessary is shown by the observation that proteins cannot be pre-cipitated by detergents (anionic or cationic) having less than 10 carbon atoms.Besides it is well known that at ordinary temperature, micelle structure exists only in solutions of ionic lipids having more than 8 carbon atoms in their chain. To satisfy all these conditions i.e. ionic interaction between lipids and proteins necessity of micellar state for the lipid and the appearance 23 Lundgren Etam and O'ConneII J . Biol. Chem. 1943 149 183. 24 Putnam and Neurath ibid. 1945 159 195. 25 Desreux and Fabry Bull. SOC. Chem. biol. 1946 28 478. 26 Pankhurst ibid. 1949 31. a8 Dervichian and Magnant Compt. rend. SOC. Biol. 1946 140 95. 28 Kiessig and Philippoff Naturwiss 1939 27 593. 30 Hughes Sawger and Vinograd J .Chem. Physic. 1945 13 131. Klotz Chem. Rev. 1947 41 373. A I 0 LIPO-PROTEIN ASSOCIATION of paraffinic character in organic solvents,2* 31 one could suggest that in the precipitate the double layer micelles of the lipid stick by their ionic faces to the ionic faces of the protein particles and thus con-stitute alternate layers of lipid and protein.32* 33 The structure proposed here might not seem to differ from that of Pankhurst and Smith.2l In fact it originates from a different point of view. The interaction of the lipid micelle as a whole has to be con-sidered at the moment of precipitation starting from the principle that no lipid molecules can adsorb in a single layer without the double layer lattice being built up and precipitation following. One could as well say that the micelle of the detergent fixes the protein particles by its two external ionic faces (and vice versa) and that the ionic lattice (Hartley) being thus formed similarly to the crystal of fatty substances, the whole precipitates.Should the “ dissolved complexes” be considered as formed of complex particles with lipid molecules sticking to the protein molecules as they are in the precipitate? To see the problem clearly one must not lose sight of a very trivial fact ionic combination in solution does not mean aggregation of oppositely charged ions in one particle. Proportions are stoichiometric whether the reaction gives rise to aggregation and precipitation or to a dissociated compound which remains in solution. It is generally admitted that the length of the lipid or detergent chain serves to increase the forces of interaction between the lipid and the protein in a discontinuous way when there are more than eight carbons in the lipid chain.24* 34p 35 Some see here a specific action.Thus two factors would determine the force of the interaction an electrostatic attraction due to oppositely charged ions and van der Waals’ forces. This is also the conclusion arrived at by Klotz 27 con-cerning the bonds between proteins and dyes. The basis of this con-clusion is the difference in behaviour between dyes having one charged group and those having two or three charged groups. However the point should be considered that with amphipatic molecules the tendency to form micelles diminishes with the increasing number of solubilizing groups.I t seems reasonable to think that the necessity of bringing in van der Waals’ forces is not concerned with the bond between the protein and the lipid but with the linkages which hold together the lipid molecules in the lipid rnicelle in the neighbourhood of the protein particle. The interaction between the lipid micelle as a whole and the protein particle is purely ionic. The “ soluble compounds ” are formed in the pH region where the protein and the lipid have the same charge. How can ionic inter-actions take place under such conditions? The answer is given by Schwert Putnam and Briggs 36 who admit that such interactions may take place in a pH range where the two components have a net charge of the same sign provided there exist ionized groups on the protein opposite in sign to that of the net charge.Klotz2’ reached the same conclusion concerning the interaction between dyes and proteins. 3l Pankhurst in Surface Chemisfry (Butterworth London 1949)~ p. 109. 32 Elkes Frazer Schulman and Stewart Proc. Roy. Soc. A 1945 184 102. 93 Palmer Schmitt and Chargaff J . Cell. Camp. Physiol. 1941 18 43. 34 McMeekin Federation Proc. 1942 I 125. 35 Boyer Ballou and Luck J . Biol. Chem. 1947 167 407. a6 Schwert Putnam and Briggs Arch. Biochem. 1944 4 371 D. G. DERVICHIAN I 1 But how is it that the bond is purely ionic when electrophoresis shows sharp boundaries indicating migration of the two associated constituents with no dissociation ? Such migration toward the same electrode of an anion and a cation have been found by Hartley Collie and Samis 37 with a small ion like Br- associated with a cationic detergent in cetylpyridinium bromide.The bromine ion mobility is so much reduced that it becomes actually negative. More bromine is carried in the reverse direction attached to the micelles than travels un-attached in the normal direction. The adherence of gegenions to the micelle is due to the attraction of the free gegenions into a dense atmosphere around the micelle.= Thus emerge the particular con-ditions which exist at the surface of the micelles of the colloidal electro-lytes due to the high concentration of packed charges. However, in protein-detergent association conditions are even more favourable than those of cetylpyridinium bromide because the electrophoresis is carried out at a pH where both substances have net charges of the same sign and both have micelle structure.One might think that the interaction is localized a t the positive ions of the protein (e.g. protein-anionic lipid interaction at an alkaline pH), only single molecules of the lipid being able to approach and not compact micelles because of the repulsion of the negative protein ions. This is where van der Waals’ forces acting between paraffinic chains in the lipid micelle must come in. This means that it requires more energy to keep paraffin chains separated in the presence of water than t o draw some of the anionic groups of the detergent near to the anionic groups of the protein. Besides if groups of the same sign always repelled one another soaps would never crystallize nor show para-crystalline phases in water.In the lamellar structure of soap the COO- groups are face to face in what Hartley calls an ionic lattice. No doubt this ionic lattice is stabilized by the presence of small Na+ ions. In the present case the small cations of saIt present in the system or even the amino groups of the protein themselves would play the same role. Whatever is the mechanism we can at least say that it is sufficient to have a few points of attraction between the positive groups of the protein and the negative groups of the lipid for the whole lipid micelle to be held in the neighbourhood of the protein molecule. If the solu-tion is acid with respect to the isoelectric point of the protein the ionic interactions become overwhelmingly important and there is precipita-tion.The same reasoning can be applied mutatis mutandis to cationic lipid-protein interaction. In coacervation of gelatin or haemoglobin by gum arabic excess of one constituent causes both constituents to pass into solution. It cannot be seen how the acid groups of gum arabic could be fixed individually to a greater or smaller number of the amino groups of the protein nor could be understood how a second layer of gum arabic could be formed making the surface soluble. Such assumptions have been made to explain the same phenomenon with The influence of the quantity of electrolyte present either on the pH of optimum precipitation or on the proportion of detergent 37 Hartley Collie and Samis Trans.Faraday Soc. 1936 32 795. 38 Hartley Aqueous SoEutions of Parafin-chain Salts (Hermann et Cie Pans, 39 Lundgren Textile Res. J. 1945 15 335-39 7936) I 2 LIPO-PROTE IN ASSOCIATION precipitated shows that the whole phenomenon is a matter of neutraliza-tion of charge in a given volume element. This explanation is similar to that given in the case of ordinary electrolytes. This spatial neutral-ization in which small and large ions take part causes either precipita-tion or changes in velocity of migration in an electric field. We saw that the presence of salt inhibits precipitation. This effect of small ions may be duplicated by the lipid ions. An excess of detergent (carrying its gegenions) produces the same inhibitory effect by per-turbing the ionic atmosphere.This appears as if the acid binding capacity of the protein were modified (see (a) and (b) above). In the experiments of Pankhurst and Smithz1 a t pH above the isoelectric pH the quantity of detergent fixed is ten times the acid binding capacity. These authors (as has Steinhardt *O) suggest that this quantity corresponds to fixation by the amide groups following shifting of their dissociation constants. This point of view can be reconciled with that presented here if instead of fixation we consider the effect of compensation of charge in each small volume element. Other models have been proposed in which the bond is made by the paraffinic end of the detergent linking itself to the paraffinic groups of the protein. Such models have been proposed by Macheboeuf and S a n d ~ r ~ l by Dervichian,42 and by Palmer.43 The accumulated proofs, direct and indirect of the ionic nature of the phenomena seems now to exclude such a possibility.In the model here proposed there would not be fixation of the deter-gent to the protein. Fixation results in precipitation just as with ordinary ionic compounds. In solution what we call a compound or a complex is dissociated but the charge can be modified by the presence of H+ or OH- ions (action of pH) or by other small ions of salts present. A reason for this effect to be so apparent is that the ions of the detergent and the protein are strongly concentrated at the surface of their re-spective micelles. The proximity of the charges introduced by the protein by the fact that they are opposite to that of the lipid could tend to ionize the detergent groups.Thus some ionic lipids insoluble in water, are made soluble as soaps are in the presence of alkaline metal ions. Lipid -Lipid Associations A whole series of associations in the bulk can be obtained in definite proportions of lipids in contact with water.44 Generally speaking, i t is the structure of the constituents which comes into play and not a particular substance (e.g. a molecule of a long-chain aliphatic com-pound associated with a steroid molecule). Consequently in such an association a molecule may replace another of a similar type. A systematic study of a great number of pairs of lipids the one soluble and the other insoluble have allowed detection of a gradation going from simple swelling to complete d i s ~ e r s i o n .~ ~ In complete 40 Steinhardt Atan. Rev. Biochem. 1945 1 4 ~ 145. 41 Macheboeuf and Sandor Bull. SOC. Chim. biol. 1932 1 4 ~ 1168. 42 Dervichian J . Chim. physique 1941 38 59 ; J . Chem. Physics 1943 I I , 43 Palmer J . Physic. Chem. 1944 48 12. 44 Dervichian and Magnant Bull. SOC. Chim. biol. 1946 28 419; Comfit. 46 Dervichian Trans. Faraday SOC. 1946 42B 180. 219. vend. SOC. Biol. 1946 140 94 D. G. DERVICHIAN I 3 dispersion the two constituents are associated in mixed miceiles. Substances insoluble like cholesteryl oleate can thus be dispersed in ~ a t e r . 4 ~ Myelinic figures formed by such associations have a clearly stratified structure 4 7 s @ composed of double lipid layers separated by layers of several mole-cules of water.In each lipid layer the different types of molecule are oriented side by side their polar groups directed toward the water and their paraffinic chains facing the paraffin chains of the adjoining layer likewise oriented toward the water. The same double layer structure is preserved in other forms of swelling and one is led to suppose that it is preserved also in the separate micelles when completely dispersed. The association cannot be explained on purely chemical grounds but from a crystallographic point of view. The principal point is that associ-ation cannot be realized except in the presence of water and that water itself must be regarded as one of the structural constituents. Thus is explained the failure of the authors who have tried to isolate complexes in the absence of water.49 The manner of association of molecules of lipids is known.Natural Lipo-Proteins A general examination of lipo-proteins shows a striking parallelism to that of protein-ionic lipid and lipid-lipid associations. We are speaking here only of lipids masked in serum or the cells and not those which are found in the form of microscopic or ultramicroscopic particles (for details see for example50). (a) Ionic Behaviour.-The lipo-proteins of serum precipitate a t a pH lower than the isoelectric pH and go into solution again when the pH is raised to 7,51 exactly like anionic lipid-protein associations. The fact that natural lipo-proteins are not dissociated in the elec-trical field 5 2 9 53 does not as thought by Cohen and Chargaff,53 plead against the assumption of a simple saline bond between proteins and phospholipids.I t was shown that not only in protein-detergent mixtures but even in cetylpyridinium bromide both constituents or ions migrate together. (b) Extraction of Lipids.-From the point of view of extraction, natural lipo-proteins show predominantly the characteristics of lipid-lipid associations. I t is not a matter of solubility accident contrary to the idea of Cha~-gaff,~* “ that substances as disparate as cephalin and cholesterol” are extracted jointly. This type of pair gives the best lipid-lipid association. Extraction by ether can only be accomplished after dehydration by cold alcohol for example (Hardy and Gardiner method). This is 46 Valette and Cavier Bull.SOC. Chim. biol. 1938 20 1256. 47 Nageotte Compt. rend. 1927 185 1021. 48 Browaes and Dervichian Compt. rend. SOC. Biol. 1946 140 136. 49Partington J . Chem. SOC. 1911 99 313 318. 60Elkes Frazer and Stewart J . Physiol. 1939 95 68. 51 Macheboeuf Bull. SOC. Chim. biol. 1929 1 I 268 and 485. 63 Cohen and Chargaff Biochem. J. 1940 136 243. 54 Chargaff Advances in Protein Chemistry (1944) I I 8. Frazer Trans. Faraduy SOC. 1941 37 125. Macheboeuf Delsal Lepine and Giuntini Ann. Inst. Pasteur 1943 69 321 I4 LIPO-PROTEIN ASSOCIATION identical with the behaviour of lipids alone or lipid-lipid associations which cannot be extracted by ether as long as they remain dispersed in water in the form of micelles. The lowering of the temperature reconstitutes the ionic lattice45 (Krafft point) even in the presence of water and makes association impossible (e.g.myelinic forms do not develop). Extraction by ether becomes then possible. This explains the effectiveness of the method of M ~ F a r l a n e . ~ ~ The presence of alcohol destroys the micelle structure 56 and prevents lipid association. This explains why the extraction of purely lipidic associations by ether is not possible a t ordinary temperature except in the presence of alcohol,57 58 and allows us to understand why the addition of alcohol to serum helps the extraction of lipids by ether.41 Macheboeuf and Tayeau 59 showed that the addition of soap to serum permits extraction by ether. In addition i t can be seen in the eurves of these authors that the maximum of extraction is realized for a soap/protein ratio of 0.4 which is exactly that found as binding capacity of proteins towards detergent^.^ There is therefore sub-stitution from the point of view of ionic interactions we have seen that fatty acids themselves associate with proteins.The quantity of extracted lipids then diminishes with increasing proportion of soap. This might be due to the association of the excess of soap with the separated lipids keeping them in solution. The action of heparin on the extraction60 is similar to that of soap. (c) Lipid-Lipid Association.-The fact that cholesterol and phospholipids are liberated simultaneously by the action of soap seems t o indicate that there is substitution of soap micelles for the mixed cholesterol-phospholipid micelles as a whole.In addition the specific extraction of cholesterol alone by substitution of molecules of similar structure (saponin with a steroid structure 61 or sodium dehydro-abietate62) reminds us of the typical specific conditions of the purely lipidic associations and seems to show that cholesterol is directly bound with the phospholipids in mixed micelles. Tayeau61 had already been led to the conclusion that cholesterol is mainly bound to the phosphatides and that phosphatides might form the link between cholesterol and the proteins. White63 in 1908 had already foreseen that cholesterol is normally present in the tissues of animal in a state of “weak combination ” with fatty acids and lecithin forming liquid crystals.The suggestion has been made that a t least a small part of the lipids were bound to the protein by stronger bonds. This conclusion is based on the fact that part of the lipids cannot be extracted by the above-mentioned methods. In fact lipids might be included in the structure of the protein. Nevertheless it should be pointed out that this behaviour is not particular to natural lipo-proteins as it is also found with ionic lipids alone or artificially associated to protein^.^ 64 s6 McFarlane Nature 1942 149 439. s6 Ward Proc. Roy. SOC. A 1940 176 412. s7 Delezenne and Fourneau Bull. SOC. chim. 1914 15 421. Baranger Ann. Physiol. 1937 13 341. 69 Macheboeuf and Tayeau Bull. SOC. Chim. biol. 1941 23 49. b o Chargaff 2% and Cohen J . Biol. Chem. 1940 136 257.61 Tayeau Bull. SOC. Chim. biol. f944 26 287. 62 Macheboeuf and Rebeyrotte ibzd. 26 475. 6 3 White Medic. Chron. 1908 p. 47. 64Long J . Amer. Chem. SOC. 1908 30 881 D. G. DERVICHIAN I 5 Conclusion and Tentative Structure of Natural Lip0 -Proteins I t is easy to conceive that with as many varied constituents as we find in the cell or in serum associations may come undone and be rebuilt in new compositions .in the course of extraction. Water being the indispensable structural constituent if one tries to " extract " by first dehydrating the system the association may vanish. In other cases the extracted complex may well be an artefact. This means that the lipids and the proteins may well be associated in the aqueous phase of the cell or the plasma differently and with some other constituents than those which come out by precipitation.The solubilization of esters of cholesterol and glycerides their simultaneous occurrence with phospholipids and their mode of ex-traction in particular their extraction by substitution of a molecule of similar form all suggest that non-ionic lipids could be associated with ionic lipids in mixed micelles which can be dispersed in aqueous solution. The identical behaviour of natural lipo-proteins and arti-ficial lipid-protein associations favours an interaction of ionic nature between protein particles and lipid micelles. This interaction can take place as in all ionic interactions in solution between dissociated com-ponents which remain separated The union of these particles gives rise to precipitation. This is not in contradiction to the evidence of associated migration in an electric field or simultaneous sedimentation 5 a in a gravitational field. Association in mixed micelles increase the solubility of lipids. In addition ionization of the protein can because of the interaction, increase the ionization of the lipid and therefore increase its solubility and dispersion. The following structure is compatible with all these properties. In solution proteins and associated lipids constitute two sets of separated micelles. Interaction takes place between these two species of micelles and all other small ions present producing neutralization of their charges in every small volume element of the solution. This state would be represented in the isotropic media of the cells or the plasma. The alternating lipid and protein layer structure of the myelinic nerve sheath 6 5 9 formed by the same type of elements but arranged regularly would represent the other extreme case. Thus the lipo-protein solution would be to the paracrystalline nerve sheath what the isotropic soap solution is to the middle-soap or neat-soap anisotropic phases.45 I wish to thank Mr. R. S. Titchen for his help in writing this paper in English. Service de Chimie physique, Institut Pasteur, Paris, 65 Palmer and Schmitt Cold Spring Harbor Spnp. Quant. Biol. 1940. 8 97. 66 Elkes and Finean in Swface Chemistry (Buttenvorths London Ig4g), p. 289

ISSN:0366-9033    

DOI:10.1039/DF9490600007

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

16 GENERAL DISCUSSION GENERAL DISCUSSION * Dr. K. G. A. Pankhurst (London) said: According to Dr. Der- vichian’s model the matrix of the complex is the detergent micelle, but we have found that it is possible to observe these phenomena at sub- micellar concentrations, e.g., with very dilute gelatin solutions a t pH 2 in the absence of inorganic salt and ca. I O - ~ M dodecyl sodium sulphate, or even with octyl sodium sulphate. Any hypothesis to explain the formation of detergent-protein complexes must account for the observa- tion that, as the detergent-protein ratio (DIP) is increased the complexes become increasingly ZipophiZic; (and may in certain circumstances be thrown out of solution as a precipitate or a coacervate) until a critical DIP is reached where water solubility is minimal, and that increasing D / P still further causes the complex to become increasingly hydrophilic.The model that I have suggested has the protein as its matrix and does not require micelle formation for its initiation. Dr. D. G. Dervichiant (Paris) said : Dr. Pankhurst thinks that inter- actions between proteins and lipids giving phase separation can occur below the critical concentration of micelle formation of the ionic lipid. I agree that interactions (i.e. charge neutralization in each element of volume) must take place between protein and lipid even if the latter is molecularly dispersed. But the point in question is that the best pre- cipitants are the molecules containing more than 8 carbon atoms, 2 4 s s4 higher concentrations of C, or Clo being required as compared to the effective concentration of dodecyl sulphate.Dr. Pankhurst has shown us that very small concentrations of octyl sulphate do give coacervation with gelatin at pH 2. This is in accord with the general conclusions on the mutual influence of pH. and proportion on precipitation. It has also been shown 2 4 ~ 2 4 9 36 that the force of interaction between the lipid and the protein is considerably and suddenly increased when there are more than eight carbon atoms in the lipid chain. If the paraffinic chain of the detergent contributed directly to strengthen the bond with the protein (by van der Waals’ forces), it would be difficult to understand the sudden increase of this binding energy with the appear- ance of the micellar dispersion above a certain chain length.The view advanced in my paper is that the necessity of bringing in van der Waals’ forces is due to the linkages which hold together the lipid molecules in the lipid micelle. This explains also the fact that with dyes having more than one charged group, the binding diminishes.2? In fact, it is well known that the tendency to form micelles diminishes with molecules having more than one charged group. Finally, the micellar structure of the lipidic constituents in natural lipo-proteins would explain particularly the solubilization of the in- soluble lipids. This might be accomplished by the formation of purely lipidic mixed micelles. Dr. J. Elkes (Birmingham) said: In keeping with Dr. Pankhurst’s observations we, also, have observed the formation of protein-detergent complexes at submicellar concentrations of detergent.This was partic- ularly apparent with a chromoprotein such as oxyhaemoglobin. Using I O - ~ M solution of sodium hexadecyl sulphate and dilute solutions of oxyhaemoglobin, the typical zoning phenomenon, showing both insoluble and soluble complexes, was regularly seen. A tentative structure for these complexes has been suggested.2 Dr. D. G. Dervichian (Paris) said : In the work to which Dr. Elkes refers the action of hexadecyl sulphate on oxyhaemoglobin has been observed at room temperature. The insoluble complexes were obtained at pH values below 6.1 and ranging down to pH 5.1. It is highly prob- 1 This Discussion. * On preceding paper. -f Reference numbers here and in my later remarks are to my paper.2 Elkes and Finean, Surface Chemistry (Butterworth, 1949). p. 281.GENERAL DISCUSSION I 7 able that at temperatures below 3ooC and at acid pH values, I O - ~ M is not a I ‘ submicellar concentration ” (i.e. below the “ critical concentra- tion of micelle formation ”) for sodium hexadecyl sulphate. That is to think that, for a given detergent, the critical concentration is independent of temperature and pH. First of all, for each detergent, there is a fairly sharply defined temperature above which the detergent is dispersed in the micellar form giving a colloidal solution and below which the system appears as a suspension of microcrystals in water. Above this tem- perature, the critical concentration of sodium hexadecyl sulphate (i.e.the maximum quantity of detergent soluble under the molecular dis- persed form, saturation producing the appearance of micelles) is certainly higher than IO-~. Below this temperature, there is no more question of micelles, the dissolved molecules are in equilibrium with precipitated microcrystals and the saturation concentration (and not the ‘ I critical concentration ”) is certainly less than I O - ~ . The evidence that in the work quoted by Dr. Elkes the detergent at room temperature was below this particular temperature is given by the indication that the detergent “ solution ” was previously heated at 40’ C before being pipetted into each tube. Similarly, at a given temperature, decrease of pH below neutrality transforms the micellar solution again t o a suspension of microcrystals.For example, the molecular solubility of potassium laurate was studied in my laboratory at 23OC, which is above the critical temperature, If for alkaline solutions the critical concentration is nearly I O - ~ , a t pH 5 the solubility falls to rather less than I O - ~ M. The experiments quoted by Dr. Elkes cannot therefore be invokevd in support of Dr. Pankhurst’s remark, particularly as far as the insoluble complexes (precipitate) are concerned. As I have admitted in my reply to Dr. Pankhurst, interactions must take place in solution between protein and lipid even if the latter is mole- cularly dispersed. In my opinion, however, this interaction corresponds simply to charge neutralization in each element of volume of the solution. In support of this idea, Elkes and Finean’s work was one of those I have quoted in my paper concerning the striking relation between pH and detergent /protein proportion.Dr. J. Elkes (Birmingham) (communicated) : Far from being unaware of the effects of either temperature or pH on the critical micellar con- centration of detergent, detergent-buffer controls (without protein) were thought necessary and put up in each and every experiment.l Moreover in the paper referred to by Dr. Dervichian,l we give the concentration of detergent used as 0.13 yo. It is at this high concentration that heating to 4ooC was necessary to ensure ‘ I solvation ”. Lower detergent con- centrations (10-4 M) were only employed in later unpublished experi- ments, and it is to these that we wish to refer.Dr. A. S . McFarlane (London) said : Dr. Dervichian has found a general similarity in natural and artificial lipo-proteins, including those of serum. I have failed entirely t o prepare artificial serum lipo-proteins using a variety of lipids-cholesterol, cholesteryl oleate, triolein, phospho- lipids, and either defatted serum, or a serum of low natural lipid con- tent. Either the emulsion is not optically clear, or if it is, as for example, when using lecithin, the lipid does not migrate with any of the protein components seen in the electrophoresis apparatus. I would like to know if Dr. Dervichian or anyone here has been successful in doing this. Dr. D. G. Dervichian (Paris) said : Non-ionic lipids cannot be dis- persed in the presence of serum. It is possible, however, to get complete dispersion by associating substances of different molecular structure in proportions which vary with the nature of each constituent.Yet these dispersions cannot be obtained by simple addition of the different con- stituents to water or serum : very intimate mixing approaching the molecular scale must be This draws attention to a rather frequent error. 45s 4618 GENERAL DISCUSSION Regarding migration in an electric field, it is certainly influenced by pH and the proportions of lipid and protein. Boundaries common to the protein and the detergent have been observed by some 24 Dr. J. A. V. Butler (London) said : I should like to make an element- ary point. Both the speakers have referred to “ protein” without specifying what protein they mean.Everyone will agree that proteins vary enormously in their properties and are highly specific in their be- haviour. Surely it is desirable always to state what protein is referred to. We can hardly expect all proteins to act similarly, even with respect to detergents. The fact that one protein behaves in one way and another in another wa.y is not very surprising. Prof. D. G. Dervichian (Paris) said : The whole interest of the com- parison of the results obtained by different authors 2-27 resides precisely in the point that the same behaviour has been observed whatever the specified protein or the ionic lipid. General and consistent conclusions can definitely be deduced concerning the related influence of pH, con- centration of electrolytes, and proportions of protein and ionic lipid.Dr. J. T. Edsall (Harvard) said: Dr. Dervichian has emphasized the ionic character of the surface of protein molecules. However, in addition to the positively and negatively charged ionic groups, there are also numerous non-polar side-chains, so that an ion such as an alkyl sulphate would presumably adhere to a protein surface with the sulphate radical in proximity to a poFitively charged group on the protein, while the alkyl side-chain could lie next to some adjoining non-polar side- chains of the protein. This seems a much more likely configuration than one in which the alkyl group projects out into the solvent, away from the protein surface. I should like to second what Dr. Butler has said about the specificity of the reactions of different proteins with lipids and related substances.I do not believe that the profound difference between serum albumin and y-globulin, for example, in their interactions with dyes and with fatty acid anions, can be explained by any simple electrostatic mechanism. y-Globulin contains numerous cationic groups, which should be capable of binding added anions ; yet it shows no specific binding of the sort so characteristic of serum albumins. Moreover, as Dubos and Davis have shown, the binding is virtually abolished by heat denaturation of the albumin, although the positively charged groups remain. Some highly specific configurations in the native protein must evidently be required for the reaction. * I doubt whether the failure of y-globulin to bind methyl orange can be explained by the pH and the isoelectric point of the protein.Klotz and Urquhart cairied out experiments both at pH 5.7, where the globulin is positively charged, and at 6.8, where it is almost isoelectric, and found no binding in either case. If there were binding at more alkaline pH values, this would be surprising, since the electrostatic effect of the negative charge on the protein would repel the anionic methyl orange, and make combination more unlikely than at an acid pH. Many anions are indeed bound by serum albumin, even when the protein is negatively charged, but the recent studies of Scatchard and his associates4 using chloride and thiocyanate, show that binding of these anions increases as the net charge on the protein becomes more positive (or less negative), and the variation with protein charge can be satisfactorily explained by the Debye-Huckel theory.As to van der Waals’ forces, it seems to me they must play a role in such phenomena as the binding of fatty acid anions by serum albumin. Dr. Luck’s extensive studies have shown that the binding becomes stronger for each additional carbon atom, in the series of anions from * I n reply to Dr. Dervichian’s remark, p. 19. 3 J . Amer. Chem. SOC., 1949, 71, 1597. 4 Scatchard, Scheinberg and Armstrong, Jr., ibid. (in press:.GENERAL DISCUSSION I9 acetate to caprylate. Moreover, the binding is strong at concentrations of free anion far below those required for micelle formation. I should not care to generalize further. Many of the phenomena discussed a t this meeting, by Dr.Dervichian and others, clearly require explanation along very different lines. Dr. D. G. Dervichian (Paris) said : The experimental evidence 27 is that methyl orange binds less to y-globulin than to serum albumin. Now these experiments have been done at pH 5.7, that is on the basic side of the isoelectric point for serum albumin and on the acid side for y-globulin. Another experimental result l4 is that cationic detergents do precipitate with y-globulin. It is obvious that specific differences exist between proteins, one of these differences being precisely the isoelectric point, and the most striking being the immunochemical specificity These singularities are very probably bound to differences in the molecular structure and configuration (this being pure interpretation). Dr.Edsall thinks that the contrast between serum albumin and y-globulin in their possibilities of association is due to differences in the possibility of binding by van der Waals’ forces and cannot be explained by simple ionic bonds. Now it is well known that the behaviour in the presence of ordinary electrolytes of albumins is also different from that of globulins (i.e. very different concentrations of (NH,) 2S04 are necessary to produce precipitation). Yet in this case it is obvious that the inter- action is purely ionic. Similarly, if denaturation of proteins virtually abolishes the binding with lipids, it also modifies profoundly the inter- action with ordinary electrolytes (e.g. precipitation). Yet no one would think of bringing in some highly specific configuration to explain the action of ordinary salts on proteins.No doubt the structure of proteins is highly specific, but it is not certain that this specificity of configuration intervenes in the interaction between proteins and ionic lipids. In fact, we were able to obtain phase separation (precipitation or coacervation) using indifferently either anionic and cationic detergents or electrolytes such as (NH,),S04. Finally, if by van der Waals’ forces the bond were in specific relation to the nature of the lateral non-polar chains, it would be difficult to understand how, with a given protein, associations could be obtained with substances as different as fatty acids, phospholipids, nucleic acids, gum arabic, or dyes, whose paraffin chains are very different from the structural point of view.Prof. E. Chargaff (New York) said : To me complexes between un- specified proteins and gum arabic, or even soaps, do not look exactly like my conception of lipo-proteins. In considering individual lipo- proteins we shall have to pay attention not only to the polar character- istics of the lipid and of the particular protein, but also to the structure of the latter. Obviously, serum albumin or globulin will not behave in the same manner as thymus histone towards lecithin or phosphatidyl serine. Simplification has, of course, its uses ; but by carrying model experiments to such lengths, I am afraid, we may end up with the proverbial “ knife without handle which has lost its blade ”. Dr. D.G. Dervichian (Paris) said : Prof. Chargaff’s remarks are highly instructive. They show how far force of habit and mere repetition of assertions often appear as argument even in scientific reasoning. From the fact that lipo-proteins are not dissociated in the electrical field, it has been concluded by Cohen and Chargaff 53 that the bond is not a simple saline bond, Now this conclusion is implicitly based on the very familiar behaviour of the very simple electrolytes in an electric field, where neither the anion ( I ‘ blade ”) nor the cation (“ handle ”) resemble the lipo- protein (“ knife ”) constituents. Thus, while it seems very sound to compare a lipo-protein to NaC1, a colloidal electrolyte such as cetyl pyrid- inium bromide (which at least has a micellar cation) or even a protein-fatty acid mixture, are considered as models very remote from lipo-proteins.20 GENERAL DISCUSSION It would be rather unfair to the authors of the twenty-six papers 2-2; from which general and consistent conclusions have been derived, to assert that their work refers to unspecified proteins.To say that natural lipo- proteins look exactly similar to the artificial mixtures of proteins and other colloidal electrolytes is certainly overlooking and overstating some of the conclusions of this paper. The unquestionable fact which comes out from a detailed analysis of all the available work done on these associations in the course of the past forty years is that, whatever the protein (serum albumin, egg albumin, casein, edestin, haemoglobin, globulin, gelatin), or the other colloidal electrolyte (lecithin, fatty acids, anionic or cationic detergents, long-chain choline esters, bilirubin, nucleic acid, gum arabic, basic or acid dyes), the behaviour as function of the pH, salts present, and proportions, is the same.It can be asserted, on the other hand, that : (a) as far as pH, action of salts and electrophoretic migration are concerned, natural lipo-proteins behave in a way parallel to that of the different artificial associations mentioned above ; (b) with regard to extraction and substitution of lipids, as well as the solubilization of the non-soluble lipids, are concerned, the behaviour of natural lipo-proteins recalls astonishingly the behaviour of artificial lipid-lipid associations. To ignore deliberately this generaliza- tion of facts would be to deny any interest in experimental results.Prof. M. Macheboeuf (Paris) said : I should like to ask Dr. Dervichian how his very interesting theory takes into account the specificity possessed by lipidic cenapses? I have been able to obtain from horse serum a cenapse which contains lecithin and esters of cholesterol only, whereas other lipids (non-esterified cholesterol, sphingomyelin, neutral fats) are found in other cenapses. How does Dr. Dervichian interpret this specifi- city, which seems to exclude certain lipid, although these are chemically closely related to those found in the cenapse ? Dr. D. G. Dervichian (Paris) said: I can only make a tentative suggestion to explain these facts. We were able to show 4 4 9 46 that, by associating lipids and similar substances, their affinity for water is strongly increased, sometimes reaching complete solubility under the form of mixed micelles.Thus, under their associated form, the lipids found in Prof. Macheboeuf’s lipo-protein might show a greater affinity for water. Furthermore, their ionization might be increased by the ionic interaction of the protein molecules, thus producing complete solubilization. There- fore, depending on the conditions of precipitation (i.e. decrease of solubility by addition of salts or variation of pH) the less soluble lipidic associations could precipitate with certain proteins while the others remain in solution. Dr. G. Popjak (London) said : It is easy to understand the postulated ionic lipid-protein association where the “ lipid ” is a soap, which readily dissociates, but does Dr.Dervichian think that in a lecithin molecule the esterified fatty acids dissociate ? Prof. D. G. Dervichian (Paris) said : Certainly not-the ions to be considered in the case of lecithin are those of the phosophorylated choline radical. Prof. E. K. Rideal (Londoz) said : We have to thank Dr. Dervichian for a most interesting paper. As I understand the matter, Dr. Dervichian is so perturbed about the flee energy change involved in a single mole- cule of lipid or detergent leaving its micelle and attaching itself to the protein by means of the polar group, leaving the non-polar group in the water, that he wishes to bring the whole micelle with him. I think we cannot give up the view that the non-polar portion of the detergent or lipid must take part in an interaction with part of the protein. This view is supported by penetration experiments, by drying, and by specificity of reaction, especially when we assume modification of structure of this portion of the molecule. We must remember that in reactions withGENERAL DISCUSSION 21 proteins it is frequently the protein carboxyl reaction which provides most of the energy. Dr. D. G . Dervichian (Paris) said : I n fact, account should be taken of the energy provided by the protein carboxyl reaction. The exis- tence of the lipids under the micellar form has to be assumed whenever it is necessary to account for extra van der Waals’ forces in the bond energy. This does not exclude the fact that single molecules would interact with the protein, as I have already admitted in my answer to Dr. Pankhurst. From my point of view, however, this interaction is again a charge neutralization in each element of volume, the two con- stituents remaining apart, and does not mean that the molecules of lipid stick on to the protein molecule.

ISSN:0366-9033    

DOI:10.1039/DF9490600016

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

21 GENERAL DISCUSSION FORMATION OF LIPO-PROTEIN MONOLAYERS PART 1,PRELIMINARY INVESTIGATION ON THE ADSORPTION OF PROTEINS ON TO LIPID MONOLAYERS BY P. DOTY * AND J. H. SCHULMAN Received 2nd June 1949 New techniques have been developed to follow the interactions of proteins with electrically charged monolayers a t an air-water interface. When steric factors prevent the protein molecules from entering the monolayer adsorption of the protein takes place and this is shown to be dependent on the sign of the electrical charge of the compounds which meet at the interface. This adsorption is essentially reversible and accounts for the flocculation and re-dispersion of emulsions stabilized by anionic or cationic detergents in contact with proteins a t different pH’s.The kinetics of penetration and ejection of proteins in contact with charged interfaces have been studied a t constant pressure. These processes are dependent on the electrical charge of the reactants and the surface pressure at which the experiments are carried out. Stoichiometric complexes are demonstrated between non-polar portions of the protein molecules and the long-chain ionic compounds and the general forces i.e. electrical forces and van der Waals’ forces involved in the association are reviewed. Whereas adsorption follows a reversible pattern monolayer expansion is essentially irreversible. The existence of “ bound protein I’ at the charged interface is demonstrated and interpreted. The bearing of these results upon the interaction of proteins with long-chain ionic compounds in bulk solution is considered.Previous work on protein monolayers suggests that radical changes of an irreversible nature take place on spreading of the protein at an air-water or oil-water interface. Force-area compression curves of the protein monolayers are rather irreproducible above certain surface pressures. This is due to the association of the reactive groups present in the large protein molecule. The forces involved in these associations are responsible for the gel state or highly viscous characteristics of protein monolayers. For the same reasons studies on monolayers obtained by spreading a solution containing protein lipids or protein- soluble surface-active agents are only of a qualitative nature and * Rockefeller Foundation Fellow ; present address Dept.of Chemistry Harvard University U.S.A. 1 Cockbain and Schulman Trans. Faraduy Soc. 1939 35 1266. LIPC-PROTEIN MOKOLAYERS 2 2 show little bearing on the behaviour of proteins in aqueous solution in the presence of long-chain ionic compounds. Injection of lipids or detergents under protein films leads to pene- tration or displacement of the protein and the experimental results are again difficult to interpret quantitatively.2 When non-surface- active agents or poorly surface-active materials are injected under- neath protein monolayers better results can be obtained. It has been shown that tanning agents possess a certain parallelism in their behaviour on protein films and on long-chain a r n i n e ~ .~ To study the interaction of proteins with lipids and detergents it is necessary to avoid structural alterations of the protein which happen by spontaneous spreading and unfolding of the protein mole- cule a t the interface. The protein is injected underneath the inter- acting monolayers maintained at a pressure equal or above 15 dynes/cm. and the reaction is followed in terms of the mechanical changes which occur through the injection. Such techniques lead to more quanti- tative information and explain the behaviour of protein solutions in contact with charged oil-water interfaces. A direct examination of the forces involved in protein-detergent association is also obtained. Further attempts are being made to study by this method immunity or pseudo-immunity reactions such as certain lipids may undergo when associating with serum proteins.Two samples of this system are cardiolipin with luetic serum and R.H. lipid with positive serum. For this purpose it was considered necessary to examine by surface techniques the interaction which insoluble lipid monolayers undergo with different serum proteins present in the underlying solution. Part I demonstrates the adsorption on to lipid monolayers such as cardolipin cephalin Iecithin and cholesterol of serum protein fractions. Part I1 analyses quantitatively the physicochemical conditions related to protein-lipid associations and establishes new types of lipo-protein complexes. easily compressible. 1945 67 10. Experimental Lipid Mono1ayers.-It has been shown that protein monolayers on compression do not stand pressures greater than 16 dynes/cm.without crumbling or forming irreversible striations. Also in mixed films with cholesterol where weak association can be anticipated the protein is ejected from the cholesterol monolayer into the underlying solution t o form an adsorbed double layer at pressures equal t o the collapse pressure of the protein.' Therefore should the lipid monolayer be compressed initially t o the collapse surface pressure of the protein and a very dilute protein solution be injected into the underlying solution a strong rise in surface pres- sure against time be noted some association must have taken place be- tween the lipid and the protein.s Care must be taken in comparing the magnitude of these surface-pressure increases with different lipids owing to the different compressibilities of the lipid films.Thus a cholesterol monolayer is condensed and very incompressible (40-39 Aa) and small decreases in area will give large changes in surface pressure. Whereas areas (350 Az - 185 Az per molecule) czphalin and lecithin are similarly a cardiolipin film is liquid expanded and easily compressible over large (a) Neurath J . Physic. Chem. 1938 42 39. ( b ) Bull J . Amer. Chem. SOC. Cockbain and Schulman Trans. Faraday Soc 1939 35 716. * Schulman and Rideal Proc. Roy. SOC. B 1937 122 46. P. DOTY AND J. H. SCHULMAN 23 Cardiolipin Monolayers .-Force-area curves of the cardiolipin mono- layers on acetate buffer pH 5-1 reveal a liquid-expanded film compressible from 350 Hi2 to 185 Hi2 at a collapse pressure of 40 dynes/cm.This assumes a molecular weight of 2200. It is interesting that this molecular weight receives confirmation from the surface-film work. Six unsaturated fatty acid radicals are considered in the molecule. The limiting area per oleyl chain is thus 31 Hi2 which is nearly identical to the limiting compression area of a single chain of oleic acid. From the monolayers work it is easier to consider the structure as three dioleyl glyceryl phosphoric acid ester units joined on one glycerine mole- cule and not as described by Pangborn with the oleic acid radicals separ- ated by the phosphoric acid and glycerine units in the ratio 2/1/1/z. Force-area curves of the cardiolipin over the whole pH range would possibly clarify this point.* Protein Solutions.-The quantity of protein injected into the under- lying buffered aqueous solutions was so chosen that its rate of adsorption on the free aqueous side of the Langmuir trough was very small over the time period of the reaction with the lipid monolayer.Before each reading on the torsion head of the Langmuir balance this free side of the trough was cleaned by waxed slides. In Part I1 where the automatic pressure measuring device was used this technique was not required since the vertical pressure on the hydrophilic plate hanging in the free side of the trough compensated for the surface pressure of the adsorbing protein (see Part 11). A very convenient concentration of the protein in the underlying solution was found to be 2 mg.for 300 ml. Various serum protein fractions were obtained by ammonium sulphate precipitation and dialysis or standard protein fractions.? Haemoglobin was obtained from laked red cells and purified by centrifuging in NaCl and dialyzing. In analysing the results the possible presence of lipids in the protein fractions was taken into consideration. lipid monolayer is compressed to I 4 dynes /cm. Procedure .-The on a Langmuir trough and the equivalent of 2 mg. protein in 10 ml. solution injected into the underlying solution and vigorously circulated. The increase in surface pressure of the lipid film with time is noted at constant area. In Part I1 the gradual expansion (penetration) of the lipid film a t constant pressure by injection of the lipid protein solution is noted with time.Fig. 1-3 shows the changes in surface pressure of the negatively charged lipid monolayer cardiolipin with time in the presence of serum protein fractions starting at surface pressures above the collapse pressure of the protein films alone. These curves show that on the acid side of the isoelectric points of the various protein fractions strong association takes place with the negatively charged lipid film. On the alkaline side strong inhibition of this effect takes place. The slow rise in surface pressure that is observed on the alkaline side is possibly due to two causes (i) a general non-specific surface solution of the protein into lipid monolayer (see Part 11) and (ii) serum lipids associated with the injected protein fraction can preferentially associate with either the film-forming lipid or the protein in solution.This is most noticeable Pangborn J . Biol. Chem. 1944 153 343 ; 1947 168 358. * See Grazer This Discussion. t Protein fractions were supplied by Prof. John Edsall and were prepared from blood collected by the American Red Cross under contract between the Committee on Medical Research of the office of Scientific Research and Develop- ment and Harvard University. Protein fractions were also kindly supplied by Armour Co. Ltd. We are grateful to Dr. Mary C . Pangborn for help in obtaining information and supplies of cardiolipin and to Prof. Blix Uppsala Sweden for cephalin samples.LIPO-PROTEIN MONOLAYERS 24 in the case of a-globulin which is known to have surface-active lipoids associated in this fraction ; whereas the albumin and y-globulin fractions do not show this phenomenon. aH. 2 ;I IH 4.2 PH3 3 Ttme in minutes FIG. I-Pressure rise of cardiolipin monolayer on injection of human albumin pH effect. P. DOTY AND J. H. SCHULMAN In Fig. 5 a curve is taken from the work of Czeczowiczka using whole horse serum protein injected under a cholesterol film. This demonstrates the competitive lipid-protein effect in solution since with a cholesterol monolayer reacting with albumin no pH effects are observed. Thus with a lipid-free protein the surface solution of protein molecules into uncharged 2.5 lipid monolayer such as cholesterol is small and independent of the pH.In Fig. 5 the rise in surface pressure of a cholesterol film is seen to be comparable with the surface-pressure rises in reacting charged lipid- protein systems. This is shown in Part I1 to be quite small when the incompressibility of the cholesterol film is taken into account. On analj sis 1 pH 4.6 30 25 2 i V C x pH 5.2 g 20 Y n U L UI Y & 15 Time in minute6 FIG. 4-Pressure rise of cephalin monolayer on injection of human albumin pH effect of the curve obtained by expansion at constant pressure it is found that simple surface solution in the cholesterol monolayer is taking place. Fig. 4 shows albumin associating with a cephalin film which behaves as a negatively charged monolayer over pH range 2-14.The albumin- cephalin association cuts out sharply at the isoelectric part of the albumin. 6 Schulman Biochem. J . 1945 39 54. LIPO-PROTEIN MONOLAYERS Time in Minutes FIG. 5-Pressure rise of cholesterol monolayer on injection of horse serum protein (I) pH effect. 26 No association takes place a t pH's more alkaline than pH 4.6. No association could be measured with the serum protein fractions with lecithin monolayers over the pH range 3-11. Cardiolipin-Luetic Sera.-Attempts were made to measure by this technique the possible specific association of luetic sera and mixed films of cardiolipin-lecithin and cholesterol in varying proportions. No association could be measured other than that given by the normal sera.This was surprising in view of the fact that in the Kahn reaction the specific adsorption and isolation of a globulin protein has been estab- lished on the mixed suspensions of the three above-mentioned lipids in the presence of luetic serum.' Globulin fractions obtained from the luetic sera also gave no positive association on the mixed lipid monolayers. A possible explanation could be that in these surface technique experi- ments the concentration of the protein in solution is about 1/200,000 whereas in the Kahn reaction association of the globulin on the mixed lipid suspension in a very strong positive luetic serum rarely exceeds a dilution of the luetic serum of more than I/IOO (on protein about I/IZOO). It is not possible at present by surface techniques to work at these high protein concentrations.It might be possible to measure these types of specific lip-protein associations by surface techniques in those systems (RH lipid) where the protein is reactive in very dilute solutions.8 Analogy with the pH-controlled Emulsion Flocculation Work.- The preceding result is even more surprising when one considers the analogy of the flocculation of emulsions of oil droplets * with the pH-controlled monolayer adsorption work described in this paper. Similar longchain ionic compounds are used in both works in the presence of protein solu- 7 Eagle Lab. diug. Syfih. (St. Louis 1937). *Price J . Amer. Chem. Soc. 1948 70 3527. Elkes Frazer Schulman and Stewart Proc. Roy.SOC. A 1945 184 104. P. DOTY ASD J. H. SCHULMAN 27 tions in concentration sufficient to cover the surface of the emulsion droplets with a monolayer of proteins. In the Kahn reaction the protein can be adsorbed specifically against the charge on the aggregate since the specific adsorption functions equally well in acid or alkaline solution. The cardiolipin-lecithin surface is negative and the protein also can be negative on the alkaline side of its isoelectric point. The repulsion forces thus arising are not sufficient to prevent specific adsorption of the protein molecules in the luetic serum. It could then be considered that specific action of the luetic protein is related to the non-polar portion of the protein molecule. In Part I1 where the irreversible penetration of the protein molecule or bound protein is considered on the alkaline range of the isoelectric point for negatively charg-d lipid monolayers in contrast to the reversible adsorption of the adsorbed protein molecules an analogy with or inter- pretation of the Kahn reaction may be found.Our thanks are due to Dr. J. H. McCoy for considerable help and encouragement and to Mrs. hI. Doty for collaboration in the experi- mental work. Department of Colloid Science T h e University C a m bridge. PART 11.-MECHANISM OF ADSORPTION SOLUTION AND PENETRATION BY R. MATALON* AND J. H. SCHULMAN Received 2nd J u n e 1949 Most of the work on the interaction of proteins with long chain ionogenic compounds such as detergents has been carried out in bulk solution.1 When studying these interactions a t interfaces two main difficulties are encountered.They are (i) spontaneous spreading and unfolding of the molecule when a protein spreads a t pressures less than 15 dynes/cm.2 (ii) the marked solubility of the detergents in water which prevents the formation of stable monolayers a t the air-water interfacees To obviate these difficulties long-chain ionic compounds of a pronounced hydrophobic character were spread at the surface kept at pressures equal to or above 15 dynes/cm. and the proteins were then injected into the underlying solution. These long-chain ionic compounds are structurally analogous to the usual detergents in that they contain a polar group and a hydrophobic tail ; the only difference is the increased length of the hydrophobic residue of the molecule which is responsible for the marked stability of the mono- layers on the surface of the water.Cardiolipin and C,aH,,SO,Na produce negatively charged mono- layers a t all pH values. Stearylcholine on the other hand gives * Oliver Gatty student. Putnam Advances in Protein Chemistry 1948 4 79. Cockbain and Schulman Trans. Faraday SOC. 1939 35 1266. Bull J . Amer. Chem. SOC. 1945 67 10. 28 LIPO-PROTEIN NOXOLAYERS positively charged monolayers provided that care is taken to exclude polyvalent anions such as p h ~ s p h a t e ~ which on adsorption on t o the monolayer can discharge it and possibly reverse its sign. The behaviour of uncharged monolayers has also been studied and in these instances cholesterol has been used.On studying the interaction of these monolayers with proteins i t is possible to distinguish three processes adsorption penetration and solubility. Adsorption is observed under certain conditions where the protein is injected below a monolayer kept a t constant area. The protein is thus prevented from entering extensively into the surface and the pressure rise observed is mainly due to the association of the polar groups of the protein with those of the monolayer. Similarly solution and penetration of the protein take place when the pressure of the monolayer is kept constant. During these latter processes spontaneous expansion of the monolayer takes place. The rate of extension determines whether the protein is entering the surface by solution in the monolayer or by molecular interaction resulting in the association of the polar groups of the reactant and van der Waals’ attraction between the long hydrocarbon residue of the monolayer and the side chains of the proteins.Experimental New techniques developed recently for studying monolayer inter- actions at air-water interfaces have been applied t o this particular study. Although monolayer interactions have been the subject of a great deal of work one practical difficulty is inherent in this method when the re- actant injected is surface active. In these conditions the pressure measured is no longer that of the interacting monolayer but is the pressure difference between this monolayer and the pressure of the film of the adsorbed solute on the free water side of the boom.Hitherto the surface pressure value was obtained by repeatedly sweeping the free water side with waxed slides to remove the adsorbed molecules. As well as disturbing the surface a certain error was inevitable a s the adsorbed layer was never completely removed by this process. The technique adopted in these experiments introduces a device which automatically cancels the pressure of the adsorbed layer and so renders the cleaning of the surface unnecessary. The principle of the compensation is that a monolayer exerts upon a surface passing vertically through it an upward pressure equal to that which i t exerts horizontally. Hence a hydrophilic plate dipping in the free water side can be so attached t o the torsion wire that the moments of the horizontal and vertical forces about the centre of the wire are opposite and equal.Furthermore the general technique frequently used for studying the phenomenon of penetration was the recording of the compression curve of the mixed film. The existence of stoichiometric complexes was deduced from the changes of slope or the kinks of the compression curve. Should the compression be carried out at a rate greater than the rate of ejection of the solute injected crumpling of the monolayer occurs thus leading t o metastable states difficult to interpret. This has been shown to be most pronounced with rigid or solid interacting monolayers. To avoid these complications the technique of spontaneous extension Schulman and Cockbain Trans.Faraday Soc. 1940 35 663. Elkes Frazer Schulman and Stewart Proc. Roy. SOC. A 1945 184 104 Matalon and Schulman J . Colloid Sci. 1949 4 89. Matalon and Schulman Trans. Faraduy SOC. 1947 43 479. R. NATALOX ASD J. H. SCHULMAK 29 or ejection of the monolayer at constant pressure first used by Schulman and Stenhagen,* has been developed.@ The Langmuir trough to which is attached the compensating plate is equipped with a constant pressure device which operates a relay. This relay controls the movement of a motor which expands or compresses the monolayer when the surface pressure acting on the boom is above or below a certain value. This value can be adjusted before setting the experi- ment.By using a platinum-mercury contact the pressure can be main- tained constant within 0-2-0-4 dyne and the surfa.ce variation of the monolayer under injection can be followed with great accuracy. Adsorption and Desorption at Constant Area. - The protein dis- solved in a buffered solution is injected into the bath which is on the positive side of the isoelectric point when the surface is covered by nega- tively charged mono1 ayers such as cardiolipin or C,2H,,S0,Na. For stearylcholine a positively charged monolayer the pH at which the protein is injected is 10 and an acetate buffer is present in the bath. The presence of the buffer permitted small and gradual variation of the pH values in the buffering range of the salts this insured a constant pH after an injection during the course of the experiments.Results The rise in pressure of the monolayer kept at constant area following this injection is recorded with time until a state of equilibrium is reached. FIG. I-Reversible adsorption of sheep haemoglobin on t o cardiolipin. (Negative interface). This is usually attained 40 min. after the injection of the protein. When the levelling of the pressure-time curve is reached small amounts of alkali or acid are injected into the trough and the pressure variation of the surface film is recorded in parallel with the pH variation. The pH is measured using indicators with an accuracy of 0-2-0-3 pH unit in the pH range 3.6 to 10. (a) PROTEIN AND MONOLAYER OPPOSITELY CHARGED (Fig. 1-5) .- When the protein is injected so that the sign of its electrical charge is reverse of that of the monolayer the pressures reached vary between 27 and 30 dyneslcm.except in the case of C2,H,,S0,Na and albumin where this pressure is about 37-38 dyneslcm. The marked increase in pressure observed in this particular system is due to the very low com- pressibility of the C22H,,S0,Na monolayer and to the marked length 8 Schulman and Stenhagen Proc. Roy. Soc. B 1938 126 356. 9 R. Matalon (unpublished work). 30 LIPO-PROTEIN MONOLAYERS of the hydrophobic portion of this molecule which increases the affinity to the few side chains of the polypeptide backbone of the protein which can enter into the monolayer. (b) PROTEIN AND MONOLAYER SIMILARLY CHARGED (Fig. 1-5) .-As soon as the pH is altered so that the sign of the protein becomes the same I I I 1 301 I 1 1 5m9 Bovine albumin1 700mt.f 2 6 8 p H 10 5my P.asma Rovine Albumin/ 700 mi. 4 FIG. *-Reversible adsorption of bovine albumin on to cardiolipin. (Negative interface). as that of the monolayers a decrease in the pressure is observed. De- sorption is quite rapid and is over in a period of 15 to 20 min. The essential feature of these experiments is their reversibility in that FIG. 3-Reversible adsorption of bovine albumin on to C, sulphate. (Negative interface). the pressure of the monolayer can be raised to its initial value after de- sorption by adjusting the pH to its original value. (c) ANOMALOUS BEHAVIOUR OF y-GLOBULIN.-h the case of y-globulin this reversibility is not so marked 0.6 yo of NaCl is used in the trough to obtain the protein in solution.R. MATALON AND J. H. SCHULMAN 31 VE7hen the 7-globulin-cardiolipin system has been slowly brought from pH 4 to pH 10 to construct curve I of Fig. 4 and when it is restored to the original pH a pressure sensibly higher than the original is found. If it is now again brought slowly over the pH range studied in curve I the same shaped curve is produced (curve 2 ) with this increase of pressure maintained throughout. FIG. ,+-Adsorption of y-globulin (0.6 % NaCl) on to cardiolipin. (Negative interface). General Behaviour of the Desorption Curves.-Although by acid or alkali injections the sign of the protein can be reversed as the isoelectric point is crossed the entire pressure variation is not completed at the reversal of the charge (Fig.I-5) but occurs gradually upon varying the pH and reaches a constant minimum value. These values are gener- ally greater than 15 dyneslcm. The actual figures recorded are System Pressure Dyneslcm. Protein 1 I Bovine albumin 17'5 18.5 Haemoglobin 7-Globulin Monolayer Cardiolipin ~,,H,,SO,Na Cardiolipin Stearylcholine Cardiolipin 21 21 24.3 (curve I) 25.5 (curve 2) The original pressure of the monolayer is 15 dynes/cm. From these results is appears that the pressure of the monolayer attained by adjusting the pH of the bath to the non-reactive protein is essentially dependent on the nature of the protein and does not seem related to the nature of the monolayer present at the interface.While with bovine albumin on cardiolipin or C2,C,,S0,Na a pressure rise of 2.5-3.5 dyneslcm. above the initial pressure of the monolayer is observed ; with haemoglobin on cardiolipin or on stearylcholine this pressure difference is 6 dynes/cm. The marked increase of 9.3-10-5 dyne/cm. observed in the case of y-globulin with cardiolipin is probably due to the high molecular weight of this protein and also possibly to the existence of traces of lipids present as an impurity in the fraction under investigation. 3 2 LIPO-PROTEIX MONOLAYERS Solution and Penetration at Constant Pressure.-In order to deter- mine the influence of electrical forces on the association of protein with long-chain ionic compounds the behaviour of a neutral molecule such as cholesterol in the presence of proteins has been studied and compared with the association of negatively charged monolayers with proteins.haem0- globin is injected at pH 4-6-4-8 under a cholesterol monolayer kept at 28 26 24 22 20 (a) SOLUTION EFFECT.-HAEMOGLOBIN-cHOLESTEROL.-when 1.2 lo p H 1 ' I I 1 8 6 I4 4 FIG. 5-Reversible adsorption of sheep haemoglobin on to stearyl-choline. (Positive interface). 14 dyneslcm. pressure an expansion is observed (Fig. 6) which after 7 min. becomes linear. This indicates that the expansion of the cholesterol is due to solution of the protein in the monolayer and not to a specific interaction between the two compounds.This conclusion is supported by the previous worku where gliadin is ejected from a cholesterol mono- layer at its own collapse pressure. FIG. 6-Surface solution of haemoglobin in a cholesterol monolayer. Seventy minutes after the injection of the haemoglobin the cholesterol monolayer had extended by 33 yo but the film was still liquid while on the free water side was a strong gel due to the formation of an adsorbed layer of the protein. (b) SOLUTION EFFECT AND PENETRATION.-(i) PrO&?in and WZOnO- Eayers oflflositely charged.-Haemoglobin injected under a film of cardio- lipin at 25 dynes/cm. pressure and at pH 4 causes rapid extension of the monolayer followed after 55 min. by a linear expansion similar to the solution effect observed on the cholesterol monolayer.Thus the curve K. MATALON AND J. H. SCHULMAN 33 obtained (Fig. 7) is a summation of rapid penetration and slow solution the two parts of which can be distinguished by projecting the linear portion of the curve back to the extensim axis. The area at which this line cuts the axis is that increase due to complete saturation of the monolayer by penetration the remaining increase is due to the process of solution. FIG. 7.-Interaction leading to complexes between haemoglobin and cardiolipin. (ii) Neutral $rotein and negative interfaces.-If haemoglobin is now injected at its isoelectric point no expansion is observed due to protein entering the C,,H,,SO,Na monolayer kept in a pressure range of 28 to 2 0 dyneslcm. for a period of 35 min.If after this period acid is injected to shift the pH to I well on the acid side of the isoelectric point very strong penetration occurs at 28 dyneslcm. pressure and IOO yo extension Surface pressure = ~~.dyne./cm. 5m9. Hoemoylobrn 700 ml. I FIG. 8.-Interaction leading to complexes between haemoglobin and G2 sulphate. of the original area is produced within 40 min. This shows that the phenomenon of penetration is controlled by electrical forces. (iii) Physical state of expanded monoZayers.-While the cholesterol monolayer remained liquid after the expansion cardiolipin and C ,H,,SO,Na become strong gels when the protein penetrates them. (iv) Influence of steric factors on penet~ation.~-When the haemoglobin is injected at pH 3 below a C,,H,,SO,Na monolayer at 31 dyneslcm.B LIPO-PROTEIN MONOLAYERS 34 pressure the maximum extension observed is 33 yo (Fig. 9 curve Ia). A further extension to 66 yo is obtained by adjusting the pressure to 28 dynes/cm. (Fig. 9. curve Ib). Finally at 25 dyneslcm. the monolayer soon Time in m,i?uL?s. FIG. g.-Surface pressure and pH variation on the kinetics of penetration and ejection of haemoglobin with C, sulphate. The existence of “ bound protein.” develops a constant linear increase showing an added solution effect (Fig. 9 curve Ic). These experiments demonstrate that the closer the packing of the chains in the monolayer the less penetration occurs. FIG. 10.-Demonstration of irreversibly “ bound protein ” in system C, sulphate-haemoglobin.(v) Existence of “ Bound Protein.”-If under this haemoglobin C2,H,,SOdNa system at pH 3 NaOH is injected to increase the pH to 10 rapid ejection o f the protein takes place from the monolayer (Fig. g 35 R. MATALON AND J. H. SCHULMAN curve 11) and the area attained once the ejection is complete exceeds the original area of the unpenetrated monolayer by 35 %. This increase in area is due to irreversibly “ bound protein ” associated with the inter- face by non-electrical forces while the protein readily ejected by pH changes is associated with the monolayer mainly by the action of electrical forces and is thus expelled when the sign of the protein is reversed. Re-expansion of the monolayer can readily be obtained if the pH is now decreased to 4.2 by a new injection of acid.In this case the com- plex protein C2,Hd5SO4Na is reformed but the rate of solubility of the protein in the monolayer has increased i.e. the slope of the linear part of the curve is steeper (Fig. 10 curve 111). It is interesting t o note that if the pH is again increased to 6-4-6-5 close to the isoelectric point no displacement of the interface is observed for a period of 11 min. and the area is constant (Fig. 10 curve IV). Re-ejection of the protein is now obtained on altering the pH t o 10. When equilibrium is reached the monolayer occupies an area 80 yo greater than its original area before penetration (Fig. 10 curve V). Although penetration of a charged monolayer does not take place at the isoelectrie point once a monolayer is penetrated only reversal of the sign of the protein will eject it.Furthermore reversing the sign of the protein causes ejection of some of the protein only. ~~ Discussion Adsorption and Desorption.-Desorption of proteins from electri- cally charged monolayers has been studied by following the variation of a h FIG. I I .-Structure of the mixed films protein-negatively charged monolayer. a. on reversible adsorption. b. on irreversible penetration. surface pressure with varying pH on either side of the isoelectric point (Fig. 1-5). The experiments carried out a t constant area show the mechanism of one typk of association between the protein and the charged inter- face but a more complete interpretation of the results is obtained when the expansion experiments are considered at the same time.A marked rise in surface pressure follows injection of proteins under electrically charged monolayers kept at constant area if the protein and the monolayer are oppositely charged. This rise is almost entirely due to polar-polar interaction van der Waals’ forces are not appreci- ably involved since as expansion experiments show the protein cannot enter the film at the pressures thus reached (Fig. IIU). LIPO-P ROTE I N MON OLAY EKS It might be expected that the polar-polar interaction between the monolayer and the protein will increase as the pH becomes more distant from the isoelectric point as it is known that the charge of the protein increases the further the pH is moved from this point.But experimentally the pressures observed on the desorption curve are almost constant in the whole range of pH where protein and spread monolayer are oppositely charged ; this is because the structure of the protein-monolayer association limits the possible rise. Indeed the maximum pressures a t which marked expansion of monolayers by proteins can occur is only slightly less than the pressures at which these penetrated monolayers are ejected and collapsed (33 dyneslcm.). For example if haemoglobin is injected under cardiolipin at pH 4 and the surface pressure maintained a t 25 dynes/cm. (5 dynes/cm. lower than the pressure observed 36 min. after the injection (30 dyneslcm.) for this particular system at constant area) a rapid extension of the monolayer will occur doubling its area in 36 min.(Fig. 7). Furthermore the maximum pressures that such a penetrated film can sustain can be determined by recompression of the extended film to its original area. This pressure is found to be 33 dynes/cm. which is 8 dynes/cm. higher than the expansion pressure (25 dynes/cm.) and 3 dynes/cm. greater than the maximum pressure (30 dynes/cm.) at- tained at constant area. It is thus impossible to follow the varying degree of affinity of the protein and monolayer when oppositely charged solely by studying the desorption curve. The expansion technique would enable these variations to be studied. No expansion of a C22H45S04Na monolayer by haemoglobin takes place at the isoelectric point at 25 dyneslcm. pressure while a very rapid expansion occurs when the protein is well on the acid side.Furthermore if expansion is done a t 28 dynes/cm. a t pH I IOO yo area increase is observed (Fig. S) while a t pH 4 66 yo only of the extension is produced (Fig. 9 curve ~ b ) . Considering again the desorption curve in the pH range where the sign of the protein becomes identical with that of the monolayer as the protein increases in charge the pressure falls in all systems investigated. But this fall is gradual and is complete only a t pH values well separated from the isoelectric point (Fig. 1-5). Again it would have been expected from the amphoteric character of proteins and from the mechanism of the adsorption process mainly polar-polar interaction that the surface pressure would have suddenly decreased to the original pressure of the monolayer (15 dynes/cm.) on crossing the isoelectric point.These experimental results indicate that desorption of the proteins involves other factors than the overcoming of electrical forces. We know that the rise in pressure observed is due to some compression of the monolayer by the few side chains of the proteins which enter it and which associate by van der Waals’ forces to the long hydrophobic chains present a t the surface. Hence complete desorption will necessitate overcoming these forces also. In fact even when maximum desorption is reached the pressure of the monolayer is still higher than its original pressure the greater the molecular weight of the protein and the higher the probability of the existence of uncharged lipids the higher is the pressure reached a t complete desorption.The rise in pressure above 15 dynes/cm. of charged monolayers 37 R. MATALON AND J. H. SCHULMAN by proteins similarly charged is not only observed in the desorption process but in the adsorption process (see part I). The mechanism of this rise may be found in the solution effect. The pressure rise obtained when the protein and monolayer are similarly charged does not mean that there is strong association between them under these conditions. In fact detailed analysis will show that this expresses only very few contacts. The important point is that as the isoelectric point is crossed the pressure falls. Now the pressure reached at maximum desorption indicates a saturation in the adsorbed layer and this saturation has been demonstrated by the expansion technique.Indeed at acid pH values the protein is extensively accumulated below the negatively charged monolayer and orientated by an attractive electrical field of force. This is shown by the marked increase in area of the monolayer when the protein is allowed to enter the surface. I t can be shown too that at and beyond the isoelectric point no penetration occurs i.e. through- out the region where the protein and monolayer are similarly charged. Solution and Penetration.-Expansion experiments show that solution and penetration of proteins take place during the extension of monolayers and it is easy to distinguish the two processes by studying the rate of extension.Solution effect is due to simple diffusion of the protein to the surface and this process is characterized by a constant rate; thus a linear extension is obtained (Fig. 6 ) . Penetration is due to a marked interaction between the mono- layer and the protein in solution. The rate of this interaction is therefore dependent on the concentration of the two reagents. For a constant concentration of the protein in solution the rate is pro- portional to the number of non-associated molecules present in the surface. Thus penetration follows a law of pseudo first-order reaction (since the concentration of the reacting protein is not varied by the reaction) and the rate of extension decreases as the monolayer expands. I t is experimentally possible to show the existence of the solution effect exclusive of any other process whilst penetration is in most cases accompanied by solution.Failure to distinguish between the two processes had led several authors dealing with the penetration phenomena to doubtful conclusions as to the conditions of existence of stoichio- metric lo complexes. In the case of haemoglobin interacting with cardiolipin (Fig. 7) or C,,H,,SO,Na (Fig. 8) 100 yo extension due t o penetration was shown to exist. This indicates that one chain of the monolayer associates with one chain of the protein (Fig. I I ~ ) . It is therefore possible to characterize stoichiometric complexes which result from “ chain interaction. ” This result is explained if the protein has available side chains orientated towards the monolayer.It appears therefore that the globular structure of proteins in solution with boundaries covered with hydrophilic groups only is being altered when in contact with long- chain ionic compounds. Such an alteration would help to explain some of the effect of detergents on the structure of proteins. Whereas in the adsorption of proteins on to charged interfaces van der Waals’ forces are of a minor importance i t would appear that these forces are responsible for the expansion of uncharged monolayers such as cholesterol. lo Schulman Stenhagen and Rideal Nature 1938 141 785. Joly Nature 1946 158 26. LIPO-PROTEIN MONOLAYERS 38 " Bound Protein. "-The effect of pH on the expansion of charged interfaces shows the effect of electrical forces while the irreversibility of the expansion demonstrates the van der Waals' forces.Indeed it has been shown that expansion of a charged monolayer does not take place at the isoelectric point and that the monolayer already expanded at convenient pH values does not contract when the pH is adjusted to the neutral region of the protein. Extra energy is needed to eject from the surface the side chains of the proteins associated by non-polar forces to the monolayer and this energy can be supplied by reversing the charge of the protein. In this case only partial ejection occurs. The " bound protein " is a constituent of the expanded monolayer which cannot be ejected by altering the pH. The nature of this " bound protein" is of great interest.Although more experiments are still needed i t would appear that the bound protein is that which has entered the surface by a mechanism which involves only the action of van der Waals' forces and which is completely independent of any polar-polar interaction. Such a mechanism is found in the solution effect and has been proved to take place simultaneously with the penetration. The probable structures of adsorbed proteins on to charged mono- layers and of the chain complexes between protein and long-chain ionic compounds as obtained by penetration have been described in Fig. 11. Analogies to Reaction in Bulk.-Previous work shows cases of reversible and irreversible changes when the proteins are adsorbed on to charged oil-water interfaces as with emulsions and then desorbed.Haemoglobin for example is regenerated as parahaematin which is less soluble and has lost its biological properties whereas snake venoms and toxins can regain their full activity on desorption.ll The present work has differentiated two mechanisms of interaction of proteins a t charged interfaces. Adsorption which is readily revers- ible involves mainly interaction of polar groups which are available on the surface of the protein molecule without involving any change of the molecular configuration whereas penetration necessitates radical alterations in the molecular structure to enable the non-polar chains to enter into the surface and associate by penetration with the non- polar portion of the film forming molecule.By analogy it could thus be assumed that where irreversible changes are observed with the emulsion technique penetration at the charged oil-water interface has taken place. Adsorptions both a t the charged oil-water and air-water interfaces are conditioned by pH and are related to the charge of the protein coming in contact with the interface. The emulsion flocculation range which has been indicated in Fig. 1-5 show that as soon as re-dispersion of the emulsion is noticed the pressure of the monolayer falls markedly and this occurs on the side of the isoelectric point where the protein and monolayer are similarly charged. Similar to the flocculation of emulsions proteins can be reversibly precipitated by oppositely charged long-chain ionic compounds.Furthermore several authors have shown that soluble lipo-protein associations can exist over the pH range where the proteins and 11 Frazer and Stewart Brit. J . Expt. Path. 1940 21 361. R. MATALON AND J. H. SCHULMAN long-chain ions are similarly charged.12 This observation is confirmed by the present work where the existence of " bound protein" is demonstrated at the non-reactive pH range of the proteins. Our thanks are due to Dr. I. S. Longmuir for haemoglobin prepara- tions and to Mr. A. Dunn for technical assistance. Department of Colloid Science The University Cam bridge. 12Steinhardt and Fugitt J . Res. Nut. Bur. Stand. 1942 29 315. Stein- hardt Fugitt and Harris ibid. 1941 26 293. 39 GENERAL DISCUSSION 21 FORMATION OF LIPO-PROTEIN MONOLAYERS PART 1,PRELIMINARY INVESTIGATION ON THE ADSORPTION OF PROTEINS ON TO LIPID MONOLAYERS BY P.DOTY * AND J. H. SCHULMAN Received 2nd June 1949 New techniques have been developed to follow the interactions of proteins with electrically charged monolayers a t an air-water interface. When steric factors prevent the protein molecules from entering the monolayer adsorption of the protein takes place and this is shown to be dependent on the sign of the electrical charge of the compounds which meet at the interface. This adsorption is essentially reversible and accounts for the flocculation and re-dispersion of emulsions stabilized by anionic or cationic detergents in contact with proteins a t different pH’s. The kinetics of penetration and ejection of proteins in contact with charged interfaces have been studied a t constant pressure.These processes are dependent on the electrical charge of the reactants and the surface pressure at which the experiments are carried out. Stoichiometric complexes are demonstrated between non-polar portions of the protein molecules and the long-chain ionic compounds and the general forces i.e. electrical forces and van der Waals’ forces involved in the association are reviewed. Whereas adsorption follows a reversible pattern monolayer expansion is essentially irreversible. The existence of “ bound protein I’ at the charged interface is demonstrated and interpreted. The bearing of these results upon the interaction of proteins with long-chain ionic compounds in bulk solution is considered.Previous work on protein monolayers suggests that radical changes of an irreversible nature take place on spreading of the protein at an air-water or oil-water interface. Force-area compression curves of the protein monolayers are rather irreproducible above certain surface pressures. This is due to the association of the reactive groups present in the large protein molecule. The forces involved in these associations are responsible for the gel state or highly viscous characteristics of protein monolayers. For the same reasons studies on monolayers obtained by spreading a solution containing protein lipids or protein-soluble surface-active agents are only of a qualitative nature and * Rockefeller Foundation Fellow ; present address Dept.of Chemistry, 1 Cockbain and Schulman Trans. Faraduy Soc. 1939 35 1266. Harvard University U.S.A 2 2 LIPC-PROTEIN MOKOLAYERS show little bearing on the behaviour of proteins in aqueous solution in the presence of long-chain ionic compounds. Injection of lipids or detergents under protein films leads to pene-tration or displacement of the protein and the experimental results are again difficult to interpret quantitatively.2 When non-surface-active agents or poorly surface-active materials are injected under-neath protein monolayers better results can be obtained. It has been shown that tanning agents possess a certain parallelism in their behaviour on protein films and on long-chain a r n i n e ~ . ~ To study the interaction of proteins with lipids and detergents, it is necessary to avoid structural alterations of the protein which happen by spontaneous spreading and unfolding of the protein mole-cule a t the interface.The protein is injected underneath the inter-acting monolayers maintained at a pressure equal or above 15 dynes/cm., and the reaction is followed in terms of the mechanical changes which occur through the injection. Such techniques lead to more quanti-tative information and explain the behaviour of protein solutions in contact with charged oil-water interfaces. A direct examination of the forces involved in protein-detergent association is also obtained. Further attempts are being made to study by this method immunity or pseudo-immunity reactions such as certain lipids may undergo when associating with serum proteins.Two samples of this system are cardiolipin with luetic serum and R.H. lipid with positive serum. For this purpose it was considered necessary to examine by surface techniques the interaction which insoluble lipid monolayers undergo with different serum proteins present in the underlying solution. Part I demonstrates the adsorption on to lipid monolayers such as cardolipin cephalin Iecithin and cholesterol of serum protein fractions. Part I1 analyses quantitatively the physicochemical conditions related to protein-lipid associations and establishes new types of lipo-protein complexes. Experimental Lipid Mono1ayers.-It has been shown that protein monolayers on compression do not stand pressures greater than 16 dynes/cm.without crumbling or forming irreversible striations. Also in mixed films with cholesterol where weak association can be anticipated the protein is ejected from the cholesterol monolayer into the underlying solution t o form an adsorbed double layer at pressures equal t o the collapse pressure of the protein.' Therefore should the lipid monolayer be compressed initially t o the collapse surface pressure of the protein and a very dilute protein solution be injected into the underlying solution a strong rise in surface pres-sure against time be noted some association must have taken place be-tween the lipid and the protein.s Care must be taken in comparing the magnitude of these surface-pressure increases with different lipids owing to the different compressibilities of the lipid films.Thus a cholesterol monolayer is condensed and very incompressible (40-39 Aa) and small decreases in area will give large changes in surface pressure. Whereas a cardiolipin film is liquid expanded and easily compressible over large areas (350 Az - 185 Az per molecule) czphalin and lecithin are similarly easily compressible. ( b ) Bull J . Amer. Chem. SOC., 1945 67 10. (a) Neurath J . Physic. Chem. 1938 42 39. Cockbain and Schulman Trans. Faraday Soc 1939 35 716. * Schulman and Rideal Proc. Roy. SOC. B 1937 122 46 P. DOTY AND J. H. SCHULMAN 23 Cardiolipin Monolayers .-Force-area curves of the cardiolipin mono-layers on acetate buffer pH 5-1 reveal a liquid-expanded film compressible from 350 Hi2 to 185 Hi2 at a collapse pressure of 40 dynes/cm.This assumes a molecular weight of 2200. It is interesting that this molecular weight receives confirmation from the surface-film work. Six unsaturated fatty acid radicals are considered in the molecule. The limiting area per oleyl chain is thus 31 Hi2 which is nearly identical to the limiting compression area of a single chain of oleic acid. From the monolayers work it is easier to consider the structure as three dioleyl glyceryl phosphoric acid ester units joined on one glycerine mole-cule and not as described by Pangborn with the oleic acid radicals separ-ated by the phosphoric acid and glycerine units in the ratio 2/1/1/z. Force-area curves of the cardiolipin over the whole pH range would possibly clarify this point.* Protein Solutions.-The quantity of protein injected into the under-lying buffered aqueous solutions was so chosen that its rate of adsorption on the free aqueous side of the Langmuir trough was very small over the time period of the reaction with the lipid monolayer.Before each reading on the torsion head of the Langmuir balance this free side of the trough was cleaned by waxed slides. In Part I1 where the automatic pressure measuring device was used this technique was not required since the vertical pressure on the hydrophilic plate hanging in the free side of the trough compensated for the surface pressure of the adsorbing protein (see Part 11). A very convenient concentration of the protein in the underlying solution was found to be 2 mg. for 300 ml. Various serum protein fractions were obtained by ammonium sulphate precipitation and dialysis or standard protein fractions.? Haemoglobin was obtained from laked red cells and purified by centrifuging in NaCl and dialyzing.In analysing the results the possible presence of lipids in the protein fractions was taken into consideration. Procedure .-The lipid monolayer is compressed to I 4 dynes /cm. on a Langmuir trough and the equivalent of 2 mg. protein in 10 ml. solution injected into the underlying solution and vigorously circulated. The increase in surface pressure of the lipid film with time is noted at constant area. In Part I1 the gradual expansion (penetration) of the lipid film a t constant pressure by injection of the lipid protein solution is noted with time. Fig.1-3 shows the changes in surface pressure of the negatively charged lipid monolayer cardiolipin with time in the presence of serum protein fractions starting at surface pressures above the collapse pressure of the protein films alone. These curves show that on the acid side of the isoelectric points of the various protein fractions strong association takes place with the negatively charged lipid film. The slow rise in surface pressure that is observed on the alkaline side is possibly due to two causes (i) a general non-specific surface solution of the protein into lipid monolayer (see Part 11) and (ii) serum lipids associated with the injected protein fraction can preferentially associate with either the film-forming lipid or the protein in solution. This is most noticeable On the alkaline side strong inhibition of this effect takes place.Pangborn J . Biol. Chem. 1944 153 343 ; 1947 168 358. * See Grazer This Discussion. t Protein fractions were supplied by Prof. John Edsall and were prepared from blood collected by the American Red Cross under contract between the Committee on Medical Research of the office of Scientific Research and Develop-ment and Harvard University. Protein fractions were also kindly supplied by Armour Co. Ltd. We are grateful to Dr. Mary C . Pangborn for help in obtaining information and supplies of cardiolipin and to Prof. Blix Uppsala Sweden for cephalin samples 24 LIPO-PROTEIN MONOLAYERS in the case of a-globulin which is known to have surface-active lipoids associated in this fraction ; whereas the albumin and y-globulin fractions do not show this phenomenon.;I IH 4.2 PH3 aH. 2 3 Ttme in minutes FIG. I-Pressure rise of cardiolipin monolayer on injection of human albumin pH effect P. DOTY AND J. H. SCHULMAN 2.5 In Fig. 5 a curve is taken from the work of Czeczowiczka using whole horse serum protein injected under a cholesterol film. This demonstrates the competitive lipid-protein effect in solution since with a cholesterol monolayer reacting with albumin no pH effects are observed. Thus with a lipid-free protein the surface solution of protein molecules into uncharged 30 pH 4.6 25 i 2 C x V g 20 U pH & L Y n UI Y 15 lipid monolayer such as cholesterol is small and independent of the pH. In Fig. 5 the rise in surface pressure of a cholesterol film is seen to be comparable with the surface-pressure rises in reacting charged lipid-protein systems.This is shown in Part I1 to be quite small when the incompressibility of the cholesterol film is taken into account. On analj sis 1 5.2 Time in minute6 FIG. 4-Pressure rise of cephalin monolayer on injection of human albumin pH effect of the curve obtained by expansion at constant pressure it is found that simple surface solution in the cholesterol monolayer is taking place. Fig. 4 shows albumin associating with a cephalin film which behaves as a negatively charged monolayer over pH range 2-14. The albumin-cephalin association cuts out sharply at the isoelectric part of the albumin. 6 Schulman Biochem. J . 1945 39 54 26 LIPO-PROTEIN MONOLAYERS No association takes place a t pH's more alkaline than pH 4.6.No association could be measured with the serum protein fractions with lecithin monolayers over the pH range 3-11. Time in Minutes FIG. 5-Pressure rise of cholesterol monolayer on injection of horse serum protein (I) pH effect. Cardiolipin-Luetic Sera.-Attempts were made to measure by this technique the possible specific association of luetic sera and mixed films of cardiolipin-lecithin and cholesterol in varying proportions. No association could be measured other than that given by the normal sera. This was surprising in view of the fact that in the Kahn reaction the specific adsorption and isolation of a globulin protein has been estab-lished on the mixed suspensions of the three above-mentioned lipids in the presence of luetic serum.' Globulin fractions obtained from the luetic sera also gave no positive association on the mixed lipid monolayers.A possible explanation could be that in these surface technique experi-ments the concentration of the protein in solution is about 1/200,000 whereas in the Kahn reaction association of the globulin on the mixed lipid suspension in a very strong positive luetic serum rarely exceeds a dilution of the luetic serum of more than I/IOO (on protein about I/IZOO). It is not possible at present by surface techniques to work at these high protein concentrations. It might be possible to measure these types of specific lip-protein associations by surface techniques in those systems (RH lipid) where the protein is reactive in very dilute solutions.8 Analogy with the pH-controlled Emulsion Flocculation Work.-The preceding result is even more surprising when one considers the analogy of the flocculation of emulsions of oil droplets * with the pH-controlled monolayer adsorption work described in this paper.Similar longchain ionic compounds are used in both works in the presence of protein solu-7 Eagle Lab. diug. Syfih. (St. Louis 1937). *Price J . Amer. Chem. Soc. 1948 70 3527. Elkes Frazer Schulman and Stewart Proc. Roy. SOC. A 1945 184 104 P. DOTY ASD J. H. SCHULMAN 27 tions in concentration sufficient to cover the surface of the emulsion droplets with a monolayer of proteins. In the Kahn reaction the protein can be adsorbed specifically against the charge on the aggregate since the specific adsorption functions equally well in acid or alkaline solution.The cardiolipin-lecithin surface is negative and the protein also can be negative on the alkaline side of its isoelectric point. The repulsion forces thus arising are not sufficient to prevent specific adsorption of the protein molecules in the luetic serum. It could then be considered that specific action of the luetic protein is related to the non-polar portion of the protein molecule. In Part I1 where the irreversible penetration of the protein molecule or bound protein is considered on the alkaline range of the isoelectric point for negatively charg-d lipid monolayers in contrast to the reversible adsorption of the adsorbed protein molecules an analogy with or inter-pretation of the Kahn reaction may be found.Our thanks are due to Dr. J. H. McCoy for considerable help and encouragement and to Mrs. hI. Doty for collaboration in the experi-mental work. Department of Colloid Science, T h e University C a m bridge. PART 11.-MECHANISM OF ADSORPTION SOLUTION AND PENETRATION BY R. MATALON* AND J. H. SCHULMAN Received 2nd J u n e 1949 Most of the work on the interaction of proteins with long chain ionogenic compounds such as detergents has been carried out in bulk solution.1 When studying these interactions a t interfaces two main difficulties are encountered. They are (i) spontaneous spreading and unfolding of the molecule when a protein spreads a t pressures less than 15 dynes/cm.2 (ii) the marked solubility of the detergents in water which prevents the formation of stable monolayers a t the air-water interfacees To obviate these difficulties long-chain ionic compounds of a pronounced hydrophobic character were spread at the surface, kept at pressures equal to or above 15 dynes/cm.and the proteins were then injected into the underlying solution. These long-chain ionic compounds are structurally analogous to the usual detergents in that they contain a polar group and a hydrophobic tail ; the only difference is the increased length of the hydrophobic residue of the molecule which is responsible for the marked stability of the mono-layers on the surface of the water. Cardiolipin and C,aH,,SO,Na produce negatively charged mono-layers a t all pH values.Stearylcholine on the other hand gives * Oliver Gatty student. Putnam Advances in Protein Chemistry 1948 4 79. Cockbain and Schulman Trans. Faraday SOC. 1939 35 1266. Bull J . Amer. Chem. SOC. 1945 67 10 28 LIPO-PROTEIN NOXOLAYERS positively charged monolayers provided that care is taken to exclude polyvalent anions such as p h ~ s p h a t e ~ which on adsorption on t o the monolayer can discharge it and possibly reverse its sign. The behaviour of uncharged monolayers has also been studied and in these instances cholesterol has been used. On studying the interaction of these monolayers with proteins, i t is possible to distinguish three processes adsorption penetration, and solubility. Adsorption is observed under certain conditions where the protein is injected below a monolayer kept a t constant area.The protein is thus prevented from entering extensively into the surface and the pressure rise observed is mainly due to the association of the polar groups of the protein with those of the monolayer. Similarly solution and penetration of the protein take place when the pressure of the monolayer is kept constant. During these latter processes spontaneous expansion of the monolayer takes place. The rate of extension determines whether the protein is entering the surface by solution in the monolayer or by molecular interaction resulting in the association of the polar groups of the reactant and van der Waals’ attraction between the long hydrocarbon residue of the monolayer and the side chains of the proteins.Experimental recently for studying monolayer inter-actions at air-water interfaces have been applied t o this particular study. Although monolayer interactions have been the subject of a great deal of work one practical difficulty is inherent in this method when the re-actant injected is surface active. In these conditions the pressure measured is no longer that of the interacting monolayer but is the pressure difference between this monolayer and the pressure of the film of the adsorbed solute on the free water side of the boom. Hitherto the surface pressure value was obtained by repeatedly sweeping the free water side with waxed slides to remove the adsorbed molecules. As well as disturbing the surface a certain error was inevitable a s the adsorbed layer was never completely removed by this process.The technique adopted in these experiments introduces a device which automatically cancels the pressure of the adsorbed layer and so renders the cleaning of the surface unnecessary. The principle of the compensation is that a monolayer exerts upon a surface passing vertically through it an upward pressure equal to that which i t exerts horizontally. Hence a hydrophilic plate dipping in the free water side can be so attached t o the torsion wire that the moments of the horizontal and vertical forces about the centre of the wire are opposite and equal. Furthermore the general technique frequently used for studying the phenomenon of penetration was the recording of the compression curve of the mixed film. The existence of stoichiometric complexes was deduced from the changes of slope or the kinks of the compression curve.Should the compression be carried out at a rate greater than the rate of ejection of the solute injected crumpling of the monolayer occurs thus leading t o metastable states This has been shown to be most pronounced with rigid or solid interacting monolayers. To avoid these complications the technique of spontaneous extension Schulman and Cockbain Trans. Faraday Soc. 1940 35 663. Elkes Frazer Schulman and Stewart Proc. Roy. SOC. A 1945 184 104 Matalon and Schulman J . Colloid Sci. 1949 4 89. Matalon and Schulman Trans. Faraduy SOC. 1947 43 479. New techniques developed difficult to interpret R. NATALOX ASD J. H. SCHULMAK 29 or ejection of the monolayer at constant pressure first used by Schulman and Stenhagen,* has been developed.@ The Langmuir trough to which is attached the compensating plate is equipped with a constant pressure device which operates a relay.This relay controls the movement of a motor which expands or compresses the monolayer when the surface pressure acting on the boom is above or below a certain value. This value can be adjusted before setting the experi-ment. By using a platinum-mercury contact the pressure can be main-tained constant within 0-2-0-4 dyne and the surfa.ce variation of the monolayer under injection can be followed with great accuracy. Adsorption and Desorption at Constant Area. - The protein dis-solved in a buffered solution is injected into the bath which is on the positive side of the isoelectric point when the surface is covered by nega-tively charged mono1 ayers such as cardiolipin or C,2H,,S0,Na.For stearylcholine a positively charged monolayer the pH at which the protein is injected is 10 and an acetate buffer is present in the bath. The presence of the buffer permitted small and gradual variation of the pH values in the buffering range of the salts this insured a constant pH after an injection during the course of the experiments. Results The rise in pressure of the monolayer kept at constant area following this injection is recorded with time until a state of equilibrium is reached. FIG. I-Reversible adsorption of sheep haemoglobin on t o cardiolipin. (Negative interface). This is usually attained 40 min. after the injection of the protein.When the levelling of the pressure-time curve is reached small amounts of alkali or acid are injected into the trough and the pressure variation of the surface film is recorded in parallel with the pH variation. The pH is measured using indicators with an accuracy of 0-2-0-3 pH unit in the pH range 3.6 to 10. (a) PROTEIN AND MONOLAYER OPPOSITELY CHARGED (Fig. 1-5) .-When the protein is injected so that the sign of its electrical charge is reverse of that of the monolayer the pressures reached vary between 27 and 30 dyneslcm. except in the case of C2,H,,S0,Na and albumin where this pressure is about 37-38 dyneslcm. The marked increase in pressure observed in this particular system is due to the very low com-pressibility of the C22H,,S0,Na monolayer and to the marked length 8 Schulman and Stenhagen Proc.Roy. Soc. B 1938 126 356. 9 R. Matalon (unpublished work) 30 LIPO-PROTEIN MONOLAYERS of the hydrophobic portion of this molecule which increases the affinity to the few side chains of the polypeptide backbone of the protein which can enter into the monolayer. (b) PROTEIN AND MONOLAYER SIMILARLY CHARGED (Fig. 1-5) .-As soon as the pH is altered so that the sign of the protein becomes the same 301 I 1 I 1 I I 1 5m9 Bovine albumin1 700mt. 4 6 8 p H 10 f 2 FIG. *-Reversible adsorption of bovine albumin on to cardiolipin. (Negative interface). as that of the monolayers a decrease in the pressure is observed. sorption is quite rapid and is over in a period of 15 to 20 min. De-The essential feature of these experiments is their reversibility in that 5my P.asma Rovine Albumin/ 700 mi.FIG. 3-Reversible adsorption of bovine albumin on to C, sulphate. (Negative interface). the pressure of the monolayer can be raised to its initial value after de-sorption by adjusting the pH to its original value. (c) ANOMALOUS BEHAVIOUR OF y-GLOBULIN.-h the case of y-globulin this reversibility is not so marked 0.6 yo of NaCl is used in the trough to obtain the protein in solution R. MATALON AND J. H. SCHULMAN 31 VE7hen the 7-globulin-cardiolipin system has been slowly brought from pH 4 to pH 10 to construct curve I of Fig. 4 and when it is restored to the original pH a pressure sensibly higher than the original is found. If it is now again brought slowly over the pH range studied in curve I, the same shaped curve is produced (curve 2 ) with this increase of pressure maintained throughout.FIG. ,+-Adsorption of y-globulin (0.6 % NaCl) on to cardiolipin. (Negative interface). General Behaviour of the Desorption Curves.-Although by acid or alkali injections the sign of the protein can be reversed as the isoelectric point is crossed the entire pressure variation is not completed at the reversal of the charge (Fig. I-5) but occurs gradually upon varying the pH and reaches a constant minimum value. These values are gener-ally greater than 15 dyneslcm. The actual figures recorded are : System Protein I Monolayer Pressure Dyneslcm. 1 Bovine albumin Haemoglobin 7-Globulin Cardiolipin Cardiolipin Stearylcholine Cardiolipin ~,,H,,SO,Na 17'5 18.5 21 21 24.3 (curve I) 25.5 (curve 2) The original pressure of the monolayer is 15 dynes/cm.From these results is appears that the pressure of the monolayer attained by adjusting the pH of the bath to the non-reactive protein is essentially dependent on the nature of the protein and does not seem related to the nature of the monolayer present at the interface. While with bovine albumin on cardiolipin or C2,C,,S0,Na a pressure rise of 2.5-3.5 dyneslcm. above the initial pressure of the monolayer is observed ; with haemoglobin on cardiolipin or on stearylcholine this pressure difference is 6 dynes/cm. The marked increase of 9.3-10-5 dyne/cm. observed in the case of y-globulin with cardiolipin is probably due to the high molecular weight of this protein and also possibly to the existence of traces of lipids present as an impurity in the fraction under investigation 3 2 LIPO-PROTEIX MONOLAYERS Solution and Penetration at Constant Pressure.-In order to deter-mine the influence of electrical forces on the association of protein with long-chain ionic compounds the behaviour of a neutral molecule such as cholesterol in the presence of proteins has been studied and compared with the association of negatively charged monolayers with proteins.globin is injected at pH 4-6-4-8 under a cholesterol monolayer kept at (a) SOLUTION EFFECT.-HAEMOGLOBIN-cHOLESTEROL.-when haem0-28 26 24 22 20 1 ' I I 1 4 6 8 lo 1.2 I4 p H FIG. 5-Reversible adsorption of sheep haemoglobin on to stearyl-choline.(Positive interface). 14 dyneslcm. pressure an expansion is observed (Fig. 6) which after 7 min. becomes linear. This indicates that the expansion of the cholesterol is due to solution of the protein in the monolayer and not to a specific interaction between the two compounds. This conclusion is supported by the previous worku where gliadin is ejected from a cholesterol mono-layer at its own collapse pressure. FIG. 6-Surface solution of haemoglobin in a cholesterol monolayer. Seventy minutes after the injection of the haemoglobin the cholesterol monolayer had extended by 33 yo but the film was still liquid while on the free water side was a strong gel due to the formation of an adsorbed layer of the protein. Eayers oflflositely charged.-Haemoglobin injected under a film of cardio-lipin at 25 dynes/cm.pressure and at pH 4 causes rapid extension of the monolayer followed after 55 min. by a linear expansion similar to the solution effect observed on the cholesterol monolayer. Thus the curve (b) SOLUTION EFFECT AND PENETRATION.-(i) PrO&?in and WZOnO K. MATALON AND J. H. SCHULMAN 33 obtained (Fig. 7) is a summation of rapid penetration and slow solution, the two parts of which can be distinguished by projecting the linear portion of the curve back to the extensim axis. The area at which this line cuts the axis is that increase due to complete saturation of the monolayer by penetration the remaining increase is due to the process of solution. FIG. 7.-Interaction leading to complexes between haemoglobin and cardiolipin.(ii) Neutral $rotein and negative interfaces.-If haemoglobin is now injected at its isoelectric point no expansion is observed due to protein entering the C,,H,,SO,Na monolayer kept in a pressure range of 28 to 2 0 dyneslcm. for a period of 35 min. If after this period acid is injected to shift the pH to I well on the acid side of the isoelectric point very strong penetration occurs at 28 dyneslcm. pressure and IOO yo extension Surface pressure = ~~.dyne./cm. 5m9. Hoemoylobrn 700 ml. I FIG. 8.-Interaction leading to complexes between haemoglobin and G2 sulphate. of the original area is produced within 40 min. This shows that the phenomenon of penetration is controlled by electrical forces. (iii) Physical state of expanded monoZayers.-While the cholesterol monolayer remained liquid after the expansion cardiolipin and C ,H,,SO,Na become strong gels when the protein penetrates them.(iv) Influence of steric factors on penet~ation.~-When the haemoglobin is injected at pH 3 below a C,,H,,SO,Na monolayer at 31 dyneslcm. 34 LIPO-PROTEIN MONOLAYERS pressure the maximum extension observed is 33 yo (Fig. 9 curve Ia). A further extension to 66 yo is obtained by adjusting the pressure to 28 dynes/cm. (Fig. 9. curve Ib). Finally at 25 dyneslcm. the monolayer soon Time in m,i?uL?s. FIG. g.-Surface pressure and pH variation on the kinetics of penetration and of “ bound protein.” ejection of haemoglobin with C, sulphate. The existence develops a constant linear increase showing an added solution effect (Fig.9 curve Ic). These experiments demonstrate that the closer the packing of the chains in the monolayer the less penetration occurs. FIG. 10.-Demonstration of irreversibly “ bound protein ” in system C, sulphate-haemoglobin. (v) Existence of “ Bound Protein.”-If under this haemoglobin C2,H,,SOdNa system at pH 3 NaOH is injected to increase the pH to 10 rapid ejection o f the protein takes place from the monolayer (Fig. g R. MATALON AND J. H. SCHULMAN 35 curve 11) and the area attained once the ejection is complete exceeds the original area of the unpenetrated monolayer by 35 %. This increase in area is due to irreversibly “ bound protein ” associated with the inter-face by non-electrical forces while the protein readily ejected by pH changes is associated with the monolayer mainly by the action of electrical forces and is thus expelled when the sign of the protein is reversed.Re-expansion of the monolayer can readily be obtained if the pH is now decreased to 4.2 by a new injection of acid. In this case the com-plex protein C2,Hd5SO4Na is reformed but the rate of solubility of the protein in the monolayer has increased i.e. the slope of the linear part of the curve is steeper (Fig. 10 curve 111). It is interesting t o note that if the pH is again increased to 6-4-6-5 close to the isoelectric point no displacement of the interface is observed for a period of 11 min. and the area is constant (Fig. 10 curve IV). Re-ejection of the protein is now obtained on altering the pH t o 10. When equilibrium is reached the monolayer occupies an area 80 yo greater than its original area before penetration (Fig.10 curve V). Although penetration of a charged monolayer does not take place at the isoelectrie point once a monolayer is penetrated only reversal of the sign of the protein will eject it. Furthermore reversing the sign of the protein causes ejection of some of the protein only. Discussion Adsorption and Desorption.-Desorption of proteins from electri-cally charged monolayers has been studied by following the variation of a h FIG. I I .-Structure of the mixed films protein-negatively charged monolayer. surface pressure with varying pH on either side of the isoelectric point a. on reversible adsorption. b. on irreversible penetration. ~~ (Fig.1-5). The experiments carried out a t constant area show the mechanism of one typk of association between the protein and the charged inter-face but a more complete interpretation of the results is obtained when the expansion experiments are considered at the same time. A marked rise in surface pressure follows injection of proteins under electrically charged monolayers kept at constant area if the protein and the monolayer are oppositely charged. This rise is almost entirely due to polar-polar interaction van der Waals’ forces are not appreci-ably involved since as expansion experiments show the protein cannot enter the film at the pressures thus reached (Fig. IIU) LIPO-P ROTE I N MON OLAY EKS It might be expected that the polar-polar interaction between the monolayer and the protein will increase as the pH becomes more distant from the isoelectric point as it is known that the charge of the protein increases the further the pH is moved from this point.But experimentally the pressures observed on the desorption curve are almost constant in the whole range of pH where protein and spread monolayer are oppositely charged ; this is because the structure of the protein-monolayer association limits the possible rise. Indeed the maximum pressures a t which marked expansion of monolayers by proteins can occur is only slightly less than the pressures at which these penetrated monolayers are ejected and collapsed (33 dyneslcm.). For example if haemoglobin is injected under cardiolipin at pH 4 and the surface pressure maintained a t 25 dynes/cm.(5 dynes/cm. lower than the pressure observed 36 min. after the injection (30 dyneslcm.) for this particular system at constant area) a rapid extension of the monolayer will occur doubling its area in 36 min. (Fig. 7). Furthermore the maximum pressures that such a penetrated film can sustain can be determined by recompression of the extended film to its original area. This pressure is found to be 33 dynes/cm. which is 8 dynes/cm. higher than the expansion pressure (25 dynes/cm.) and 3 dynes/cm. greater than the maximum pressure (30 dynes/cm.) at-tained at constant area. It is thus impossible to follow the varying degree of affinity of the protein and monolayer when oppositely charged, solely by studying the desorption curve. The expansion technique would enable these variations to be studied.No expansion of a C22H45S04Na monolayer by haemoglobin takes place at the isoelectric point at 25 dyneslcm. pressure while a very rapid expansion occurs when the protein is well on the acid side. Furthermore if expansion is done a t 28 dynes/cm. a t pH I IOO yo area increase is observed (Fig. S) while a t pH 4 66 yo only of the extension is produced (Fig. 9 curve ~ b ) . Considering again the desorption curve in the pH range where the sign of the protein becomes identical with that of the monolayer, as the protein increases in charge the pressure falls in all systems investigated. But this fall is gradual and is complete only a t pH values well separated from the isoelectric point (Fig. 1-5).Again it would have been expected from the amphoteric character of proteins and from the mechanism of the adsorption process mainly polar-polar interaction that the surface pressure would have suddenly decreased to the original pressure of the monolayer (15 dynes/cm.) on crossing the isoelectric point. These experimental results indicate that desorption of the proteins involves other factors than the overcoming of electrical forces. We know that the rise in pressure observed is due to some compression of the monolayer by the few side chains of the proteins which enter it, and which associate by van der Waals’ forces to the long hydrophobic chains present a t the surface. Hence complete desorption will necessitate overcoming these forces also. In fact even when maximum desorption is reached the pressure of the monolayer is still higher than its original pressure the greater the molecular weight of the protein and the higher the probability of the existence of uncharged lipids the higher is the pressure reached a t complete desorption.The rise in pressure above 15 dynes/cm. of charged monolayer R. MATALON AND J. H. SCHULMAN 37 by proteins similarly charged is not only observed in the desorption process but in the adsorption process (see part I). The mechanism of this rise may be found in the solution effect. The pressure rise obtained when the protein and monolayer are similarly charged does not mean that there is strong association between them under these conditions. In fact detailed analysis will show that this expresses only very few contacts.The important point is that as the isoelectric point is crossed the pressure falls. Now the pressure reached at maximum desorption indicates a saturation in the adsorbed layer and this saturation has been demonstrated by the expansion technique. Indeed at acid pH values the protein is extensively accumulated below the negatively charged monolayer and orientated by an attractive electrical field of force. This is shown by the marked increase in area of the monolayer when the protein is allowed to enter the surface. I t can be shown too that at and beyond the isoelectric point no penetration occurs i.e. through-out the region where the protein and monolayer are similarly charged. Solution and Penetration.-Expansion experiments show that solution and penetration of proteins take place during the extension of monolayers and it is easy to distinguish the two processes by studying the rate of extension.Solution effect is due to simple diffusion of the protein to the surface and this process is characterized by a constant rate; thus a linear extension is obtained (Fig. 6 ) . Penetration is due to a marked interaction between the mono-layer and the protein in solution. The rate of this interaction is therefore dependent on the concentration of the two reagents. For a constant concentration of the protein in solution the rate is pro-portional to the number of non-associated molecules present in the surface. Thus penetration follows a law of pseudo first-order reaction (since the concentration of the reacting protein is not varied by the reaction) and the rate of extension decreases as the monolayer expands.I t is experimentally possible to show the existence of the solution effect exclusive of any other process whilst penetration is in most cases accompanied by solution. Failure to distinguish between the two processes had led several authors dealing with the penetration phenomena to doubtful conclusions as to the conditions of existence of stoichio-metric lo complexes. In the case of haemoglobin interacting with cardiolipin (Fig. 7) or C,,H,,SO,Na (Fig. 8) 100 yo extension due t o penetration was shown to exist. This indicates that one chain of the monolayer associates with one chain of the protein (Fig. I I ~ ) . It is therefore possible to characterize stoichiometric complexes which result from “ chain interaction.” This result is explained if the protein has available side chains orientated towards the monolayer. It appears therefore that the globular structure of proteins in solution with boundaries covered with hydrophilic groups only is being altered when in contact with long-chain ionic compounds. Such an alteration would help to explain some of the effect of detergents on the structure of proteins. Whereas in the adsorption of proteins on to charged interfaces van der Waals’ forces are of a minor importance i t would appear that these forces are responsible for the expansion of uncharged monolayers such as cholesterol. Joly Nature, 1946 158 26. lo Schulman Stenhagen and Rideal Nature 1938 141 785 38 LIPO-PROTEIN MONOLAYERS " Bound Protein."-The effect of pH on the expansion of charged interfaces shows the effect of electrical forces while the irreversibility of the expansion demonstrates the van der Waals' forces. Indeed it has been shown that expansion of a charged monolayer does not take place at the isoelectric point and that the monolayer already expanded at convenient pH values does not contract when the pH is adjusted to the neutral region of the protein. Extra energy is needed to eject from the surface the side chains of the proteins associated by non-polar forces to the monolayer and this energy can be supplied by reversing the charge of the protein. In this case only partial ejection occurs. The " bound protein " is a constituent of the expanded monolayer which cannot be ejected by altering the pH.The nature of this " bound protein" is of great interest. Although more experiments are still needed i t would appear that the bound protein is that which has entered the surface by a mechanism which involves only the action of van der Waals' forces and which is completely independent of any polar-polar interaction. Such a mechanism is found in the solution effect and has been proved to take place simultaneously with the penetration. The probable structures of adsorbed proteins on to charged mono-layers and of the chain complexes between protein and long-chain ionic compounds as obtained by penetration have been described in Fig. 11. Analogies to Reaction in Bulk.-Previous work shows cases of reversible and irreversible changes when the proteins are adsorbed on to charged oil-water interfaces as with emulsions and then desorbed. Haemoglobin for example is regenerated as parahaematin which is less soluble and has lost its biological properties whereas snake venoms and toxins can regain their full activity on desorption.ll The present work has differentiated two mechanisms of interaction of proteins a t charged interfaces. Adsorption which is readily revers-ible involves mainly interaction of polar groups which are available on the surface of the protein molecule without involving any change of the molecular configuration whereas penetration necessitates radical alterations in the molecular structure to enable the non-polar chains to enter into the surface and associate by penetration with the non-polar portion of the film forming molecule. By analogy it could thus be assumed that where irreversible changes are observed with the emulsion technique penetration at the charged oil-water interface has taken place. Adsorptions both a t the charged oil-water and air-water interfaces are conditioned by pH and are related to the charge of the protein coming in contact with the interface. The emulsion flocculation range which has been indicated in Fig. 1-5 show that as soon as re-dispersion of the emulsion is noticed the pressure of the monolayer falls markedly, and this occurs on the side of the isoelectric point where the protein and monolayer are similarly charged. Similar to the flocculation of emulsions proteins can be reversibly precipitated by oppositely charged long-chain ionic compounds. Furthermore several authors have shown that soluble lipo-protein associations can exist over the pH range where the proteins and 11 Frazer and Stewart Brit. J . Expt. Path. 1940 21 361 R. MATALON AND J. H. SCHULMAN 39 long-chain ions are similarly charged.12 This observation is confirmed by the present work where the existence of " bound protein" is demonstrated at the non-reactive pH range of the proteins. Our thanks are due to Dr. I. S. Longmuir for haemoglobin prepara-tions and to Mr. A. Dunn for technical assistance. Department of Colloid Science, The University, Cam bridge. 12Steinhardt and Fugitt J . Res. Nut. Bur. Stand. 1942 29 315. Stein-hardt Fugitt and Harris ibid. 1941 26 293

ISSN:0366-9033    

DOI:10.1039/DF9490600021

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

R. MATALON AND J. H. SCHULMAN L Lo R R R R 2 GENERAL DISCUSSION * Dr. J. Glazer (Cambridge) said Doty Matalon and Schulman have described and characterized the different types of affinity exhibited by 2 CH ,-CH-CH 0 I Lo Lo CH 2-CH-CH I I 0 I L A ? A Lo 0 0 I I Lo I 1 I I R R CHZ-CH-CH 0 I 0 I I 9 0 O t P - 0 Xa R R R co co CO 0 f NaQ I00 O t P - 0 Na 0 I I CH,- R R R R r f I 1 I 1 co co co co I I CH2-CH-CH2 (after Doty and Schulman) b b b b l o I 0 I Na@ Na@ R I CO I CO I 0 I CH- a certain proteins towards charged monolayers one of which was cardio- lipin. Doty and Schulman observed that monolayers of cardioplipin on acetate buffer (pH 5-1) collapsed at an area of 185 Hiz per molecule thereby providing strong evidence for the existence of six unsaturated long chains in the molecule each of which is known to occupy ca.30 Hi2 at the collapse point. They suggested that the monolayer behaviour seemed more consistent with a structure A involving three glyceryl phosphoric acid units joined to one glycerine molecule rather than the linear structure B proposed by Pangb0rn.l (See structural diagrams A and B.) (after Pangborn) I STRUCTURE B * On two preceding papers. IPangborn J. Biol. Chem. 1947 168 351. I00 O t P - 0 Na 0 I 1 CH STRUCTURE A 39 I I I 0 I I -CH GENERAL DISCUSSION 40 In choosing between these basic structures the fact that no mono- glyceryl triphosphates have ever been isolated from natural sources or prepared synthetically makes it rather unlikely that the structure A is correct.The method of molecular models shows that while such a structure is consistent with a globular type of molecule in which the long- chain units radiate out symmetrically from the central glyceryl tri- phosphate core it would be quite impossible for a molecule having this structure to unfold so as to produce a stable monolayer. Furthermore as will be shown below the Pangborn structure B is fully consistent with the monolayer properties of cardiolipin although it would seem that a modification involving intramolecular electrostatic cross-linking between adjacent phosphate groups is necessary to explain several of its properties.2 0 . 8 c - - - ! 100 0 A more detailed investigation has now been made of the monolayer properties of cardiolipin using a Langmuir-Adam trough which permitted simultaneous measurements of surface pressure and surface potential. The results are shown in Fig. I. The phospholipid was spread from ethyl alcohol solution (0.76 mg./ml.) on to substrates of varying pH and containing various salts. It was found that cardiolipin does not spread completely on distilled water whereas the presence of salts in the aqueous substrate results in complete spreading. When cardiolipin is spread on distilled water and the sub- strate is then injected with phosphate buffer the cardiolipin remains on the surface in its unfolded form. A subsequent spread of cardiolipin on to the same phosphate substrate results in complete spreading the resulting monolayer having an area (at the collapse point) of 180 As per molecule.Complete spreading was found to take place on M/IOO HC1 M/zo phosphate buffer (pH 7-4) M/IOO CaC1 (pH 8.5) M/7 NaCl (pH 8.0) and M/20 borate buffer (pH 10-I) whereas incomplete spreading took pIace on distilled water and M/5000 HC1 substrates. It can be seen - 2 0 0 2 0 0 5 0 0 5 0 0 6 2 306 306 400 400 I I I 2 0 0 I I 3. I. SUBSTRATE 0.8s)b NaCl 2. . Phoaphat. buffer - N/lOO HCI pH 7.4 5 0 0 6 0 0 300 400 AREA/MOLECULE AtL FIG. I. GENERAL DISCUSSION therefore that the ethyl alcohol present in the spreading solution is responsible for the initial spreading process while the presence of electro- lytes in t h j aqueous substrate is necessary for the stabilization of the completely spread monolayer at the surface.(It should be mentioned that the instability of the cardiolipin monolayer is due to the spontaneous accumulation of three-dimensional islands or conglomerates in the sur- face. This type of spontaneous collapse is in general observed when the intermolecular attraction of the monolayer exceeds the adhesional attraction between the monolayer and the aqueous substrate.) The nature of this stabilization is a matter for discussion but it seems reason- able to ascribe it to the disruption by the dissolved electrolyte of inter- phosphate cross-links in the cardiolipin molecule. It is suggested that cardiolipin under normal conditions (i.e.in the folded state) contains the following type of intra-molecular cross-linking involving a " sodium ion " bridge as in structure c the function of the positive sodium ion being to link together a formally negatively charged oxygen ion with a semi-polar negative oxygen atom. Should these cross-links be sufficiently strong to overcome the affinity of the sodium ion and phosphate group for the aqueous substrate then the molecule will not unfold completely to form a monolayer ; this is found experimentally on distilled water and M/5000 HC1. The presence of sufficient electrolyte in the. aqueous sub- strate serves to disrupt the cross-links by increasing the adhesion of the ionic part of the cardiolipin to the aqueous phase as a result of ion-ion attraction.-0 -0 0 I 0 I -o 3 40 0 I 4+v4. I S @ O+P-0 . Na . O+P-0 . Na. W P - 0 . Na l e e I 9 8 0 I STRUCTURE C -0 0 0 0-P-0. Substrate I m 6 e b e 1 . .Cd . . . 0-P+O. w I 0.85 yo NaCl . Phosphate buffer (pH 7.4) . I % CaC1 M/IOO HC1 STRUCTURE D Reference to Fig. I shows that cardiolipin forms liquid-expanded monolayers on aqueous substrates containing electrolytes. The surface characteristics are summarized in the following Table. The limiting Lim. Area at low Pressure A2 per moI. ] 350 3 50 310 Dipole at low Pressure e l . .Na Q . . .O-P+O 0 I Area a t Collapse A2 per mol. b+#w (Milli-D) I 80 184 2300 I 800 24.50 1850 I 60 I75 .areas both at zero pressure and at the collapse pressure are best char- acterized by discontinuities in the surface dipole (rather than the surface pressure) since this function is appreciably linear over considerable ranges. From a consideration of both the above Table and Fig. I it is clear that the nature of the electrolyte exerts only minor effects on the B * Dipole a t Collapse (milli-D ) I 400 I 300 1500 1320 GENERAL DISCUSSION 42 monolayer characteristics. Nevertheless these effects are real and definite. With the exception of the CaCl substrate (see below) the monolayer collapses at an area of 180 & 5 A2 per molecule. This confirms the observation of Doty and Schulman on acetate buffer pH 5.1 and cor- responds well to the area occupied by six close-packed unsaturated long chains at the air-water interface (i.e.30 Aa per long chain) ; triolein for example occupies 95 Hi2 per molecule a t the collapse point. Further- more the limiting area a t low pressure of cardiolipin is with the exception of M/roo HC1 GU. 350 larger than that of triolein (43 Az per oleyl chain) and is attributable per molcule. The latter area is appreciably to the existence oi the strong negative charge in the monolayer which as a result of mutual repulsion produces a more expanded film. It is significant that on a substrate of appreciable acidity such as M/IOO HC1 where this negative charge is somewhat discharged (cf. a-glyceryl phosphoric acid pKt = 1-40) the monolayer is more condensed in the low-pressure region.The presence of a divalent cation such as calcium is seen to effect marked condensation of the film the area at the collapse point being only 160 per A 2 mol. This effect is characteristic of calcium substrates in contact with negatively charged monolayers such as fatty acids. Pangborn 2 noticed that it was never possible completely to replace the sodium of cardiolipin by cadmium ; she remarked that the cadmium salt of cardiolipin was always found to contain one atom of sodium together with one atom of cadmium. This is readily understood in terms of the above postulated cross-links where a divalent cation such as cadmium is able to act as a bridge between two formally negatively charged oxygen ions as in structure D.This divalent type of cross- link is to be distinguished from the monovalent sodium type in structure c since the former is partially covalent in character while the latter retains the whole of its electrostatic character. The tendency to assume this type of linkage at the air-water interface may very well account for the observed condensation when calcium ions are present in the sub- strate. This condensation with consequent loss of electrostatic charge is further characterized by the increased positive surface dipole cor- responding to the partial removal of a negatively charged layer in the surface. The above experiments show therefore that the structure of cardio- lipin when spread completely at the air-water interface is fully con- sistent with the structure B it being understood that the polyglyceryl phosphate skeleton is lying in the interface with the unsaturated long chains pointing away from the aqueous phase.Prof. F. Haurowitz (Bloomington Indiana U.S.A.) said It is very difficult to understand the formation of ten layers of unfolded denatured haemoglobin around the lipid droplets. The parahaematin spectrum cannot be considered as a proof for unfolding of the protein molecules. Parahaematin spectra would also be observed if haem were detached temporarily from the globin surface and the binding surface of the globin molecule altered slightly so that the oi iginal globin-haem bond would not be reconstituted. Miss M. Pangborn (New Yo&) said The evidence presented by Dr.Doty and Schulman and by Dr. Glazer on the structure of cardio- lipin is most helpful. The linear structure of the polyglycerophosphoric ester which I suggested in 1947 represented merely the simplest way cf accounting for the analytical composition and hydrolytic products found ; there was no evidence at that time regarding the position of the linkages. The report of the composition of the Cd salt which still contained Na probably should not be used as an argument for structural considerations. This finding was quoted from my first paper when the purification methods were rather inadequate. The fatty acid in cardiolipin is linoleic rather GENERAL DISCUSSION 43 than oleic and I wonder whether the properties of the monolayer are affected by the second double bond.Prof. J. R. Marrack (London) said It is probable that the antibody ir syphilitic serum will combine with cardiolipin even when the serum is highly diluted although no floccules are formed. The essential point is the amount of cardiolipin per cm.a of surface. The concentration of antibody in the experiments of Dr. Doty and Dr. Schulman must be a small fraction of a microgram per ml. ; this may be too little to have an appreciable effect on the layer of caxdiolipin even if it does combine. It is unlikely that effects would be detected when whole serum is used as th inert proteins of serum exceed the antibody in a ratio of over IOO to I . Failure of antibodies to combine with antigens spread on a surface may be du- to distortion of the spatial arrangement on which specific combination depends.Dr. J. Glazer (Cambridge) said I n reply to Miss Pangborn the mono- layer compression curves of linoleic and oleic acids are practically identical. I am of course aware that the fatty acid content of tardiolipin is linoleic rather than oleic but the absence of any monolayer information con- cerning glyceryl trilinoleate forces comparison with the corresponding trioleate. The mon olayer similarity between linoleic and oleic acid suggests that this procedure is not unreasonable. Regarding the composition of the cadmium salt of cardiolipin I agree that caution should be exercised. It would be of interest to know if more recent analytical results are yet available since it seems more than coincidental that the Na/Cd ratio should be unity.Prof. A. C. Frazer (Birmingham) said No association can be demon- strated between lecithin and protein using the techniques described by Dr. Schulman and Dr. Matalon. There is however evidence of a close association between lecithin and protein in the blood and this association markedly alters the properties and behaviour of the individual com- ponents. This association can be demonstrated by the effect of leci- thinase on chylomicron stability and the separation of lipid and protein elements in the Nagler reaction. Lipids or proteins by themselves give rise to quite different characteristics in artificial emulsions. Presumably different types of association are being studied under these different con- ditions.Failure to obtain association of added lecithin with plasma protein might be due to the fact that lecithin is already present in natural plasma proteins. There is no reason to suppose that the destruction of I his natural association by fractionating procedures should be readily reversible by the simple addition of lecithin. Dr. A. S. McFarlane (London) saia I can confirm that lecithin does not combine with serum proteins. No-one disputes Prof. Frazer’s point that lecithin is bound to proteins in natural serum but in attempts to emulsify lecithin by various methods with normal or lipid-poor sera Mrs. Davey and I find that the lecithin always migrates in the electro- phoresis cell separately from the serum proteins.This applies to egg and brain lecithins. Miss N. M. Czeczowicka (London) said Is there any evidence that the “ residual protein bound in lipid ” is not just surface-denatured protein and therefore cannot be resolubilized ? Dr. R. Matalon (Cambridge) said Surface denaturation of the protein consists of the spontaneous unfolding of the protein and this cannot take place under the experimental condition, as the spontaneous spreading of the protein is prevented by the high pressure which is established in the monolayer. In reply to Prof. Frazer the presence of lecithin in serum proteins could be explained by the solubility mechanism which has been described in our paper for cholesterol and haemoglobin. Dr. J. H. Schulman (Cambridge) said In reply to Prof.Frazer by surface methcds lecithin monolayers have been shown to be non-reactive COMBINATION OF ANIONS WITH PROTEINS 44 to serum proteins over the pH range 3-10 but can be readily shown to interact with snake venom injected into the underlying solution to form a lysolecithin monolayer. In reply to Prof. Haurowitz the desorbed haemoglobin from the emulsion surface not only shows the pure haematin spectrum but floccu- lates at the isoelectric point of haemoglobin. Further the desorbed monodisperse protein solution shows the colour change to reddish brown on reduction by sodium bisulphate on warming and on pH change to alkali solution.2 The restabilization of the emulsion on desorption of the protein is readily explained by the reversible adsorption of the haemo- globin from a negatively charged surface on changing the pH of the solution to a pH above the isoelectric point of the protein.Elkes Frazer Schulman and Stewart Proc. Roy. SOC. A 1945 184 IOZ. R. MATALON AND J. H. SCHULMAN 39 GENERAL DISCUSSION * Dr. J. Glazer (Cambridge) said Doty Matalon and Schulman have described and characterized the different types of affinity exhibited by R R R R R R Lo Lo L Lo Lo Lo I I L A ? A 0 0 I I CHZ-CH-CH I 1 CH ,-CH-CH 2 I I CH 2-CH-CH 2 I 0 I I00 I 9 0 0 I I00 0 O t P - 0 Na O t P - 0 Xa O t P - 0 Na I 0 I 0 I 0 1 CH I CH,-STRUCTURE A (after Doty and Schulman) R R r l o I 1 co co b b I Na@ R CO CH-I I R R co co I I 0 f a NaQ f I 0 0 R R I I CH2-CH-CH2 R I I I CO 0 -CH, STRUCTURE B (after Pangborn) certain proteins towards charged monolayers one of which was cardio-lipin.Doty and Schulman observed that monolayers of cardioplipin on acetate buffer (pH 5-1) collapsed at an area of 185 Hiz per molecule, thereby providing strong evidence for the existence of six unsaturated long chains in the molecule each of which is known to occupy ca. 30 Hi2 at the collapse point. They suggested that the monolayer behaviour seemed more consistent with a structure A involving three glyceryl phosphoric acid units joined to one glycerine molecule rather than the linear structure B proposed by Pangb0rn.l (See structural diagrams A and B.) * On two preceding papers. IPangborn J. Biol. Chem.1947 168 351 40 GENERAL DISCUSSION In choosing between these basic structures the fact that no mono-glyceryl triphosphates have ever been isolated from natural sources or prepared synthetically makes it rather unlikely that the structure A is correct. The method of molecular models shows that while such a structure is consistent with a globular type of molecule in which the long-chain units radiate out symmetrically from the central glyceryl tri-phosphate core it would be quite impossible for a molecule having this structure to unfold so as to produce a stable monolayer. Furthermore, as will be shown below the Pangborn structure B is fully consistent with the monolayer properties of cardiolipin although it would seem that a modification involving intramolecular electrostatic cross-linking between adjacent phosphate groups is necessary to explain several of its properties.2 0 0 306 400 5 0 0 6 2 0 . 8 c 2 - - ! I I I I I -I. SUBSTRATE 0.8s)b NaCl 2. . Phoaphat. buffer 3. - N/lOO HCI pH 7.4 0 100 2 0 0 300 400 5 0 0 6 0 0 AREA/MOLECULE AtL -FIG. I. A more detailed investigation has now been made of the monolayer properties of cardiolipin using a Langmuir-Adam trough which permitted simultaneous measurements of surface pressure and surface potential. The results are shown in Fig. I. The phospholipid was spread from ethyl alcohol solution (0.76 mg./ml.) on to substrates of varying pH and containing various salts. It was found that cardiolipin does not spread completely on distilled water, whereas the presence of salts in the aqueous substrate results in complete spreading.When cardiolipin is spread on distilled water and the sub-strate is then injected with phosphate buffer the cardiolipin remains on the surface in its unfolded form. A subsequent spread of cardiolipin on to the same phosphate substrate results in complete spreading the resulting monolayer having an area (at the collapse point) of 180 As per molecule. Complete spreading was found to take place on M/IOO HC1 M/zo phosphate buffer (pH 7-4) M/IOO CaC1 (pH 8.5) M/7 NaCl (pH 8.0) and M/20 borate buffer (pH 10-I) whereas incomplete spreading took pIace on distilled water and M/5000 HC1 substrates. It can be seen GENERAL DISCUSSION therefore that the ethyl alcohol present in the spreading solution is responsible for the initial spreading process while the presence of electro-lytes in t h j aqueous substrate is necessary for the stabilization of the completely spread monolayer at the surface.(It should be mentioned that the instability of the cardiolipin monolayer is due to the spontaneous accumulation of three-dimensional islands or conglomerates in the sur-face. This type of spontaneous collapse is in general observed when the intermolecular attraction of the monolayer exceeds the adhesional attraction between the monolayer and the aqueous substrate.) The nature of this stabilization is a matter for discussion but it seems reason-able to ascribe it to the disruption by the dissolved electrolyte of inter-phosphate cross-links in the cardiolipin molecule.It is suggested that cardiolipin under normal conditions (i.e. in the folded state) contains the following type of intra-molecular cross-linking involving a " sodium ion " bridge as in structure c the function of the positive sodium ion being to link together a formally negatively charged oxygen ion with a semi-polar negative oxygen atom. Should these cross-links be sufficiently strong to overcome the affinity of the sodium ion and phosphate group for the aqueous substrate then the molecule will not unfold completely to form a monolayer ; this is found experimentally on distilled water and M/5000 HC1. The presence of sufficient electrolyte in the. aqueous sub-strate serves to disrupt the cross-links by increasing the adhesion of the ionic part of the cardiolipin to the aqueous phase as a result of ion-ion attraction.0 -0 l e e I 9 8 -0 I S @ I I O+P-0 . Na . O+P-0 . Na. W P - 0 . Na I 0 0 -o 4+v4. STRUCTURE C 0 -0 I m 6 e b e 1 Q e l 0-P-0. . .Cd . . . 0-P+O. . .Na . . .O-P+O I I I 0 0 w 0 b+#w STRUCTURE D Reference to Fig. I shows that cardiolipin forms liquid-expanded monolayers on aqueous substrates containing electrolytes. The surface characteristics are summarized in the following Table. The limiting Substrate 0.85 yo NaCl . Phosphate buffer (pH 7.4) . I % CaC1, M/IOO HC1 . Lim. Area at low Pressure A2 per moI. ] 350 3 50 3 40 310 Dipole at low Pressure (Milli-D) 2300 I 800 24.50 1850 Area a t Collapse A2 per mol. I 80 I 60 I75 184 Dipole a t Collapse (milli-D ) I 400 I 300 1500 1320 areas both at zero pressure and at the collapse pressure are best char-acterized by discontinuities in the surface dipole (rather than the surface pressure) since this function is appreciably linear over considerable ranges.From a consideration of both the above Table and Fig. I it is clear that the nature of the electrolyte exerts only minor effects on the B 42 GENERAL DISCUSSION monolayer characteristics. Nevertheless these effects are real and definite. With the exception of the CaCl substrate (see below) the monolayer collapses at an area of 180 & 5 A2 per molecule. This confirms the observation of Doty and Schulman on acetate buffer pH 5.1 and cor-responds well to the area occupied by six close-packed unsaturated long chains at the air-water interface (i.e.30 Aa per long chain) ; triolein, for example occupies 95 Hi2 per molecule a t the collapse point. Further-more the limiting area a t low pressure of cardiolipin is with the exception of M/roo HC1 GU. 350 The latter area is appreciably larger than that of triolein (43 Az per oleyl chain) and is attributable to the existence oi the strong negative charge in the monolayer which, as a result of mutual repulsion produces a more expanded film. It is significant that on a substrate of appreciable acidity such as M/IOO HC1 where this negative charge is somewhat discharged (cf. a-glyceryl phosphoric acid pKt = 1-40) the monolayer is more condensed in the low-pressure region. The presence of a divalent cation such as calcium is seen to effect marked condensation of the film the area at the collapse point being only 160 per A 2 mol.This effect is characteristic of calcium substrates in contact with negatively charged monolayers such as fatty acids. Pangborn 2 noticed that it was never possible completely to replace the sodium of cardiolipin by cadmium ; she remarked that the cadmium salt of cardiolipin was always found to contain one atom of sodium together with one atom of cadmium. This is readily understood in terms of the above postulated cross-links where a divalent cation such as cadmium is able to act as a bridge between two formally negatively charged oxygen ions as in structure D. This divalent type of cross-link is to be distinguished from the monovalent sodium type in structure c since the former is partially covalent in character while the latter retains the whole of its electrostatic character.The tendency to assume this type of linkage at the air-water interface may very well account for the observed condensation when calcium ions are present in the sub-strate. This condensation with consequent loss of electrostatic charge, is further characterized by the increased positive surface dipole cor-responding to the partial removal of a negatively charged layer in the surface. The above experiments show therefore that the structure of cardio-lipin when spread completely at the air-water interface is fully con-sistent with the structure B it being understood that the polyglyceryl phosphate skeleton is lying in the interface with the unsaturated long chains pointing away from the aqueous phase.Prof. F. Haurowitz (Bloomington Indiana U.S.A.) said It is very difficult to understand the formation of ten layers of unfolded denatured haemoglobin around the lipid droplets. The parahaematin spectrum cannot be considered as a proof for unfolding of the protein molecules. Parahaematin spectra would also be observed if haem were detached temporarily from the globin surface and the binding surface of the globin molecule altered slightly so that the oi iginal globin-haem bond would not be reconstituted. Miss M. Pangborn (New Yo&) said The evidence presented by Dr. Doty and Schulman and by Dr. Glazer on the structure of cardio-lipin is most helpful. The linear structure of the polyglycerophosphoric ester which I suggested in 1947 represented merely the simplest way cf accounting for the analytical composition and hydrolytic products found ; there was no evidence at that time regarding the position of the linkages.The report of the composition of the Cd salt which still contained Na probably should not be used as an argument for structural considerations. This finding was quoted from my first paper when the purification methods were rather inadequate. The fatty acid in cardiolipin is linoleic rather per molcule GENERAL DISCUSSION 43 than oleic and I wonder whether the properties of the monolayer are affected by the second double bond. Prof. J. R. Marrack (London) said It is probable that the antibody ir syphilitic serum will combine with cardiolipin even when the serum is highly diluted although no floccules are formed.The essential point is the amount of cardiolipin per cm.a of surface. The concentration of antibody in the experiments of Dr. Doty and Dr. Schulman must be a small fraction of a microgram per ml. ; this may be too little to have an appreciable effect on the layer of caxdiolipin even if it does combine. It is unlikely that effects would be detected when whole serum is used as th inert proteins of serum exceed the antibody in a ratio of over IOO to I . Failure of antibodies to combine with antigens spread on a surface may be du- to distortion of the spatial arrangement on which specific combination depends. Dr. J. Glazer (Cambridge) said I n reply to Miss Pangborn the mono-layer compression curves of linoleic and oleic acids are practically identical.I am of course aware that the fatty acid content of tardiolipin is linoleic rather than oleic but the absence of any monolayer information con-cerning glyceryl trilinoleate forces comparison with the corresponding trioleate. The mon olayer similarity between linoleic and oleic acid suggests that this procedure is not unreasonable. Regarding the composition of the cadmium salt of cardiolipin I agree that caution should be exercised. It would be of interest to know if more recent analytical results are yet available since it seems more than coincidental that the Na/Cd ratio should be unity. Prof. A. C. Frazer (Birmingham) said No association can be demon-strated between lecithin and protein using the techniques described by Dr.Schulman and Dr. Matalon. There is however evidence of a close association between lecithin and protein in the blood and this association markedly alters the properties and behaviour of the individual com-ponents. This association can be demonstrated by the effect of leci-thinase on chylomicron stability and the separation of lipid and protein elements in the Nagler reaction. Lipids or proteins by themselves give rise to quite different characteristics in artificial emulsions. Presumably different types of association are being studied under these different con-ditions. Failure to obtain association of added lecithin with plasma protein might be due to the fact that lecithin is already present in natural plasma proteins.There is no reason to suppose that the destruction of I his natural association by fractionating procedures should be readily reversible by the simple addition of lecithin. Dr. A. S. McFarlane (London) saia I can confirm that lecithin does not combine with serum proteins. No-one disputes Prof. Frazer’s point that lecithin is bound to proteins in natural serum but in attempts to emulsify lecithin by various methods with normal or lipid-poor sera Mrs. Davey and I find that the lecithin always migrates in the electro-phoresis cell separately from the serum proteins. This applies to egg and brain lecithins. Miss N. M. Czeczowicka (London) said Is there any evidence that the “ residual protein bound in lipid ” is not just surface-denatured protein and therefore cannot be resolubilized ? Dr.R. Matalon (Cambridge) said Surface denaturation of the protein consists of the spontaneous unfolding of the protein and this cannot take place under the experimental condition, as the spontaneous spreading of the protein is prevented by the high pressure which is established in the monolayer. In reply to Prof. Frazer the presence of lecithin in serum proteins could be explained by the solubility mechanism which has been described in our paper for cholesterol and haemoglobin. Dr. J. H. Schulman (Cambridge) said In reply to Prof. Frazer by surface methcds lecithin monolayers have been shown to be non-reactiv 44 COMBINATION OF ANIONS WITH PROTEINS to serum proteins over the pH range 3-10 but can be readily shown to interact with snake venom injected into the underlying solution to form a lysolecithin monolayer. In reply to Prof. Haurowitz the desorbed haemoglobin from the emulsion surface not only shows the pure haematin spectrum but floccu-lates at the isoelectric point of haemoglobin. Further the desorbed monodisperse protein solution shows the colour change to reddish brown on reduction by sodium bisulphate on warming and on pH change to alkali solution.2 The restabilization of the emulsion on desorption of the protein is readily explained by the reversible adsorption of the haemo-globin from a negatively charged surface on changing the pH of the solution to a pH above the isoelectric point of the protein. Elkes Frazer Schulman and Stewart Proc. Roy. SOC. A 1945 184 IOZ

ISSN:0366-9033    

DOI:10.1039/DF9490600039

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

COMBINATION OF ANIONS WITH PROTEINS 44 THE COMBINATION OF FATTY ACID ANIONS WITH PROTEINS 7 BY J. MURRAY LUCK Received 20th May 1949 Anions of the aliphatic and aromatic carboxylates sulphonates and sul- phates are bound by the serum albumins more markedly than by other proteins reported upon t o date. The number of ions bound per mole of serum albumin as well as the contribution of the ion to the thermal stability of aqueous solu- tions of serum albumin is a function of the length of the side chain. Maximal effects in the case of fatty acid anions are observed with chains of six to nine carbon atoms. Protection against the denaturation of serum albumin by urea is also conferred by rhe family of anions mentioned. It is concluded that the binding of fatty acid anions a t pH 7-5 t o 8.0 is due to electrostatic attraction by the positively charged guanidine and lysine residues and van der Waals' forces between the side chains of the added anion and the side chains of leucine isoleucine valine and phenylalanine.The present paper is devoted to recent work on the binding of ions by proteins. It is largely restricted to the binding of fatty acid ions by serum albumin and possibly with undue attention to studies that have proceeded in the author's laboratory. The first of these restric- tions is deliberately imposed by a desire not to wander too far from the general subject of the Discussion and also by the rather curious speci- ficity of the phenomenon itself to which we shall presently return. But first let us address to ourselves a very pertinent question what experimental findings necessitate the conclusion that ions are bound by proteins ? Some of the evidence comes from ultrafiltration and dialysis-equilibrium studies 3* 4 9 5 9 effects on absorption 1 Boyer Ballou and Luck J .BioE. Chem. 1947 167 407. Greenberg and Gunther ibid. 1929-30 85 491. 3 Klotz Walker and Pivan J . Amer. Chem. SOC. 1946 68 1486. 5 Klotz Triwush and Walker J . Amer. Chem. Soc. 1948 70 2935. 6 Klotz ibid. 1946 68 2299. 4 Teresi and Luck J . BioZ. Chem. 1948 174 653 ; 1949 177 383. 7 Irvine and Irvine Fed. Proc. 1949 8 209. J. MURRAY LUCK and binding studies on dyes,* 9 7 lo on indicators 11-14 and on synthetic In addition Kendall 2o has described a serum albumin which contained 2 yo fatty acid-somewhat more than the crystallized albumins prepared by the ethanol procedure.Macheboeuf’s studies 21 on protein-fatty acid complexes are well known and add to the body of direct evidence. 45 Equally convincing evidence consists in part of an observation by Scatchard and Black 22 that serum albumin solutions rendered iso- ionic by exhaustive dialysis against water undergo an increase in pH of as much as 1-65 pH units by addition of various neutral inorganic salts. This is suggestive of anion binding and of a considerable change in the ionic properties of the molecule. Studies recently reported by Longsworth and Jacobsen 23 and by Velick likewise give evidence of protein-anion combinations sometimes of a clearly competitive char- acter.The electrophoretic mobility of serum albumin determined after equilibration with sodium salts of the lower fatty acids increases with increase in chain length of the added This is strongly sug- gestive of an anion-albumin association. The results however do not permit of an indubitable conclusion since phosphate which also in- creases the net negative charge of serum albumin is found by Teresi,26 using another method to bind with serum albumin to a degree less than would be predicted from the electrophoretic findings. Some years ago we observed by means of a so-called cloud-point technique that the thermal stability of serum albumin in aqueous solution was much increased by fatty acid anions,l 27’ 28 29 the effect increasing with increase of chain length.I doubt that I would be inclined to cite this as conclusive evidence of fatty acid binding were it not that this method of study shows the same effects of chain length and side-chain polar groups as ultra-filtration,l and stabilization of serum albumin against urea denaturation as studied by visc~simetry.~~ 31 Further indirect evidence that points to the same conclusion is found in many current studies akin to those of Davis and Dubos32 in which it was observed that the salutary effects of serum albumin on the growth of 8 Chapman Greenberg and Schmidt J . Biol. Chem. 1927 72 707. 13 Marshall and Vickers Bull. Johns Hopkins Hosp. 1938 34 I. 11 de Haan J . Physiol. 1922 56 444. l2 Grollman J . Biol. Chem. 1925 64 141.s Rawlins and Schmidt ibid. 1929 82 709 ; 1930 88 271. 10 Stern J . Physic. Chem. 1930 34 973 980. 14 Smith and Smith J . Biol. Chem. 1938 124 107. l6 Lundgren Elam and O’Connell ibid. 1943 149 183. 16 Lundgren and O’Connell Ind. Eng. Chem. 1g44,36 370. 17 Putnam and Neurath J . Amer. Chem. Soc. 1944 66 692 rggz. 18 Neurath and Putnam J . Biol. Chem. 1945 160 397. 19 Lundgren J . Textile Res. 1945 15 335. 20 Kendall J . Biol. Chem. 1941 138 97. 21 Macheboeuf and Tayeau Bull. SOC. Chem. Biol. 1941 23 49. za E.g. Scatchard and Black J . Physic. Chem. 1949 53 88. 23 Longsworth and Jacobsen ibid. 1949 53 126. 24 Velick ibid. 1949 53 135. 25 Ballou Boyer and Luck J . Biol. Chem. 1945 159 III. 26 Teresi (unpublished observation). 27 Ballou Boyer Luck and Lum J .Clin. Iravest. 1944 23 454. 28 Ballou Boyer Luck and Lum J . Biol. Chem. 1944 153 589. 2s Boyer Lum Ballou Luck and Rice ibid. 1946 162 181. 30 Boyer Ballou and Luck ibid. 1946 162 199. 31 Duggan and Luck ibid. 1948 172 205. aa Davis and Dubos J . Expt. Men. 1947. 86 215. COMBINATION OF ANIONS WITH PROTEINS 46 the tubercle bacillus in vitro were due to the albumin fixation of oleic acid (and perhaps other unsaturated acids) present as a contaminant in the nutrient medium. Related no doubt is the protective effect of serum albumin against haemolysis in vitro by various fatty acids. Our own studies 1 of this phenomenon restricted to sodium caprylate were pursued with the hope that the quantitative findings would agree with other methods and would permit the application of this simple and rapid technique to other proteins other fatty acids and other ions.However the protective action of caprylate was greater than that which would have been predicted from the " combined caprylate " content of the medium independently determined by ultra-filtration. Many observations 239 241 33-38 have been made in electrophoretic studies of effects sometimes specific of buffer anions on the mobility and iso-electric or iso-ionic point of a protein. Although in many of the cases reported a protein-anion interaction is in evidence the observa- tions are not readily interpreted; the size and valency of the anion the ionic strength and pH of the solution the nature of the protein component competition between the various ionic species present and simultaneous proton binding by the COO- groups of the protein are recognized as important variables.Of the methods mentioned for studying fatty acid binding the ultra- filtration and dialysis-equilibrium procedures are among the most satisfactory. Boyer,l formerly in our laboratory has used the first of these to considerable advantage in studying the binding of butyrate caproate caprylate caprate and acetyltryptophan. Noda 39 extended its use to mandelate. Results obtained by this method are amenable to quantitative interpretation since a simple mass action expression is found to be applicable. Higher concentrations of protein and ion may be employed than in the usual type of dialysis-equilibrium study this in turn sometimes permits the use of analytical methods which are insufficiently sensitive for application to the low concentrations used in dialysis-equilibrium investigations..Specifically we are not yet able to carry out binding studies with unlabelled aliphatic anions by the latter method owing t o their lack of absorption in the visible or ultraviolet but we are able to do so by simple acid-base titration with the higher protein concentrations characteristic of ultra-filtration studies. I suppose it may also be argued that the protein concentrations used in the latter more nearly approach serum protein values and that results obtained may therefore be more significant in connection with the transport function now commonly assigned to serum albumin.4o The dialysis-equilibrium method was introduced to this field of investigation quite some years ago.v. Muralt 41 developed a mathe- matical treatment based upon the law of mass action applicable to the binding of hydrogen ions and clearly capable of extension to the multiple binding of other ions where a series of association constants is involved. This extension was effected and the treatment somewhat 34 Moyer and Moyer J . Biol. Chem. 1940 132 373. 33 Moyer Trans. Faraday Soc. 1940 36 248. 36 Davis and Cohn J. Amer. Chem. SOC. 1939 61 2092. 37 Longsworth Ann. N . Y . Acad. Sci. 1941 41 267. Alberty J . Physic. Chem. 1949 53 114. 35 Sookne and Harris J . lies. Nut. Bur. Stand. 1939 23 299. 38 Noda unpublished observations see Luck ibid.1947 51 229. Davis Amer. Scientist 1946 34 611. 41 v. Muralt .J Amer. Chem. SOC. 1930 52 3518. J. MURRAY LUCK 47 simplified by Klotz and used in dialysis-equilibrium studies on pro- teins by various investigators. The results obtained lend themselves readily to conventional thermodynamic treatment despite the present inadequacy of all attempts to describe adequately the binding centres and to define precisely the character of the bonds that are formed. The equations developed by KlotzQ" permit an evaluation of the role of statistical and electrostatic factors in binding and the determin- ation in most cases of the number of ions bound per mole of protein and the bond energies. Some of Klotz's most interesting findings are derived from the binding of methyl orange and azosulphathiazole but it seems probable that some of his conclusions are applicable also to fatty acid binding.However it appears that van der Waals' forces play a much more important role with fatty acids than with azo compounds-a point to which we shall presently return. It may now be of interest to inquire whether the ions bound by the serum albumins at least have any characteristic and distinguishing qualities. If we restrict the problem to systems in solution and exclude ion-albumin complexes of very low solubility in water such as some of the metallic salts one or two generalizations appear to be inescap- able. First of all i t is increasingly apparent that the serum albumins have a singularly conspicuous capacity to bind non-polar anions.The aliphatic and aromatic carboxylates sulphonates and sulphates are strongly bound if the side chain is sufficiently long and is virtually free of polar groups. The introduction of hydroxy groups or amino groups reduces the binding. Mandelate is bound slightly as compared with its homologue phenylacetate. a-Amino acids are bound to a negligible extent if at all but the acetylated amino acids such as acetyltryptophan are quite appreciably bound and may for example displace methyl orange.6 Binding is also in evidence in the case of a number of organic ions which lack the marked non-polar properties of the other ions mentioned e.g. 2 4-dichlorophenolate 2 4-dinitro- phenolate the three mononitrophenolates picrate and trichloroacetate.The binding of organic anions by serum albumin takes place over a wide pH range although most of our studies have been carried out at pH 7.5 t o 8.2. In this region the positive charges are localized in the guanidine and lysine side chains of the protein molecule. The binding of inorganic ions t o which increasing attention is now being given,22 is deliberately omitted from this paper. If it be assumed for the moment that the binding of organic anions is partly electrostatic it would seem reasonable to expect that organic cations of side chain structure similar to the organic anions would also be bound the side-chain free carboxyl groups of aspartic and glutamic acids are fully ionized a t pH 7.8 and the number of such groups is about as great as the number of basic groups-130 t o I35 per mole of serum albumin ; the low dipole moment of the protein suggests furthermore that the positively and negatively charged groups are fairly evenly distributed over the surface of the molecule.We have completed however an extensive study Qf many aliphatic mono- amines from C to CI2 and of several di-amines without observing any comparable phenomenon viscosity studies failed t o reveal any appreciable stabilization against urea denaturation and cloud-point studies revealed a heightened susceptibility to heat denaturation. We 48 Klotz Arch. Biochem. 1946 9 rog. Is Luck and Welsh (unpublished observations). COMBINATION OF ANIONS WITH PROTEINS 44 Klotz Sun Fvancisco Meeting Amer. Cham.Soc. March 29 1949. 48 have not employed with amines the quantitative dialysis-equilibrium techniques and shall have to postpone such studies until we have several radioactive amines or suitable microanalytical methods for the unlabelled substances. The next question I would like to consider is whether the ions bound by serum albumin especially the organic non-polar anions are bound by other proteins to a comparable degree. Our own findings indicate that bovine serum albumin binds about 25 ions per mole in the case of the more strongly associated anion^,^ crystalline p-lacto- globulin 26 about 2 and crystalline /3-amylase 26 none. Klotz ** divides the proteins he has studied into three groups on the basis of their relative binding capacities serum albumin and /3-lactoglobulin in the first ovalbumin and conalbumin in the second and pepsin trypsin chymotrypsin ribonuclease and insulin in the third (no binding).Although serum albumin and b-lactoglobulin are grouped together Klotz recognizes that the latter is much inferior to serum albumin in binding capacity. Davis and Dubos report 32 that the protective action of serum albumin against oleic acid as observed in cultivation of the tubercle bacillus was evidenced by p-lactoglobulin to a slight degree and n.as not displayed by other proteins that were tried. A study cjf the spectral shifts due to complex formation between proteins and azo dyes has been carried out by Klotz6 By displacement analysis the competitive effects of a number of simple anions have been in- vestigated.Germane to our present point is the observation that spectral shifts were not observed when serum albumin was replaced by gelatin or y-globulin evidently binding did not occur. Our own cloud-point studies cause us to exclude serum y-globulin insulin diphtheria toxin diphtheria antitoxin and papain though we recognize that the evidence by this method is suggestive and not conclusive. I t appears then that we are concerned with a phenomenon which is peculiar to the serum albumins. In seeking an explanation for this specificity it is necessary next t o inquire into the mechanism of binding. For example it would be pertinent to ask ourselves whether electrostatic forces expressed by a straightforward salt linkage between the anions and positively charged groups on the protein play an essential role Part of the answer is t o be found in the behaviour of certain amides and esters.Here again we have only the indirect evidence contributed by thermal stability studies. Ethyl butyrate butyramide caproamide monocaproin mono- caprylin monocaprin and triacetin all have quite small stabilizing effects with solutions of crystalline human serum albumin; none has an effect as great as even butyrate and most of them are about as effective as chloride.29 The results with the monoglycerides are not uriequivocal because of the polarity of the glycerol residue we would now expect such compounds to be less effective for this reason alone. With caproamide however no such property is in evidence and a simple comparison with caproate obliges us to conclude that the substance must be ionic if it is to be bound.* Inferentially we next con- clude that binding with positively charged groups is essentially what happens.* This may not be rigorously true since very small amounts of aliphatic alcohols (C to C1,) benzene toluene chloroform and ethylene dichloride when used to facilitate the crystallization of serum albumin have been found by 49 J. MURRAY LUCK The direct evidence of participation of basic groups is found in studies on acetylated serum albumin and formaldehyde-treated protein. Teresi 46 finds that elimination of the free €-amino groups of lysine by this treatment reduces the number of ions bound per mole of protein in the case of m-nitrophenolate and p-nitrophenolate.The three o-nitrophenolates (mono- di- and tri-nitro) suffered no reduction in binding by treatment of the protein with dormaldehyde or acetylation. This suggests that the binding of these last-mentioned substances may be a function of other groups e.g. guanidine residues. To test the pre- ceding hypothesis Teresi is now investigating the behaviour of guanidin- ated serum albumin which if the hypotheses be true should bind in- creased quantities of the ions mentioned. Klotz has also found” that the acetylation of serum albumin greatly reduces its capacity to bind methyl orange. The role of van der Waals’ forces into which we might next inquire is important. That they play a conspicuous part is clearly evident from the work of Boyerl on the effect of chain length on the binding of aliphatic carboxylates and from the indirect evidence afforded by cloud-point studies increase of chain length in the homologous series (up to C,,,) of aliphatic carboxylates increases the capacity of these substances to stabilize solutions of serum albumin against thermal or urea denaturation.The role of van der Waals’ forces is lessened as we turn to the azo dyes or to ions with side-chain polar groups. With the alkyl sulphates and serum albumin the role of van der Waals’ forces and Sonenberg ** with octyl decyl and dodecyl sulphates. is not clearly established as witness the confusing results of Karush I t seems improbable that quantitative evaluations of the relative contributions of electrostatic forces and van der Waals’ forces are of general significance since the values are determined by the nature of the ion under consideration.The contribution of hydrogen bonding as between side-chain groups and carboxyl hydroxy and imino groups in the albumin molecule has not been determined. Indeed to con- sider the nature of the binding forces satisfactorily and to determine for stated anions the contribution of each (electrostatic van der Waals’ and hydrogen bonds) we would have to know much about the topography of the protein surface that is still in the unknown the distribution of the quaternary nitrogens and their distance from such influential neighbours as the free carboxyls of glutamic and aspartic acid the hydroxyl groups of serine threonine and tyrosine and the non-polar side chains of such amino acids as leucine isoleucine valine and phenyl- alanine.The binding energies for various ions in combination with serum albumin are expressed in Table I. The maximum possible number of ions bound per mole of protein are also presented as n values3 where n is given by the equation k = n/K. AF is calculated from the first equilibrium constant (kJ in the series of reactions P + A z= PA ; PA + A = PA ; . . . ; PA,- + A = PA ; . . . ; PA,, + A = PA,. Cohn et aZ.45 to be associated with the crystalline product. Whether or not these substances are combined with the albumin in solution is undetermined. Bilirubin is also bound though i t appears that the binding capacity of serum a-globulin is appreciably greater than albumin.47 45 Cohn Hughes and Weare J .Amer. Chem. SOC. 1947 69 1753. 48 Teresi Portland Meeting Amcr. Chem. SOC. September 1948. 47Nicholas J . Amer. Chem. SOC. 1949 71 1230. Karush and Sonenberg ibid 1949 71 1369. 50 COMBINATION OF ANIONS WITH PROTEINS From the nitrophenolate series it would appear that increase in ion- ization increases as might be expected the binding energy; this is further increased by the introduction of a second or third nitro group. The behaviour of o-nitrophenolate is unlike that of the m- and p-isomers both in respect t o n and - AF,. At this point it may be worth asking ourselves whether in the serum albumin-anion complex the protein is present in the native €orm or the open extended form characteristic of the denatured mole- cule.Much seems t o depend upon the nature of the anion. For example with caprylate as used in many of our studies there seems to be no doubt that the association complex is built up from native serum albumin in the closed configuration. So also with dodecyl sulphate in low concentrations. This is proven by viscosity studies even in the presence of high concentrations of urea and for caprylate also by rate of digestion with papain. With certain other ions however TABLE I.-BINDING ENERGIES WITH SERUM ALBUMIN Anion A-Form per cent. . . €hloride Sulphanilamide . Salicylate . o-Nitrophenolate . . Reference 3ond Energies (-AFl) Calories per mole m-Nitrophenolate . .$-Nitrophenolate 43 73 - - - 75 91 16 91 I00 I00 - I00 I00 - - . 2 4-Dinitrophenolate . Phenyl acetate . Picrate Phenoxyacetate . Cinnamate . Methyl orange . Dodecyl sulphate . notably mandelate no protection is conferred against urea denatura- tion the relative viscosity of the solution is no less than in the absence of mandelate. The same seems to be true of benzoate benzene sulphon- ate phenylacetate and acetyltryptophan. In these cases we are drawn to the conclusion that while under ordinary circumstances the substances mentioned may combine with albumin in its native state under conditions that favour protein denaturation partial opening out of the molecule proceeds combination with the anion next takes place to give an anion-protein complex of greater solubility than the denatured protein alone.True it is that these observations were made upon albumin solutions of about 2% i t is not proven that the same con- clusions would be applicable to the o.zo/ solutions used in Teresi’s quantitative dialysis-equilibrium studies. In conclusion I think i t desirable to speculate upon the specificity of serum albumin in so far as the binding of anions is concerned. Serum albumin is a molecule of low dipole moment with over 130 groups (guanidine and lysine) that carry a positive charge at pH 7.5 t o 8.0 and an almost equal number of negatively charged groups arising 43 43 4 4 4 4 n 43 43 43 4 4 4 4 4 4 4 J.MURRAY LUCK from the ionization of the free carboxyl groups of aspartic and glutamic acid residues. The low dipole moment of the protein suggests that these ionized groups are uniformly distributed over the surface. From the reported dimensions of the serum albumin molecule 38 A x 150 A assuming it to be a prolate ellipsoid Teresi has calculated the average distances that separate centre to centre positive and negative charges. Whether the charges are hexagonally distributed over the surface or at the corners of squares is of comparatively little consequence. The value works out to about 7.1 A in either event and would be appreci- ably greater if haK of the chargeswere buried beneath the surface as may indeed be the case. In so far as this distance is maintained between neighbouring oppositely charged groups the probability of interaction is slight.It seems not improbable however by virtue of the number of ionized groups involved that some if not many of these would be contiguous to the non-polar side-chains of leucine isoleucine valine and phenyl- alanine. Since van der Waals’ forces play an important role in the binding of fatty acids we propose the hypothesis that the binding capacity of serum albumin is a dual function of the large number of positively charged groups and the close juxtaposition of some of these to non-polar side chains of certain amino acids especially leucine isa- leucine valine and phenylalanine. In using this hypothesis as a basis for predicting what other proteins would be of high binding capacity we consider that many positively charged groups would be essential ; so also would it appear important that the proteins be of low dipole moment otherwise some of the groups might be unavailable because of an inappropriate distribution over the surface of the molecule.Conceivably proteins with an appreciable excess of positively charged groups might be of high anion-binding capacity provided that the groups concerned were well distributed over the surface of the mole- cule and the number of non-polar amino acid residues were not too low (as in the protamines). Klotz 43 has recently proposed the theory that the binding capacity of a protein is a direct function of the number of positively charged groups and an inverse function of the number of carboxyl and hydroxy groups.Since hydrogen bonding between the carboxyl and hydroxy groups is involved the theory tacitly infers that the aspartic glutamic tyrosine serine and threonine residues are so oriented as t o permit this hydrogen bonding to take place. In the absence of any evidence that such is the case at least on the scale implicit in the theory we are unable to regard it as an acceptable explanation of the facts. The theory also minimizes the role of van der Waals’ forces. On the basis of binding studies with methyl orange a t two different temperatures Klotz has concluded on thermodynamic grounds that van der Waals’ forces are of minor significance. In our experience methyl orange is atypical and conclusions drawn therefrom are not applicable to the binding of fatty acids in so far as determination of the role of van der Waals’ forces is concerned.The author is most grateful to Dr. J. D. Teresi for permission to report upon his current investigations and for his generous assistance in preparation of this manuscript. Prof. I. M. Klotz generously made available an advance copy of his latest paper on the binding of organic ions by proteins. We are also indebted to Dr. A. K. Balls for a sample 5 2 ADSORPTION OF PARAFFIN-CHAIN SALTS of crystalline /3-amylase to Dr. W. G. Gordon for a sample of crystalline /3-lactoglobulin and to the Armour Laboratories for the crystalline bovine serum albumin used in our studies. The Rockefeller Foundation through a grant-in-aid has made possible these investigations.Department of Chemistry Stanford University Stunford California. 44 COMBINATION OF ANIONS WITH PROTEINS THE COMBINATION OF FATTY ACID ANIONS WITH PROTEINS BY J. MURRAY LUCK Received 20th May 1949 Anions of the aliphatic and aromatic carboxylates sulphonates and sul-phates are bound by the serum albumins more markedly than by other proteins reported upon t o date. The number of ions bound per mole of serum albumin, as well as the contribution of the ion to the thermal stability of aqueous solu-tions of serum albumin is a function of the length of the side chain. Maximal effects in the case of fatty acid anions are observed with chains of six to nine carbon atoms. Protection against the denaturation of serum albumin by urea is also conferred by rhe family of anions mentioned.It is concluded that the binding of fatty acid anions a t pH 7-5 t o 8.0 is due to electrostatic attraction by the positively charged guanidine and lysine residues and van der Waals' forces between the side chains of the added anion and the side chains of leucine, isoleucine valine and phenylalanine. The present paper is devoted to recent work on the binding of ions by proteins. It is largely restricted to the binding of fatty acid ions by serum albumin and possibly with undue attention to studies that have proceeded in the author's laboratory. The first of these restric-tions is deliberately imposed by a desire not to wander too far from the general subject of the Discussion and also by the rather curious speci-ficity of the phenomenon itself to which we shall presently return.But first let us address to ourselves a very pertinent question : what experimental findings necessitate the conclusion that ions are bound by proteins ? and dialysis-equilibrium studies 3* 4 9 5 9 effects on absorption 7 Some of the evidence comes from ultrafiltration 1 Boyer Ballou and Luck J . BioE. Chem. 1947 167 407. Greenberg and Gunther ibid. 1929-30 85 491. 3 Klotz Walker and Pivan J . Amer. Chem. SOC. 1946 68 1486. 4 Teresi and Luck J . BioZ. Chem. 1948 174 653 ; 1949 177 383. 5 Klotz Triwush and Walker J . Amer. Chem. Soc. 1948 70 2935. 6 Klotz ibid. 1946 68 2299. 7 Irvine and Irvine Fed. Proc. 1949 8 209 J. MURRAY LUCK 45 and binding studies on dyes,* 9 7 lo on indicators 11-14 and on synthetic In addition Kendall 2o has described a serum albumin which contained 2 yo fatty acid-somewhat more than the crystallized albumins prepared by the ethanol procedure.Macheboeuf’s studies 21 on protein-fatty acid complexes are well known and add to the body of direct evidence. Equally convincing evidence consists in part of an observation by Scatchard and Black 22 that serum albumin solutions rendered iso-ionic by exhaustive dialysis against water undergo an increase in pH of as much as 1-65 pH units by addition of various neutral inorganic salts. This is suggestive of anion binding and of a considerable change in the ionic properties of the molecule. Studies recently reported by Longsworth and Jacobsen 23 and by Velick likewise give evidence of protein-anion combinations sometimes of a clearly competitive char-acter.The electrophoretic mobility of serum albumin determined after equilibration with sodium salts of the lower fatty acids increases with increase in chain length of the added This is strongly sug-gestive of an anion-albumin association. The results however do not permit of an indubitable conclusion since phosphate which also in-creases the net negative charge of serum albumin is found by Teresi,26 using another method to bind with serum albumin to a degree less than would be predicted from the electrophoretic findings. Some years ago we observed by means of a so-called cloud-point technique that the thermal stability of serum albumin in aqueous solution was much increased by fatty acid anions,l 27’ 28 29 the effect increasing with increase of chain length.I doubt that I would be inclined to cite this as conclusive evidence of fatty acid binding were it not that this method of study shows the same effects of chain length and side-chain polar groups as ultra-filtration,l and stabilization of serum albumin against urea denaturation as studied by visc~simetry.~~ 31 Further indirect evidence that points to the same conclusion is found in many current studies akin to those of Davis and Dubos32 in which it was observed that the salutary effects of serum albumin on the growth of 8 Chapman Greenberg and Schmidt J . Biol. Chem. 1927 72 707. s Rawlins and Schmidt ibid. 1929 82 709 ; 1930 88 271. 10 Stern J . Physic.Chem. 1930 34 973 980. 11 de Haan J . Physiol. 1922 56 444. l2 Grollman J . Biol. Chem. 1925 64 141. 13 Marshall and Vickers Bull. Johns Hopkins Hosp. 1938 34 I. 14 Smith and Smith J . Biol. Chem. 1938 124 107. l6 Lundgren Elam and O’Connell ibid. 1943 149 183. 16 Lundgren and O’Connell Ind. Eng. Chem. 1g44,36 370. 17 Putnam and Neurath J . Amer. Chem. Soc. 1944 66 692 rggz. 18 Neurath and Putnam J . Biol. Chem. 1945 160 397. 19 Lundgren J . Textile Res. 1945 15 335. 20 Kendall J . Biol. Chem. 1941 138 97. 21 Macheboeuf and Tayeau Bull. SOC. Chem. Biol. 1941 23 49. za E.g. Scatchard and Black J . Physic. Chem. 1949 53 88. 23 Longsworth and Jacobsen ibid. 1949 53 126. 24 Velick ibid. 1949 53 135. 25 Ballou Boyer and Luck J . Biol. Chem. 1945 159 III.26 Teresi (unpublished observation). 27 Ballou Boyer Luck and Lum J . Clin. Iravest. 1944 23 454. 28 Ballou Boyer Luck and Lum J . Biol. Chem. 1944 153 589. 2s Boyer Lum Ballou Luck and Rice ibid. 1946 162 181. 30 Boyer Ballou and Luck ibid. 1946 162 199. 31 Duggan and Luck ibid. 1948 172 205. aa Davis and Dubos J . Expt. Men. 1947. 86 215 46 COMBINATION OF ANIONS WITH PROTEINS the tubercle bacillus in vitro were due to the albumin fixation of oleic acid (and perhaps other unsaturated acids) present as a contaminant in the nutrient medium. Related no doubt is the protective effect of serum albumin against haemolysis in vitro by various fatty acids. Our own studies 1 of this phenomenon restricted to sodium caprylate, were pursued with the hope that the quantitative findings would agree with other methods and would permit the application of this simple and rapid technique to other proteins other fatty acids and other ions.However the protective action of caprylate was greater than that which would have been predicted from the " combined caprylate " content of the medium independently determined by ultra-filtration. Many observations 239 241 33-38 have been made in electrophoretic studies of effects sometimes specific of buffer anions on the mobility and iso-electric or iso-ionic point of a protein. Although in many of the cases reported a protein-anion interaction is in evidence the observa-tions are not readily interpreted; the size and valency of the anion, the ionic strength and pH of the solution the nature of the protein component competition between the various ionic species present and simultaneous proton binding by the COO- groups of the protein are recognized as important variables.Of the methods mentioned for studying fatty acid binding the ultra-filtration and dialysis-equilibrium procedures are among the most satisfactory. Boyer,l formerly in our laboratory has used the first of these to considerable advantage in studying the binding of butyrate, caproate caprylate caprate and acetyltryptophan. Noda 39 extended its use to mandelate. Results obtained by this method are amenable to quantitative interpretation since a simple mass action expression is found to be applicable. Higher concentrations of protein and ion may be employed than in the usual type of dialysis-equilibrium study : this in turn sometimes permits the use of analytical methods which are insufficiently sensitive for application to the low concentrations used in dialysis-equilibrium investigations..Specifically we are not yet able to carry out binding studies with unlabelled aliphatic anions by the latter method owing t o their lack of absorption in the visible or ultraviolet but we are able to do so by simple acid-base titration with the higher protein concentrations characteristic of ultra-filtration studies. I suppose it may also be argued that the protein concentrations used in the latter more nearly approach serum protein values and that results obtained may therefore be more significant in connection with the transport function now commonly assigned to serum albumin.4o The dialysis-equilibrium method was introduced to this field of investigation quite some years ago.v. Muralt 41 developed a mathe-matical treatment based upon the law of mass action applicable to the binding of hydrogen ions and clearly capable of extension to the multiple binding of other ions where a series of association constants is involved. This extension was effected and the treatment somewhat 33 Moyer Trans. Faraday Soc. 1940 36 248. 34 Moyer and Moyer J . Biol. Chem. 1940 132 373. 35 Sookne and Harris J . lies. Nut. Bur. Stand. 1939 23 299. 36 Davis and Cohn J. Amer. Chem. SOC. 1939 61 2092. 37 Longsworth Ann. N . Y . Acad. Sci. 1941 41 267. 38 Noda unpublished observations see Luck ibid. 1947 51 229. 41 v. Muralt .J Amer.Chem. SOC. 1930 52 3518. Alberty J . Physic. Chem. 1949 53 114. Davis Amer. Scientist 1946 34 611 J. MURRAY LUCK 47 simplified by Klotz and used in dialysis-equilibrium studies on pro-teins by various investigators. The results obtained lend themselves readily to conventional thermodynamic treatment despite the present inadequacy of all attempts to describe adequately the binding centres and to define precisely the character of the bonds that are formed. The equations developed by KlotzQ" permit an evaluation of the role of statistical and electrostatic factors in binding and the determin-ation in most cases of the number of ions bound per mole of protein and the bond energies. Some of Klotz's most interesting findings are derived from the binding of methyl orange and azosulphathiazole, but it seems probable that some of his conclusions are applicable also to fatty acid binding.However it appears that van der Waals' forces play a much more important role with fatty acids than with azo compounds-a point to which we shall presently return. It may now be of interest to inquire whether the ions bound by the serum albumins at least have any characteristic and distinguishing qualities. If we restrict the problem to systems in solution and exclude ion-albumin complexes of very low solubility in water such as some of the metallic salts one or two generalizations appear to be inescap-able. First of all i t is increasingly apparent that the serum albumins have a singularly conspicuous capacity to bind non-polar anions.The aliphatic and aromatic carboxylates sulphonates and sulphates are strongly bound if the side chain is sufficiently long and is virtually free of polar groups. The introduction of hydroxy groups or amino groups reduces the binding. Mandelate is bound slightly as compared with its homologue phenylacetate. a-Amino acids are bound to a negligible extent if at all but the acetylated amino acids such as acetyltryptophan are quite appreciably bound and may for example, displace methyl orange.6 Binding is also in evidence in the case of a number of organic ions which lack the marked non-polar properties of the other ions mentioned e.g. 2 4-dichlorophenolate 2 4-dinitro-phenolate the three mononitrophenolates picrate and trichloroacetate. The binding of organic anions by serum albumin takes place over a wide pH range although most of our studies have been carried out at pH 7.5 t o 8.2.In this region the positive charges are localized in the guanidine and lysine side chains of the protein molecule. The binding of inorganic ions t o which increasing attention is now being given,22 is deliberately omitted from this paper. If it be assumed for the moment that the binding of organic anions is partly electrostatic it would seem reasonable to expect that organic cations of side chain structure similar to the organic anions would also be bound the side-chain free carboxyl groups of aspartic and glutamic acids are fully ionized a t pH 7.8 and the number of such groups is about as great as the number of basic groups-130 t o I35 per mole of serum albumin ; the low dipole moment of the protein suggests, furthermore that the positively and negatively charged groups are fairly evenly distributed over the surface of the molecule.We have completed however an extensive study Qf many aliphatic mono-amines from C to CI2 and of several di-amines without observing any comparable phenomenon viscosity studies failed t o reveal any appreciable stabilization against urea denaturation and cloud-point studies revealed a heightened susceptibility to heat denaturation. We 48 Klotz Arch. Biochem. 1946 9 rog. Is Luck and Welsh (unpublished observations) 48 COMBINATION OF ANIONS WITH PROTEINS have not employed with amines the quantitative dialysis-equilibrium techniques and shall have to postpone such studies until we have several radioactive amines or suitable microanalytical methods for the unlabelled substances.The next question I would like to consider is whether the ions bound by serum albumin especially the organic non-polar anions are bound by other proteins to a comparable degree. Our own findings indicate that bovine serum albumin binds about 25 ions per mole in the case of the more strongly associated anion^,^ crystalline p-lacto-globulin 26 about 2 and crystalline /3-amylase 26 none. Klotz ** divides the proteins he has studied into three groups on the basis of their relative binding capacities serum albumin and /3-lactoglobulin in the first ovalbumin and conalbumin in the second and pepsin trypsin, chymotrypsin ribonuclease and insulin in the third (no binding).Although serum albumin and b-lactoglobulin are grouped together Klotz recognizes that the latter is much inferior to serum albumin in binding capacity. Davis and Dubos report 32 that the protective action of serum albumin against oleic acid as observed in cultivation of the tubercle bacillus was evidenced by p-lactoglobulin to a slight degree and n.as not displayed by other proteins that were tried. A study cjf the spectral shifts due to complex formation between proteins and azo dyes has been carried out by Klotz6 By displacement analysis the competitive effects of a number of simple anions have been in-vestigated. Germane to our present point is the observation that spectral shifts were not observed when serum albumin was replaced by gelatin or y-globulin evidently binding did not occur.Our own cloud-point studies cause us to exclude serum y-globulin insulin, diphtheria toxin diphtheria antitoxin and papain though we recognize that the evidence by this method is suggestive and not conclusive. I t appears then that we are concerned with a phenomenon which is peculiar to the serum albumins. In seeking an explanation for this specificity it is necessary next t o inquire into the mechanism of binding. For example it would be pertinent to ask ourselves whether electrostatic forces expressed by a straightforward salt linkage between the anions and positively charged groups on the protein play an essential role Part of the answer is t o be found in the behaviour of certain amides and esters.Here again we have only the indirect evidence contributed by thermal stability studies. Ethyl butyrate butyramide caproamide monocaproin mono-caprylin monocaprin and triacetin all have quite small stabilizing effects with solutions of crystalline human serum albumin; none has an effect as great as even butyrate and most of them are about as effective as chloride.29 The results with the monoglycerides are not uriequivocal because of the polarity of the glycerol residue we would now expect such compounds to be less effective for this reason alone. With caproamide however no such property is in evidence and a simple comparison with caproate obliges us to conclude that the substance must be ionic if it is to be bound.* Inferentially we next con-clude that binding with positively charged groups is essentially what happens.44 Klotz Sun Fvancisco Meeting Amer. Cham. Soc. March 29 1949. * This may not be rigorously true since very small amounts of aliphatic alcohols (C to C1,) benzene toluene chloroform and ethylene dichloride when used to facilitate the crystallization of serum albumin have been found b J. MURRAY LUCK 49 The direct evidence of participation of basic groups is found in studies on acetylated serum albumin and formaldehyde-treated protein. Teresi 46 finds that elimination of the free €-amino groups of lysine by this treatment reduces the number of ions bound per mole of protein in the case of m-nitrophenolate and p-nitrophenolate. The three o-nitrophenolates (mono- di- and tri-nitro) suffered no reduction in binding by treatment of the protein with dormaldehyde or acetylation.This suggests that the binding of these last-mentioned substances may be a function of other groups e.g. guanidine residues. To test the pre-ceding hypothesis Teresi is now investigating the behaviour of guanidin-ated serum albumin which if the hypotheses be true should bind in-creased quantities of the ions mentioned. Klotz has also found” that the acetylation of serum albumin greatly reduces its capacity to bind methyl orange. The role of van der Waals’ forces into which we might next inquire is important. That they play a conspicuous part is clearly evident from the work of Boyerl on the effect of chain length on the binding of aliphatic carboxylates and from the indirect evidence afforded by cloud-point studies increase of chain length in the homologous series (up to C,,,) of aliphatic carboxylates increases the capacity of these substances to stabilize solutions of serum albumin against thermal or urea denaturation.The role of van der Waals’ forces is lessened as we turn to the azo dyes or to ions with side-chain polar groups. With the alkyl sulphates and serum albumin the role of van der Waals’ forces is not clearly established as witness the confusing results of Karush and Sonenberg ** with octyl decyl and dodecyl sulphates. I t seems improbable that quantitative evaluations of the relative contributions of electrostatic forces and van der Waals’ forces are of general significance since the values are determined by the nature of the ion under consideration.The contribution of hydrogen bonding as between side-chain groups and carboxyl hydroxy and imino groups in the albumin molecule has not been determined. Indeed to con-sider the nature of the binding forces satisfactorily and to determine, for stated anions the contribution of each (electrostatic van der Waals’, and hydrogen bonds) we would have to know much about the topography of the protein surface that is still in the unknown the distribution of the quaternary nitrogens and their distance from such influential neighbours as the free carboxyls of glutamic and aspartic acid the hydroxyl groups of serine threonine and tyrosine and the non-polar side chains of such amino acids as leucine isoleucine valine and phenyl-alanine. The binding energies for various ions in combination with serum albumin are expressed in Table I.The maximum possible number of ions bound per mole of protein are also presented as n values3 where n is given by the equation k = n/K. AF is calculated from the first equilibrium constant (kJ in the series of reactions P + A z= PA ; PA + A = PA ; . . . ; PA,, + A = PA,. Cohn et aZ.45 to be associated with the crystalline product. Whether or not these substances are combined with the albumin in solution is undetermined. Bilirubin is also bound though i t appears that the binding capacity of serum a-globulin is appreciably greater than albumin.47 PA,- + A = PA ; . . . ; 45 Cohn Hughes and Weare J . Amer. Chem. SOC. 1947 69 1753. 48 Teresi Portland Meeting Amcr. Chem.SOC. September 1948. 47Nicholas J . Amer. Chem. SOC. 1949 71 1230. Karush and Sonenberg ibid 1949 71 1369 50 COMBINATION OF ANIONS WITH PROTEINS From the nitrophenolate series it would appear that increase in ion-ization increases as might be expected the binding energy; this is further increased by the introduction of a second or third nitro group. The behaviour of o-nitrophenolate is unlike that of the m- and p-isomers both in respect t o n and - AF,. At this point it may be worth asking ourselves whether in the serum albumin-anion complex the protein is present in the native €orm or the open extended form characteristic of the denatured mole-cule. Much seems t o depend upon the nature of the anion. For example with caprylate as used in many of our studies there seems to be no doubt that the association complex is built up from native serum albumin in the closed configuration.So also with dodecyl sulphate in low concentrations. This is proven by viscosity studies even in the presence of high concentrations of urea and for caprylate also, by rate of digestion with papain. With certain other ions however, TABLE I.-BINDING ENERGIES WITH SERUM ALBUMIN Anion €hloride . . Sulphanilamide . Salicylate . o-Nitrophenolate . . m-Nitrophenolate . $-Nitrophenolate . 2 4-Dinitrophenolate . Picrate . Phenyl acetate . Phenoxyacetate . Cinnamate . Methyl orange . Dodecyl sulphate . A-Form per cent. ---75 91 16 43 73 91 I00 I00 ---I00 I00 3ond Energies (-AFl) Calories per mole n Reference 43 43 43 4 4 4 4 4 4 4 4 4 4 4 43 43 notably mandelate no protection is conferred against urea denatura-tion the relative viscosity of the solution is no less than in the absence of mandelate.The same seems to be true of benzoate benzene sulphon-ate phenylacetate and acetyltryptophan. In these cases we are drawn to the conclusion that while under ordinary circumstances the substances mentioned may combine with albumin in its native state, under conditions that favour protein denaturation partial opening out of the molecule proceeds combination with the anion next takes place to give an anion-protein complex of greater solubility than the denatured protein alone. True it is that these observations were made upon albumin solutions of about 2% i t is not proven that the same con-clusions would be applicable to the o.zo/ solutions used in Teresi’s quantitative dialysis-equilibrium studies.In conclusion I think i t desirable to speculate upon the specificity of serum albumin in so far as the binding of anions is concerned. Serum albumin is a molecule of low dipole moment with over 130 groups (guanidine and lysine) that carry a positive charge at pH 7.5 t o 8.0, and an almost equal number of negatively charged groups arisin J. MURRAY LUCK from the ionization of the free carboxyl groups of aspartic and glutamic acid residues. The low dipole moment of the protein suggests that these ionized groups are uniformly distributed over the surface. From the reported dimensions of the serum albumin molecule 38 A x 150 A, assuming it to be a prolate ellipsoid Teresi has calculated the average distances that separate centre to centre positive and negative charges.Whether the charges are hexagonally distributed over the surface or at the corners of squares is of comparatively little consequence. The value works out to about 7.1 A in either event and would be appreci-ably greater if haK of the chargeswere buried beneath the surface, as may indeed be the case. In so far as this distance is maintained between neighbouring oppositely charged groups the probability of interaction is slight. It seems not improbable however by virtue of the number of ionized groups involved that some if not many of these would be contiguous to the non-polar side-chains of leucine isoleucine valine and phenyl-alanine.Since van der Waals’ forces play an important role in the binding of fatty acids we propose the hypothesis that the binding capacity of serum albumin is a dual function of the large number of positively charged groups and the close juxtaposition of some of these to non-polar side chains of certain amino acids especially leucine isa-leucine valine and phenylalanine. In using this hypothesis as a basis for predicting what other proteins would be of high binding capacity we consider that many positively charged groups would be essential ; so also would it appear important that the proteins be of low dipole moment otherwise some of the groups might be unavailable because of an inappropriate distribution over the surface of the molecule.Conceivably proteins with an appreciable excess of positively charged groups might be of high anion-binding capacity provided that the groups concerned were well distributed over the surface of the mole-cule and the number of non-polar amino acid residues were not too low (as in the protamines). Klotz 43 has recently proposed the theory that the binding capacity of a protein is a direct function of the number of positively charged groups and an inverse function of the number of carboxyl and hydroxy groups. Since hydrogen bonding between the carboxyl and hydroxy groups is involved the theory tacitly infers that the aspartic glutamic, tyrosine serine and threonine residues are so oriented as t o permit this hydrogen bonding to take place. In the absence of any evidence that such is the case at least on the scale implicit in the theory we are unable to regard it as an acceptable explanation of the facts. The theory also minimizes the role of van der Waals’ forces. On the basis of binding studies with methyl orange a t two different temperatures, Klotz has concluded on thermodynamic grounds that van der Waals’ forces are of minor significance. In our experience methyl orange is atypical and conclusions drawn therefrom are not applicable to the binding of fatty acids in so far as determination of the role of van der Waals’ forces is concerned. The author is most grateful to Dr. J. D. Teresi for permission to report upon his current investigations and for his generous assistance in preparation of this manuscript. Prof. I. M. Klotz generously made available an advance copy of his latest paper on the binding of organic ions by proteins. We are also indebted to Dr. A. K. Balls for a sampl 5 2 ADSORPTION OF PARAFFIN-CHAIN SALTS of crystalline /3-amylase to Dr. W. G. Gordon for a sample of crystalline /3-lactoglobulin and to the Armour Laboratories for the crystalline bovine serum albumin used in our studies. The Rockefeller Foundation through a grant-in-aid has made possible these investigations. Department of Chemistry, Stanford University, Stunford California

ISSN:0366-9033    

DOI:10.1039/DF9490600044

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

ADSORPTION OF PARAFFIN-CHAIN SALTS 5 2 THE ADSORPTION OF PARAFFIN-CHAIN SALTS TO PROTEINS PART V. THE INFLUENCE OF SIZE OF ION ON THE BINDING OF AMPHIPATHIC ANIONS AND CATIONS TO GELATIN SOC. Chian. biol. 1948. 30 398. BY KENNETH G. A. PANKHURST Received 19th May 1949 A study has been made of the primary adsorption of a variety of amphi- pathic ions to gelatin and it has been shown that ionic attraction between the charged side chains of the protein and the amphipathic ions irrespective of the size of the ion depends on the net charge on the protein and the charge on the amphipathic ions. Adsorption of amphipathic ions can also occur a t some or all of the keto-imide groups of the main chain of the protein molecule by an ion-dipole association. The presence of inorganic electrolyte encourages such adsorption whereas hydrogen ions prevent it.Only when the heads of the amphipathic ions are sufficiently small (ca. 20-25 Aa cross-sectional area) than ca. 45 A2 in cross-sectional area are too large to be adsorbed at any of the are they able to penetrate to all the keto-imide groups. Ions with heads larger backbone sites. Ions of intermediate size are able to penetrate only to a. limited number of these sites. Previous work has shown that adsorption of dodecyl sodium sulphate (DSS) to gelatin takes place in two consecutive stages.l First DSS anions are adsorbed with their polar groups towards the gelatin until a primary monolayer is built up and secondly when this is complete a further layer is formed with the polar groups of the detergent anions orientated outwards.Thus on the addition of DSS to a gelatin sol adsorption complexes are formed which become increasingly hydro- phobic as the detergent/protein ratio increases and then having reached a maximum become more hydrophilic as the secondary layer is formed. In certain circumstances e.g. at low pH values or in the isoelectric zone in the presence of inorganic salt the adsorption complexes become sufficiently hydrophobic to be thrown out of solution as oil-soluble coacervates. The number of DSS anions per unit of gelatin required for the formation of a complete primary monolayer has been shown t o be dependent on pH and the presence of inorganic electrolyte and adsorption at two distinct types of site in the protein molecule have been suggested to explain the results.2 The cationic side chains alone Pankhurst and Smith Trans.Faraday Sac. 1944 40 565. Joly Bull. a Pankhurst and Smith Trans. Faraday Sac. 1945 41 630. K. G. A. PANKHURST 5 3 are responsible for the coulombic binding of DSS anions a t low pH values in the absence of inorganic salt and a stoichiometric equivalence has been demonstrated between the number of these groups and the number of DSS anions bound in the primary layer. This number decreases as the protein is deaminated.3 Indeed the completion of the primary layer can be used to determine the number of cationic side chains in the protein molecule4 in the same way as dye anions have been used.5 Increasing the pH and the addition of inorganic electrolyte permits the fixation of many more DSS anions in the primary layer indicating that fresh sites in the protein molecule become available for the primary adsorption of DSS anions.At pH values at and above the isoelectric point in the presence of a high concentration of inorganic salt (e.g. M NaCl) primary adsorption of DSS anions is maximal the number bound being practically equivalent to the total number of amino acid residues in the protein. The most likely sites for such adsorption to occur are the keto-imide groups in the protein backbone adsorption being by an ion-dipole mechanism. If this is so the degree to which these groups are capable of taking part in such a process is dependent on pH and inorganic salt hydrogen ions reducing and inorganic salt increasing their polarity.In the present work a study has been made of the primary adsorption of a number of different anionic and cationic paraffin-chain salts to test this hypothesis. Experimental Materials .-The primary alkyl sulphates were prepared from care- fully fractionated alcohols (b.p. range of less than 5" C) by treatment with concentrated sulphuric acid below 40" C added over a period of an hour. The reaction mixtures were neutralized dried extracted with dry methyl alcohol and the alkyl sulphates crystallized not less than three times from methyl alcohol. Sodium contents were all within I yo of theory. A secondary isomer of DSS (sodium dodecyl 5-sulphate) was pre- pared by sulphating the corresponding alcohol prepared from re-distilled butyl bromide and n-caprylaldehyde via the Grignard 1 eaetion with chlorsulphonic acid.This was crystallized three times from methyl alcohol. The sodium content was found to be 8-7 yo whereas C,H . CH . C,H, OSO,Na gives 7-98 yo. Titration against standard cetyl trimethylammonium bromide6 gave a molecular weight of 268 €or which Na = 8.6 yo. (I Epton Trans. Faraday SOG. 1948 44 226. I The alkyl naphthalene sulphonate was commercial Perminal W (ex. I.C.I.) which contained 64 yo of inorganic salt. When used additions of Flavianic acid (2 4-dinitro I-naphthol 7-sulphonic acid) (ex. R.D.H.) was used without further purification and gave a titration with NaOH Cetyl pyridinium bromide and cetyl trimethylammonium bromide were prepared from fractionated cetyl bromide (see Adam and Pank- NaCl were made to give a constant inorganic salt concentration.equivalent to 99.3 yo purity. hurst 7. 3 Harris Pankhurst and Smith Trans. Faruduy SOC. 1g47,43 506. Pankhurst in Szlrface Chemistry (Butterworth London 1949) p. rog. 6 Fraenkel-Conrat and Cooper J. Biol. Chem. 1944 154 238. 7 Adam and Pankhurst ibid. 1946 42 523. ADSORPTION OF PARAFFIN-CHAIN SALTS 54 Cetylamine hydrochloride was prepared from purified ethyl palmitate via the acid acid chloride amide and nitrile as described by Adam and Dyer.8 Gelatin was Coignet Gold Leaf and all quantities refer to the moisture- free protein. Measurements of the size of the detergent ions were made from Fisher- Hirschfelder atom models kindly lent by the Wellcome Foundation.Insoluble Complexes.-For each pH value and inorganic salt con- centration a series of mixtures was prepared each being identical as regards pH added salt and gelatin concentration (0'5 yo) but with varying concentrations of detergent so as completely to cover the range of water insolubility. These were left overnight at 35" C to allow the insoluble complexes to separate. The supernatant liquors were then analyzed for nitrogen (micro-Kjeldahl) and the complex of minimum water solu- bility i.e. that at which the supernatant nitrogen was minimal deter- mined. It was found unnecessary to analyze for detergent ion in the supernatant liquor as i t had been found that except for relatively high concentrations of detergent (above ca.0.1 M) all of the detergent reacts with all of the protein the supernatant liquor being a saturated solution of the separated complex. This method can be used even if the detergent contains nitrogen since when the primary layer is complete both protein and detergent in the supernatant liquor are minimal and the required detergent /protein ratio is that of the two initial reactant concentrations.* Soluble Complexes.-Viscosity measurements (Ostwald viscometer) of a series of mixtures of detergent and constant protein in solution show a pronounced fall to a minimum as the detergentlprotein ratio in- creases and then a rise.@ The ratio corresponding to minimum viscosity was taken as that at which the primary layer was complete the solute here being least lyophilic (cf.the effect of the addition of a poor solvent to a lyophilic sol lo). Results The main experimental results are shown in Fig. I in which the deter- gent /protein ratio (mmole /g.) corresponding to the complete primary monolayer is plotted vertically against pH for a variety of anionic and cationic detergents. Anionic Detergents.-At pH 2 in the absence of inorganic electrolyte the binding of alkyl sodium sulphates is equivalent to the total cationic side chains of the protein (0.87 mmole/g.ll). The addition of inorganic electrolyte increases the number of anions bound at this pH (e.g. M NH,NO increases the fixation of DSS fourfold). At pH 5.5 in the presence of M NaCl primary alkyl sulphates are bound to the extent of 10 (& 0.5) mmole/g.the alkyl naphthalene sul- phonate-6.2 mmole /g. the secondary alkyl sulphate-4-6 mmole lg. and the flavianate not at all. The binding of the primary alkyl sulphates in the presence of M NaCl is constant between pH 5.5 and 10. Cationic Detergents.-At pH I 2.5 cetyl pyridinium bromide and cetyl trimethylammonium bromide are adsorbed to the extent of 1-7 and 1-3 mmole/g. respectively and as the pH is reduced adsorption decreases until a t about pH 4 it reaches zero. A few experiments with cetylamine however showed that at pH 5-5 and 11 between 8 and 10 mmolelg. were adsorbed. A more accurate estimate of the ratio was not possible with this compound owing to its low solubility. At pH 2 cetylamine hydrochloride forms no complexes in the absence of inorganic salt.8 Adam and Dyer J. Chem. SOC. 1925 7 2 . @ Pankhurst Bull. SOC. Chim. biol. (in press). 10 Alfrey Bartovics and Mark J. Amer. Chem. SOC. 1942 64 1557. 11 Bowes and Kenten Biochem. J. 1948 43 358. K. G. A. PANKHURST 55 AN primary dkyl su@uh.s I a Discussion It has been suggested that two types of link are involved in the formation of protein-detergent complexes (a) ion-ion ( b ) ion-dipole. I t would be expected that the former type would operate according to the net charge on the protein i.e. maximum binding of detergent anions would occur a t low pH values where the protein has its maximum positive charge and maximum binding of detergent cations would occur at high pH values the protein having its maximum negative charge.The experimental evidence with all the anions shows that a t about pH 2 the number bound is equivalent to the sum of the ionized lysine hydroxylysine arginine and histidine side chains. Recent R. C. M. Smith with sulphonates of naphthalene also confirms (MJNaCl) 0+ dkyl naphthalene sulphonale (M Na CI) *4 secondary olkyl sulphate 4 0 (M NoCI) r FIG. I. this.12 Studying complexes formed with the a-monosulphonate I 3- disulphonate and I 3 5-trisulphonate he found that a t low pH values whereas the monosulphonate bound is equivalent to the cationic sites on the protein side chains the di- and tri-sulphonates combine in decreasing amounts expressed in mmole/g. in the ratio 3/2/1.Were ionic forces solely responsible for the adsorption of long chain anions one would expect that as the pH is raised the number bound would decrease. Although no detailed study of this has been made in the absence of inorganic electrolyte i t has been showng that at pH 5 - 5 in the absence of salt soluble complexes are formed containing about 0.8 mrnole/g. and that if inorganic salt is added this ratio in- creases t o ca. 10 mmole/g. (observed when NaCl is 0.75 M and over). It is thus concluded that even though raising the pH may cause a re- duction in the number of anions bound purely ionically this effect is masked since other sites to which detergent anions are capable of being l2 Smith Nature 1949 164 447. ADSORPTION OF PARAFFIN-CHAIN SALTS 56 adsorbed come into operation.Furthermore the presence of inorganic salt tends to enhance the availability of such other sites even at low pH values. The maximum number of bound detergent anions which has been observed is 10 (& 0.5) mmole/g. for the primary alkyl sulphate anions at pH 5-5 in the presence of ca. M NaC1 which is very close to the number of amino acid residues in the protein ( I O . ~ ) and suggests that the backbone keto-imide groups can act as sites for the adsorption of detergent anions presumably by an ion-dipole mechanism. The results with the secondary isomer of DSS the alkyl naphthalene sulphonate and flavianic acid are interesting in that under conditions where the primary sulphates are maximally adsorbed (pH 5-5 in the presence of M NaCl) they are bound in much smaller amounts the latter forming no complex a t all soluble or insoluble.With cationic detergents it would be expected that ionic forces and ion-dipole association would also be operative the former becoming increasingly effective a t higher pH values and the latter at all pH values on the alkaline side of the isoelectric point. With cetyl pyridinium bromide and cetyl trimethylammonium bromide however at pH 12.5 the binding is equivalent to 1-7 and 1.3 mmole/g. respectively and is independent of the presence of inorganic electrolyte. This is only slightly in excess of the total anionic side chains of the protein (1.24 mmole/g.ll) and can readily be accounted for since some hydrolysis of the protein occurs under these conditions of pH and temperature rendering available fresh ionized carboxyl groups which are capable for adsorbing long chain cations.There is no evidence of the binding of these long chain cations to any other site in the protein molecule. As the pH is lowered the number of cations bound decreases and on the acid side of the isoelectric point no combination takes place a t all. With cetylamine however a t pH 11 in the presence of M NaCl al- though a very precise estimate of the number of molecules bound was not possible it was evident that between 8 and 10 mmole/g. were ad- sorbed indicating some adsorption a t the keto-imide backbone groups presumably by dipole-dipole association. It has therefore to be explained why some detergent anions and cations are capable of being adsorbed a t the keto-imide groups of the main chains of the protein whereas others under comparable con- ditions are not.The most likely reason seems to be connected with the size of the amphipathic ions particularly the cross-sectional area of the head group. The primary alkyl sulphates and the primary amine having the smallest cross-sectional area appear to be able to penetrate between the protein side chains and approach sufficiently closely to the keto-imide groups to enable ion-dipole association to become effective whereas the flavianate anion and the cetyl pyridinium and cetyl tri- methylammonium cations being the largest are incapable of penetrat- ing to these groups a t all. The secondary sulphate and the alkyl naphthalene sulphonate have intermediate molecular dimensions and appear to be able to penetrate into about half of the keto-imide groups.Table I shows the dimensions of the head groups of the various detergent ions and an estimate of their penetrability into the backbone of the gelatin molecule. This explanation is somewhat similar to that given by Schulman and Armstrong13 to explain the effect of various ionic groups in haemolytic 19 Schulman and Armstrong in Surfuce Chemistry (Butterworth London) 1949 P. 275. K. G. A. PANKHURST Amphipathic Ion Primary alkyl sulphate . Primary alkyl amine . 7-alkyl naphthalene 3-sulphonate . . . Secondary alkyl sulphate Flavianate (z 4-dinitro I-naphthol 7-sulphonate) . HO<>NO SO,’ \5 .. . . . . . . . ‘I Aikyl trimethylammonium . . hlkyl pyridinium . . . * Assuming primary sulphates and amines can penetrate to all the keto-imide groups. Minimum section 57 pEstimated enetrability Backbone * into the TABLE I End Group (Yo) Cross- 20 25 20 \ < . . . . . . . . . . SO,’ i . . . . . . . . . \ / / < \ . . . . . . . . . NHs+. . . . . . . . . 20 35 25 40 25 !j . . . . . . . . NO I00 I00 62 46 0 \ c> . . . . . . so; I . . . . . . . . . . . . Me-N-Me + \ Me . . . 47 25 20 49 72 0 0 GENERAL DISCUSSION 58 and enzymatic activity. They point out that the charge centres of the - NH,+ and - OS03- groups can approach a dipole such as the - OH of cholesterol more closely than those of - NMe3+ and - SO3- respectively.With proteins however the steric effect of the side chains on penetration to the backbone is probably even more important. Thus the primary sulphate is able to penetrate more completely than the more bulky secondary sulphate. It is concluded that two types of mechanism are responsible for the primary adsorption of amphipathic anions and cations to gelatin. First a purely coulombic ion-ion attraction the extent of which de- pends on the net charge on the protein and the number of charged groups on the amphipathic ion and is independent of the size of the ion. Secondly an ion-dipole association between the amphipathic ions and the keto-imide groups in the backbone of the protein.This mechanism is inhibited by hydrogen ions encouraged by inorganic electrolyte and is susceptible to the size and shape of the amphipathic ion. My thanks are due to the Director and Council of the British Leather Manufacturers’ Research Association for permission to publish these results and also to Mr. K. G. E. Wyatt for assistance with the experi- mental work. The British Leather Manufacturers’ Research Association London S.E.I. 5 2 ADSORPTION OF PARAFFIN-CHAIN SALTS THE ADSORPTION OF PARAFFIN-CHAIN SALTS TO PROTEINS PART V. THE INFLUENCE OF SIZE OF ION ON THE BINDING OF AMPHIPATHIC ANIONS AND CATIONS TO GELATIN BY KENNETH G. A. PANKHURST Received 19th May 1949 A study has been made of the primary adsorption of a variety of amphi-pathic ions to gelatin and it has been shown that ionic attraction between the charged side chains of the protein and the amphipathic ions irrespective of the size of the ion depends on the net charge on the protein and the charge on the amphipathic ions.Adsorption of amphipathic ions can also occur a t some or all of the keto-imide groups of the main chain of the protein molecule by an ion-dipole association. The presence of inorganic electrolyte encourages such adsorption whereas hydrogen ions prevent it. Only when the heads of the amphipathic ions are sufficiently small (ca. 20-25 Aa cross-sectional area) are they able to penetrate to all the keto-imide groups. Ions with heads larger than ca. 45 A2 in cross-sectional area are too large to be adsorbed at any of the backbone sites.Ions of intermediate size are able to penetrate only to a. limited number of these sites. Previous work has shown that adsorption of dodecyl sodium sulphate (DSS) to gelatin takes place in two consecutive stages.l First DSS anions are adsorbed with their polar groups towards the gelatin until a primary monolayer is built up and secondly when this is complete, a further layer is formed with the polar groups of the detergent anions orientated outwards. Thus on the addition of DSS to a gelatin sol, adsorption complexes are formed which become increasingly hydro-phobic as the detergent/protein ratio increases and then having reached a maximum become more hydrophilic as the secondary layer is formed.In certain circumstances e.g. at low pH values or in the isoelectric zone in the presence of inorganic salt the adsorption complexes become sufficiently hydrophobic to be thrown out of solution as oil-soluble coacervates. The number of DSS anions per unit of gelatin required for the formation of a complete primary monolayer has been shown t o be dependent on pH and the presence of inorganic electrolyte and adsorption at two distinct types of site in the protein molecule have been suggested to explain the results.2 The cationic side chains alone Pankhurst and Smith Trans. Faraday Sac. 1944 40 565. Joly Bull. SOC. Chian. biol. 1948. 30 398. a Pankhurst and Smith Trans. Faraday Sac. 1945 41 630 K. G. A. PANKHURST 5 3 are responsible for the coulombic binding of DSS anions a t low pH values in the absence of inorganic salt and a stoichiometric equivalence has been demonstrated between the number of these groups and the number of DSS anions bound in the primary layer.This number decreases as the protein is deaminated.3 Indeed the completion of the primary layer can be used to determine the number of cationic side chains in the protein molecule4 in the same way as dye anions have been used.5 Increasing the pH and the addition of inorganic electrolyte permits the fixation of many more DSS anions in the primary layer indicating that fresh sites in the protein molecule become available for the primary adsorption of DSS anions. At pH values at and above the isoelectric point in the presence of a high concentration of inorganic salt (e.g.M NaCl) primary adsorption of DSS anions is maximal the number bound being practically equivalent to the total number of amino acid residues in the protein. The most likely sites for such adsorption to occur are the keto-imide groups in the protein backbone adsorption being by an ion-dipole mechanism. If this is so the degree to which these groups are capable of taking part in such a process is dependent on pH and inorganic salt hydrogen ions reducing and inorganic salt increasing their polarity. In the present work, a study has been made of the primary adsorption of a number of different anionic and cationic paraffin-chain salts to test this hypothesis. Experimental Materials .-The primary alkyl sulphates were prepared from care-fully fractionated alcohols (b.p.range of less than 5" C) by treatment with concentrated sulphuric acid below 40" C added over a period of an hour. The reaction mixtures were neutralized dried extracted with dry methyl alcohol and the alkyl sulphates crystallized not less than three times from methyl alcohol. Sodium contents were all within I yo of theory. A secondary isomer of DSS (sodium dodecyl 5-sulphate) was pre-pared by sulphating the corresponding alcohol prepared from re-distilled butyl bromide and n-caprylaldehyde via the Grignard 1 eaetion with chlorsulphonic acid. This was crystallized three times from methyl alcohol. The sodium content was found to be 8-7 yo whereas C,H . CH . C,H,, I OSO,Na gives 7-98 yo. Titration against standard cetyl trimethylammonium bromide6 gave a molecular weight of 268 €or which Na = 8.6 yo.The alkyl naphthalene sulphonate was commercial Perminal W (ex. I.C.I.) which contained 64 yo of inorganic salt. When used additions of NaCl were made to give a constant inorganic salt concentration. Flavianic acid (2 4-dinitro I-naphthol 7-sulphonic acid) (ex. R.D.H.) was used without further purification and gave a titration with NaOH equivalent to 99.3 yo purity. Cetyl pyridinium bromide and cetyl trimethylammonium bromide were prepared from fractionated cetyl bromide (see Adam and Pank-hurst 7. 3 Harris Pankhurst and Smith Trans. Faruduy SOC. 1g47,43 506. 6 Fraenkel-Conrat and Cooper J. Biol. Chem. 1944 154 238. (I Epton Trans. Faraday SOG. 1948 44 226. 7 Adam and Pankhurst ibid.1946 42 523. Pankhurst in Szlrface Chemistry (Butterworth London 1949) p. rog 54 ADSORPTION OF PARAFFIN-CHAIN SALTS Cetylamine hydrochloride was prepared from purified ethyl palmitate via the acid acid chloride amide and nitrile as described by Adam and Dyer.8 Gelatin was Coignet Gold Leaf and all quantities refer to the moisture-free protein. Measurements of the size of the detergent ions were made from Fisher-Hirschfelder atom models kindly lent by the Wellcome Foundation. Insoluble Complexes.-For each pH value and inorganic salt con-centration a series of mixtures was prepared each being identical as regards pH added salt and gelatin concentration (0'5 yo) but with varying concentrations of detergent so as completely to cover the range of water insolubility.These were left overnight at 35" C to allow the insoluble complexes to separate. The supernatant liquors were then analyzed for nitrogen (micro-Kjeldahl) and the complex of minimum water solu-bility i.e. that at which the supernatant nitrogen was minimal deter-mined. It was found unnecessary to analyze for detergent ion in the supernatant liquor as i t had been found that except for relatively high concentrations of detergent (above ca. 0.1 M) all of the detergent reacts with all of the protein the supernatant liquor being a saturated solution of the separated complex. This method can be used even if the detergent contains nitrogen since when the primary layer is complete both protein and detergent in the supernatant liquor are minimal and the required detergent /protein ratio is that of the two initial reactant concentrations.* Soluble Complexes.-Viscosity measurements (Ostwald viscometer) of a series of mixtures of detergent and constant protein in solution, show a pronounced fall to a minimum as the detergentlprotein ratio in-creases and then a rise.@ The ratio corresponding to minimum viscosity was taken as that at which the primary layer was complete the solute here being least lyophilic (cf.the effect of the addition of a poor solvent to a lyophilic sol lo). Results The main experimental results are shown in Fig. I in which the deter-gent /protein ratio (mmole /g.) corresponding to the complete primary monolayer is plotted vertically against pH for a variety of anionic and cationic detergents. Anionic Detergents.-At pH 2 in the absence of inorganic electrolyte, the binding of alkyl sodium sulphates is equivalent to the total cationic side chains of the protein (0.87 mmole/g.ll).The addition of inorganic electrolyte increases the number of anions bound at this pH (e.g. M NH,NO, increases the fixation of DSS fourfold). At pH 5.5 in the presence of M NaCl primary alkyl sulphates are bound to the extent of 10 (& 0.5) mmole/g. the alkyl naphthalene sul-phonate-6.2 mmole /g. the secondary alkyl sulphate-4-6 mmole lg., and the flavianate not at all. The binding of the primary alkyl sulphates in the presence of M NaCl is constant between pH 5.5 and 10. Cationic Detergents.-At pH I 2.5 cetyl pyridinium bromide and cetyl trimethylammonium bromide are adsorbed to the extent of 1-7 and 1-3 mmole/g.respectively and as the pH is reduced adsorption decreases until a t about pH 4 it reaches zero. A few experiments with cetylamine however showed that at pH 5-5 and 11 between 8 and 10 mmolelg. were adsorbed. A more accurate estimate of the ratio was not possible with this compound owing to its low solubility. At pH 2, cetylamine hydrochloride forms no complexes in the absence of inorganic salt. 8 Adam and Dyer J. Chem. SOC. 1925 7 2 . @ Pankhurst Bull. SOC. Chim. biol. (in press). 10 Alfrey Bartovics and Mark J. Amer. Chem. SOC. 1942 64 1557. 11 Bowes and Kenten Biochem. J. 1948 43 358 K. G. A. PANKHURST 55 Discussion It has been suggested that two types of link are involved in the formation of protein-detergent complexes (a) ion-ion ( b ) ion-dipole.I t would be expected that the former type would operate according to the net charge on the protein i.e. maximum binding of detergent anions would occur a t low pH values where the protein has its maximum positive charge and maximum binding of detergent cations would occur at high pH values the protein having its maximum negative charge. The experimental evidence with all the anions shows that a t about pH 2 the number bound is equivalent to the sum of the ionized lysine hydroxylysine arginine and histidine side chains. Recent R. C. M. Smith with sulphonates of naphthalene also confirms AN primary dkyl su@uh.s (MJNaCl) I a a 0+ dkyl naphthalene sulphonale 4 (M Na CI) 0 *4 secondary olkyl sulphate (M NoCI) r FIG.I. this.12 Studying complexes formed with the a-monosulphonate I 3-disulphonate and I 3 5-trisulphonate he found that a t low pH values whereas the monosulphonate bound is equivalent to the cationic sites on the protein side chains the di- and tri-sulphonates combine in decreasing amounts expressed in mmole/g. in the ratio 3/2/1. Were ionic forces solely responsible for the adsorption of long chain anions one would expect that as the pH is raised the number bound would decrease. Although no detailed study of this has been made, in the absence of inorganic electrolyte i t has been showng that at pH 5 - 5 in the absence of salt soluble complexes are formed containing about 0.8 mrnole/g. and that if inorganic salt is added this ratio in-creases t o ca. 10 mmole/g.(observed when NaCl is 0.75 M and over). It is thus concluded that even though raising the pH may cause a re-duction in the number of anions bound purely ionically this effect is masked since other sites to which detergent anions are capable of being l2 Smith Nature 1949 164 447 56 ADSORPTION OF PARAFFIN-CHAIN SALTS adsorbed come into operation. Furthermore the presence of inorganic salt tends to enhance the availability of such other sites even at low pH values. The maximum number of bound detergent anions which has been observed is 10 (& 0.5) mmole/g. for the primary alkyl sulphate anions at pH 5-5 in the presence of ca. M NaC1 which is very close to the number of amino acid residues in the protein ( I O . ~ ) and suggests that the backbone keto-imide groups can act as sites for the adsorption of detergent anions presumably by an ion-dipole mechanism.The results with the secondary isomer of DSS the alkyl naphthalene sulphonate and flavianic acid are interesting in that under conditions where the primary sulphates are maximally adsorbed (pH 5-5 in the presence of M NaCl) they are bound in much smaller amounts the latter forming no complex a t all soluble or insoluble. With cationic detergents it would be expected that ionic forces and ion-dipole association would also be operative the former becoming increasingly effective a t higher pH values and the latter at all pH values on the alkaline side of the isoelectric point. With cetyl pyridinium bromide and cetyl trimethylammonium bromide however at pH 12.5 the binding is equivalent to 1-7 and 1.3 mmole/g.respectively and is independent of the presence of inorganic electrolyte. This is only slightly in excess of the total anionic side chains of the protein (1.24 mmole/g.ll) and can readily be accounted for since some hydrolysis of the protein occurs under these conditions of pH and temperature, rendering available fresh ionized carboxyl groups which are capable for adsorbing long chain cations. There is no evidence of the binding of these long chain cations to any other site in the protein molecule. As the pH is lowered the number of cations bound decreases and on the acid side of the isoelectric point no combination takes place a t all. With cetylamine however a t pH 11 in the presence of M NaCl al-though a very precise estimate of the number of molecules bound was not possible it was evident that between 8 and 10 mmole/g.were ad-sorbed indicating some adsorption a t the keto-imide backbone groups, presumably by dipole-dipole association. It has therefore to be explained why some detergent anions and cations are capable of being adsorbed a t the keto-imide groups of the main chains of the protein whereas others under comparable con-ditions are not. The most likely reason seems to be connected with the size of the amphipathic ions particularly the cross-sectional area of the head group. The primary alkyl sulphates and the primary amine, having the smallest cross-sectional area appear to be able to penetrate between the protein side chains and approach sufficiently closely to the keto-imide groups to enable ion-dipole association to become effective, whereas the flavianate anion and the cetyl pyridinium and cetyl tri-methylammonium cations being the largest are incapable of penetrat-ing to these groups a t all.The secondary sulphate and the alkyl naphthalene sulphonate have intermediate molecular dimensions and appear to be able to penetrate into about half of the keto-imide groups. Table I shows the dimensions of the head groups of the various detergent ions and an estimate of their penetrability into the backbone of the gelatin molecule. This explanation is somewhat similar to that given by Schulman and Armstrong13 to explain the effect of various ionic groups in haemolytic 19 Schulman and Armstrong in Surfuce Chemistry (Butterworth London), 1949 P.275 K. G. A. PANKHURST TABLE I Minimum Cross-section 57 pEstimated enetrability into the Backbone * Amphipathic Ion Primary alkyl sulphate . Primary alkyl amine . 7-alkyl naphthalene 3-sulphonate . . . Secondary alkyl sulphate . Flavianate (z 4-dinitro I-naphthol 7-sulphonate) . Aikyl trimethylammonium . hlkyl pyridinium . . . End Group \ < . . . . . . . . . . . . . . . . . . . SO,’ i \ / \ . . . . . . . . . / < NHs+. . . . . . . . . \5 . . . . . . . . . !j ‘I SO,’ . . . . . . . . NO, \ HO<>NO . . . c> I so; . . . . . . . . . . . . Me-N-Me . . . . . . + \ Me 20 25 20 20 35 25 40 25 47 25 20 49 72 (Yo) I00 I00 62 46 0 0 0 * Assuming primary sulphates and amines can penetrate to all the keto-imide groups 58 GENERAL DISCUSSION and enzymatic activity. They point out that the charge centres of the - NH,+ and - OS03- groups can approach a dipole such as the - OH of cholesterol more closely than those of - NMe3+ and - SO3-respectively. With proteins however the steric effect of the side chains on penetration to the backbone is probably even more important. Thus, the primary sulphate is able to penetrate more completely than the more bulky secondary sulphate. It is concluded that two types of mechanism are responsible for the primary adsorption of amphipathic anions and cations to gelatin. First a purely coulombic ion-ion attraction the extent of which de-pends on the net charge on the protein and the number of charged groups on the amphipathic ion and is independent of the size of the ion. Secondly an ion-dipole association between the amphipathic ions and the keto-imide groups in the backbone of the protein. This mechanism is inhibited by hydrogen ions encouraged by inorganic electrolyte and is susceptible to the size and shape of the amphipathic ion. My thanks are due to the Director and Council of the British Leather Manufacturers’ Research Association for permission to publish these results and also to Mr. K. G. E. Wyatt for assistance with the experi-mental work. The British Leather Manufacturers’ Research Association, London S.E.I

ISSN:0366-9033    

DOI:10.1039/DF9490600052

出版商:RSC    

年代:1949

数据来源: RSC

摘要:

58 GENERAL DISCUSSION GENERAL DISCUSSION * Prof. F. Haurowitz (Bloomington, Indiana, U.S.A.) said : The re- action between proteins and cationic detergents takes place slowly. When we mixed 8 mg. horse haemoglobin with z mg. desogen (tolyldodecyl- trimethylammonium chloride) in z ml. of 0.05 M borate-phosphate-citrate buffer (Stenhagen-Teorell), we obtained after 30 min. 7-5 mg. precipitate containing 27 desogen molecules per haemoglobin molecule. After 24 hr. a second precipitate was formed ; its amount was 0.63 mg. and the ratio desogen/haemoglobin was 324 /I. The slow secondary reaction consists, apparently, of the crystallization of the detergent on the surface of the primary haemoglobin-detergent complex. Dr. K. G. A. Pankhurst (London) said: Apropos Dr. Haurowitz’s remarks, we have also found from viscosity studies that the protein- detergent reaction, under conditions which we believe to involve the backbone -NH-CO- groups, takes place slowly, although coulombic binding is very rapid.in his flow birefringence work. I t is not to be expected that the ease of penetration to the -NH-CO- groups will be the same for all proteins since this will depend largely on the sequence of amino acid residues in the main chain and other structural features, which may account for some of the specificity to which Dr. Edsall has referred. In gelatin, the apparent inaccessibility of the -NH-CO- groups to large ions may well be due to the high proportion of proline and hydroxy-proline residues, causing frequent twists in the main chain.3 Dr.J. H. Schulman (Cambridge) said: Pankhurst’s table giving the This has also been observed by Joly * On two preceding papers. Joly, ibid., 1948, 30, 398. Pankhurst, Bull. SOC. Cham. biol., 1949, 31, 703. A4r.tbury, ,I. Int. SOC. Leath. Tr. Chenz., 1940, 24, 69.GENERAL DISCUSSION 59 dimensions of the head group and hydrocarbon chains do not agree with the results obtained from surface chemical techniques. Thus the -SO,- polar group can be readily squeezed to 20 Hi2 as seen from F-A curves of C,, sulphate monolayers. The -N+(CH,), polar group can be con- tracted to 31 A2 as seen from F--A plots of C,, N(CH,),HCl monolayers and the long-chain pyridium hydrochloride packs, with an area per molecule of possibly 40 pi2. It is possible to consider an explanation for Pankhurst’s results on the ease of hydration of these polar groups and also the hydrophobic- hydrophilic balance change of the protein molecule on adsorption of the compounds. This balance is changing on adsorption of the long-chain compounds and will act as a barrier to the approach of further molecules.This can be demonstrated especially with long-chain trimethylammonium salt compounds, which can adsorb on to the ionized carboxyl groups and make the protein molecule hydrophobic. This hydrophobic barrier would prevent the approach of further trimethylammonium ions to the protein molecule. Dr. K. G. A. Pankhurst (London) said : I do not think that the areas given by Dr. Schulman, derived from monolayer measurements, are applic- able in the case under discussion since considerable overlap and inter- locking of the head groups may take place as the film is compressed.The areas quoted in my paper were derived from measurements of scale atom models and represent the projected area of single molecules. This is not ideal since no account is taken of hydration. It is, I think, sig- nificant that whether one takes Schulman’s figures or those which I have given, the same conclusion is drawn, that under conditions where one would expect adsorption to take place at the backbone -NH-CO- groups, the larger the ion, the less is its penetrability, The suggestion that highly hydrated ions such as -N+-Me, act as a hydrophobic barrier when already adsorbed at the negatively charged side-chain sites does not, I think, explain all the facts since under iso- electric conditions where there is no evidence for coulombic binding of either anions or cations, the larger ions are still incapable of penetrating to the backbone -NH-CO- groups even when inorganic salt is present, i.e.when smaller ions such as -SO’, and -NH,+ are strongly adsorbed. Dr. R. Matalon (Cambridge) said : How do the number of molecules of homologous alkyl sulphates taken up by the protein (gelatin) compare, when these detergents are used at the same concentration and below the micellar state ? In other words is it possible to establish for this present study of protein-detergent association an effect similar to the Traube effect ? Then it might help to explain the nature of the protein-detergent binding in the bulk state. Dr.K. G. A. Pankhurst (London) said : In answer to Dr. Matalon, although increasing the length of hydrocarbon chain from C, to C,, has no effect on the primary binding capacity of long chain sulphates, the detergent /protein ratio corresponding to the beginning of complex separa- tion does decrease by a factor of about 1.5 to 2 per CH, group. Prof. E. K. Rideal (London) said : I find the problem of interaction of detergents with native proteins much more difficult to understand than the reaction with fibrous proteins. It seems clear that there are three distinct steps in this interaction, the first which occurs with extremely dilute solutions of detergents seems to be highly specific for the protein and it appears that on these proteins there are a few, say 4 or 5 , readily accessible groups.If the chain is not too long this is the only reaction involved. With longer chains wTe get the second stage, namely, a reaction with all the available amino groups of the protein, and finally, with still longer chains and higher concentrations, we get the solubilizing adsorption which may be on the -CO-NH groups, but equally well may be the solution of the chains in the now hydrophobic protein surface, requiring +60 GENERAL DISCUSSION the -SO, groups in the surrounding medium. It seems likely that stage z or 3 is associated with denaturation or unfolding. Dr. K. G. A. Pankhurst (London) said : I do not think there is much evidence to suggest that the -NH-CO- groups are centres solely for solubilizing adsorption as suggested by Prof.Rideal. Rather do I think that the evidence points to their being centres for primary ad- sorption (with the lipophilic tails of the detergent ions outwards) in the same general fashion as the charged side chains, except that they have a different dependence on pH and inorganic salt. I think that solubilization is brought about entirely by the formation of a second layer, attached to the first by van der Waals’ forces, with the ionic groups outermost. Prof. J. Murray Luck (Stanford, California) said : The work I have reported is difficult to compare with the other studies reported this afternoon. The fatty acids we used contained less than ten carbon atoms. They were monodisperse and not micellar and it is certain that the binding of a micelle is different from the binding of single ions.With few exceptions we have restricted our studies to well-defined proteins. Gelatin we have avoided, partly because it is not well characterized, but also because it is quite unsuitable for the viscosity, thermal stability, and dialysis-equilibrium studies in which we were interested. As far as the binding studies are concerned, the method used rests upon the establishment of a thermodynamic equilibrium between the reactants. The method does not permit the complete titration of positive groups and /or other binding centres for which it appears to be necessary to permit the formation of a precipitate and the development of a polyphase system. I am not prepared to say which method is the more meaningful in so far as biological implications are concerned : this is still a matter of mere opinion. It is also necessary to emphasize the specificity of the phenomenon.The serum albumins alone bind the fatty acid anions ; a t least the binding capacity of other proteins is virtually negligible under the conditions of our experiments. This is the experience of workers in other laboratories as well. As for dodecyl sulphate, it is well to recognize that this substance plays a dual role. In very low concentrations it is actually a good stabilizer of serum albumin, protecting it even more effectively than caprylate against urea denaturation. The viscosity-concentration curve rises sharply, however, from this polnt on, and the denaturating property of the detergent soon comes into evidence. Dr. Duggan found the critical point of inflection, where the stabilizing property was maximal, was a t a mole ratio of detergentlprotein = 9.Dr. M. Joly (Paris) said : The interaction between protein and deter- gent does not necessarily involve the denaturation of the protein. For instance, if sodium dodecyl sulphate a t very low concentration is added to a solution of tobacco mosaic virus, we observe by streaming bire- fringence a change of shape of the virus particles, but the biological properties of the virus do not change. The protein virus is not denatured by the sodium dodecylsulphate at a concentration far below the critical concentration of micelle formation. Dr. H. L. Booij (Leiden) said : Some ten years ago Mrs. Teunissen- van Zijp, in our laboratory, made some experiments on the influence of organic anions on the charge of a positive protein (clupein).When comparing the concentrations of fatty acid anions needed to reach the state of zero-charge, it was found that between C, and G2 the influence of the length of the carbon chain was very pronounced (C6, 0.6 mole/l. ; C,, 0-1 mole/l. : C1,, 0-01 mole/l. ; CIz, 0.003 molell.). As in this homo- logous series the interacting negative groups of the fatty acid anions and the positive groups of the protein are the same throughout, these experi- ments suggest the important role of the van der Waals’ forces betweenGENERAL DISCUSSION 61 the carbon chains and the protein. So I quite agree with Prof. Luck that the binding capacity of proteins generally depends on Coulomb forces between oppositively charged groups and van der Waals’ forces between carbon chains, and non-polar side chains of the amino acid residues of the protein.In other organic molecules (e.g. with OH groups) hydrogen bonds may be expected, too. Dr. J. T. Edsall (Harvavd) said : I should like once more to emphasize the specificity of the stabilizing action of fatty acid anions on serum albumin, which Dr. Luck has discussed. Oncley, Melin and Gross, in our laboratory, showed during the war that y-globulin is not at all stabil- ized against heat denaturation by these reagents; in fact, it becomes somewhat less heat-stable in their presence. On the other hand, reagents such as glycine, or the salts of some dicarboxylic acids, do significantly stabilize y-globulin, although the effects are far less dramatic than those of fatty acid anions on serum albumin. It might be of interest to study the stereochemical specificity of some of these reactions with albumin.For instance, would the binding con- stants be different for d- and Z-mandelate, or for cis- and trans-cinnamates ? Considering the great importance of steric factors in immunologic reactions, one might expect to find something a little similar here. Dr. 0. Hoffmann-Ostenhof (Vieutna) said : In connection with the experiments reported it may be of interest that the interaction of cation detergents with protein can be well demonstrated by measuring the in- activation of enzyme activity by these compounds. Enzymes are, as far as it is known, proteins and are rather unspecifically inhibited by cationic detergents.Experiments performed in my laboratory with various enzyme preparations (urease, papain, catalase, phosphomono- sterases) have shown that cationic detergents of various chain length (C, to CIS) have qualitatively the same effect on enzyme action ; quan- titatively there is a marked difference, the C1, compound being about two hundred times a stronger inhibitor of the action of the enzymes named than the C, compound. Dr. J. A. V. Butler (London) said: I would like to ask Prof. Luck whether the “ bond free energies ” he gives do not really refer to dis- placement reaction. They are based on the equilibrium of the anions between a dialysis bag containing the protein and an outer buffer solution. When an anion enters the bag it will be accompanied by an equal quan- tity of a positive ion, or will displace an equivalent quantity of negative ions.So what is ieally measured is either the sum of quantities which refer tc the distribution of both positive and negative ions, or a difference represepting the displacement of one anion by another. Sine proteins hold water very tenaciously and it has been found thpt each polar side group binds at least one water molecule with a considerable energy, I would also like to ask if he thinks that the anions are bound on top of this bound water, or do they displace it ? It would be very interesting to have the bonding energies (AH) imtead of the free energies which are quoted. Have any temperature coefficients been measured ? Dr. J. T. Edsall (Harvard) said : Dr.Butler asks whether any AH values have been measured for these reactions. Klotz and Urquhart * reported AH as - 2100 cal./mole for the binding of methyl orange by bovine serum albumin and as - 2000 cal./mole for azosulphathiazol. Still more recently, Scatchard, Scheinberg and Armstrong have obtained AH values for the binding of chloride and thiocyanate by albumin. These values are extremely low, not more than a few hundred cal./mole, and could indeed be regarded as zero within the experimental error of the measurements. Prof. J. Murray Luck (Stanford, CaEifornia) replied : I don’t know how to answer Dr. Butler’s questions about bond energies and water. J . Amer. Chem. SOL., 1949, 71, 847. 6 Ibid., 1950 (in press).62 LIPO-PROTEIN CENAPSES O F HORSE SERUM As for the former, I quite agree that the bond energies mentioned in the paper may not be as amenable to the precise interpretations that apply to small molecules and very simple systems. Competition of other anions derived for example from salt and buffer, must confuse things somewhat so that the bond energy is really a net value representative of the affinity between positively charged groups on the protein and the added anion, but in the presence of certain competing anions.It is relevant to mention that the bond energies are calculated only for the first ion that is bound. In so far as statistical factors predominate and inter- ference with the adding of the remaining ions is negligible the cal- culated bond energy is probably about the same for all of the ions that are bound.There is, however, another qualification that should be made. The value is obviously statistical and presupposes that the first ion to be bound is not preferentially directed in each reacting protein molecule to a guanidine group, a lysine group, or some certain amino acid side chain. I shall have to skip over the water question : I don’t know what effect, if any, hydration of the positively charged groups would have. Prof. J. R. Squire (Birmingham) said: As we have heard today various examples in which the body lipo-protein differs in behaviour from those studied experimentally in vitro, it seems appropriate to give a more encouraging example of the relevance and fundamental importance of such combinations in human economy. With Dr. C. Ricketts and Dr. E. Topley of the Medical Research Council Units at the Birmingham Accident Hospital, studies have been made of the self-sterilizing power of human skin against pathogens such as haemolytic streptococci. These organisms are destroyed by oleic acid which is found free only with other lipids in quantities corresponding to a layer 0.5 to 1.0 p thick on the skin. As previously shown by Dubos, the bactericidal power of oleic acid is inhibited by serum albumin. We have shown that the self-sterilizing power of skin against streptococci is correspondingly inhibited by serum albumin. As described by Prof. Murray Luck, the combination is quanti- tative (even though in our experiments concentrations leading to micelle formation were employed) and is specific-for example, neither the keratin surface of skin nor added peptone interferes with self-sterilization. Breaches of the skin surface leading to its contamination with serum proteins would be expected to allow the multiplication of pathogenic organisms; this finding is the rule in dermatitis and other conditions. The complete understanding of lipid-protein interaction is clearly of the greatest importance to many aspects of biology and medicine.

ISSN:0366-9033    

DOI:10.1039/DF9490600058

出版商:RSC    

年代:1949

数据来源: RSC