摘要:

DISCUSSIONS OF THE FARADAY SOCIETY No. 13, 1953 THE PHYSICAL CHEMISTRY OF PROTEINS THE FARADAY SOCIETY Agents .for the Society’s Publications : The Aberdeen University Press Ltd. 6 Upper Kirkgate A berdeenThe Faraday Society reserves the copyright of all Communications published in the '' Discussions If PUBLISHED . . . 1953 PRINTED IN GREAT BRITAIN AT THE UNIVERSITY PRESS A B E R D E E NA GENERAL DISCUSSION ON The Physical Chemistry of Proteins A GENERAL DISCUSSION on The Physical Chemistry of Proteins was held in the Zoological Department, Downing Street, Cambridge (by kind permission of Prof. J. Gray) on the 6th, 7th and 8th August, 1952. The Chairman of the Colloid and Biophysics Committee, Dr. J. H. Schulman, O.B.E., was in the Chair and about 400 members and visitors were present.Among the distinguished overseas members and visitors welcomed by the Chairman were the following:- Dr. L.-G. Allgen (Sweden), Mr. W. McD. Armstrong (Eire), Mr. J. P. W. van Baal (Holland), Mr. R. C. Backus (U.S.A.), Dr. E. Barbu (France), Dr. J. J. Blum (U.S.A.), Dr. P. Bocquet (France), Prof. C. J. F. Bottcher (Holland), Miss J. Botts (U.S.A.), Dr. A. Bussard (France), Dr. A. S. Cummin (U.S.A.), Mr. J. Drenth (Holland), Mr. W. Drenth (Holland), Dr. B. Drake (Sweden), Dr. E. L. Durrum (U.S.A.), Prof. and Mrs. J. T. Edsall (U.S.A.), Prof. H. Eilers (Holland), Dr. E. Ellenbogen (U.S.A.), Dr. F. Elliott (Belgium), Mr. P. Flodin (Sweden), Dr. E. Fredericq (Belgium), Mr. H. C. Freeman (Australia), Dr. G. Frick (Sweden), Dr. S. L. Friess (U.S.A.), Mr.and Mrs. Gelotte (Sweden), Dr. R. J. Goldberg (U.S.A.), Prof. and Mrs. E. Gorter (Holland), Mr. P. Gros (France),Dr. K. H. Gustavson (Sweden), Dr. G. Hamoir (Belgium), Prof. A. Baird Hastings (U.S.A.), Prof. F. Haurowitz (U.S.A.), Miss A. Haurowitz (U.S.A.), Dr. T. L. Hill (U.S.A.), Dr. M. Joly (France), Dr. C. Julen (Sweden), Dr. F. Karush (U.S.A.), Dr. D. Kertesz (France), Dr. Katchalsl (Israel), Dr. R. L. Kenyon (U.S.A.), Dr. H. B. Klevens (France), Prof. I. M. and Mrs. Klotz (U.S.A.), Prof. R. Lontie (Belgium), Prof. J. Murray Luck (U.S.A.), Dr. and Mrs. B. Malmstrom (Sweden), Prof. K. Meyer (U.S.A.), Prof. W. J. Moore (U.S.A.), Dr. M. Morales (U.S.A.), Prof. and Mrs. A. Monroy (Italy), Dr. L. Nanninga (Holland), Prof. and Mrs. Neurath (U.S.A.), Dr.and Mrs. Nottingham (New Zealand), Prof. J. L. Oncley (U.S.A.), Dr. L. Ouellet (U.S.A.), Dr. A. B. Pardee (U.S.A.), Prof. and Mrs. Pauling (U.S.A.), Dr. K. 0. Pedersen (Sweden), Dr. Gertrude Perlmann (U.S.A.), Dr. E. M. Petri (Holland), Dr. Mary Petermann (U.S.A.), Dr. J. Pouradier (France), Prof. P. Putzeys (Belgium), Dr. S. Ranzi (Italy), Prof. R. I. Razouk (Egypt), Prof. and Mrs. 0. K. Rice (U.S.A.), Dr. and Mrs. L. Robert (France), Dr. L. A. Sluyterman (Holland), Prof. A. SLhoberl (Germany), Dr. A. J. 34 OVERSEAS MEMBERS AND VISITORS Staverman (Holland), Dr. G. J. M. Sprokel (Holland), Prof. D. M. and Mrs. Surgenor (U.S.A.), Dr. D. B. Taylor (U.S.A.), Mr. G. P. Talwar (France), Mr. M. van den Tempe1 (Holland), Miss M. G. Ter Horst (Holland), Mr. J.H. Verhoog (Holland), Mr. A. J. de Vries (Holland), Dr. L. A. Wall (U.S.A.), Mr. J. W. Williams (U.S.A.), and Prof. L. Zervas (Greece). A special welcome was given to Dr. J. T. Edsall (Harvard University) who gave the 6th Spiers Memorial Lecture, entitled “ The molecular shapes of certain proteins and some of their interactions with other substances ”.CONTENTS Sixth Spiers Memorial Lecture- The Molecular Shapes of certain Proteins and some of their Interactions with other Substances. By John T. Edsall . I. Experimental Techniques- Zone Electrophoresis in Filter Paper and other Media. By Arne Tiselius . Polarjzation of the Fluorescence of Labelled Protein Molecules. By G. Weber The Application of Solubility Measurements to the Study of Complex Protein Solutions and to the Isolation of Individual Proteins.By J. S. Falconer, D. J. Jenden and D. B. Taylor . GENERAL DrscussIoN.-Dr. P. Flodin, Dr. R. Consden, Dr. E. L. Durrum, Dr. D. L. Mould, Dr. E. Barbu, Dr. L. Robert, Dr. A. G. Ogston, Dr. K. 0. Pedersen, Dr. B. Robert, Dr. G. S . Adair, Dr. L. Nanninga, Dr. D. B. Taylor . 11. Low Molecular Weight Proteins- Thermodynamics of the Association of Insulin Molecules. By Paul Doty and George E. Myers . Molecular-kinetic Properties of Trypsin and Related Proteins. By Leon W. Cunningham, Jr., Frank Tietze, N. Michael Green and Hans Neurath . Electrophoretic Studies of Enzymatically Modified Ovalbumin and Casein. By Gertrude E. Psrlmann . The Globular-fibrous Protein Transformation. By E. Barbu and M. Joly . GENERAL DIscussIoN.-Prof.H. Neurath, Dr. A. Wassermann, Dr. P. Doty, Prof. F. Haurowitz, Dr. L. Nanninga, Prof. W. T. Astbury, Dr. E. Barbu, Dr. W. E. F. Naismith, Mr. A. J. Hyde, Dr. M. Joly 111. High Molecular Weight Systems- The Physicochemical Examination of the Conarachin Fraction of the By P. Johnson and Groundnut Globulins (Arachis hypugaea). W. E. F. Naismith . The Effects of certain Ions and Neutral Molecules on the Conversion of Fibrinogen to Fibrin. By Sidney Shulman . 5 PAGE 9 29 33 40 46 51 58 67 77 93 98 1096 CONTENTS Electrophoretic Study of the Muscle Structural Proteins. Energetics and Molecular Mechanisms in Muscle Action- By G. Hamoir . Part I.-Outline of a Theory of Muscle Action, and some of its Experimental Basis. By Manuel Morales and Jean Botts . Part I1 .-S t a t is t ical Thermodynamical Treatment of Contractile Systems.By Terrell L. Hill . Aspects of Polymerization in Proteins of the Muscle Fibril. By T.-C. Tsao and K. Bailey . Electrochemical Properties of Lupin Seed Protein. By Miss E. M. Petri and A. J. Staverman . GENERAL DIscussIoN.-Dr. G. A. Gilbert, Dr. P. Johnson, Dr. L. Nanninga, Dr. K. H. Gustavson, Dr. Staverman, Dr. M. G. ter Horst, Dr. G. Hamoir, Dr. A. Wassermann, Dr. A. G. Ogston, Prof. W. T. Astbury, Dr. J. T. Edsall, Dr. M. Morales . IV. Protein Interactions- Aggregation of Globular Proteins. By Linus Pauling . The Interaction of Plasma Proteins with Heavy Metals and with Alkaline Earths, with Specific Anions and Specific Steroids, with Specific Polysaccharides and with the formed elements of the Blood.By Edwin J. Cohn, Douglas M. Surgenor, Karl Schmid, W. H. Batchelor, Henry C . Tsliker and Eva H. Alameri . Protein Interactions with Organic Molecules. By Irving M. Klotz and Janet Ayers . The Influence of Salt on the Size and Shape of a Protein-detergent Complex. By B. S. Harrap and J. H. Schulman . Complexes of Heparin and Proteins. By E. Gorter and L. Nanninga Nucleic Acid and Protein Interactions-an Electrophoretic Study of Calf Thymus Deoxypentose Nucleoprotein and of Tobacco Mosaic Virus. By Muriel Fleming and D. 0. Jordan . Antigen-antibody Reactions in Theory and Practice. By Richard J. Goldberg and J. W. Williams . GENERAL DrscussIoN.-Dr. J. A. V. Butler, Dr. J . T. Edsall, Prof. A Schoberl, Dr. B. Robert, Dr. L. Robert, Dr. Bo G. Malmstrom, Dr.K. H. Gustavson, Prof. J. Murray Luck, Mr. B. P. Brand, Dr. B. A. Pethica, Dr. K. Walton, Dr. D. Hamer, Dr. G. A. Gilbert, Prof. K. Meyer, Prof. E. Gorter and Dr. L. Nanninga, Prof. J. W. Williams, Dr. C. G. Pope, Dr. S. McGavin and Dr. J. Iball, Prof, S. Haurowitz, Prof. J. R. Marrack, Dr. R. J. Goldberg PAGE 116 125 132 145 151 159 170 176 189 197 205 21 7 224 23 1CONTENTS 7 V . Conjugated Proteins (Nucleo- and Muco-Proteins)- PAGE Conjugated Proteins. By M. Stacey . . 245 Tuberculin Proteins. By Florence B. Seibert . . 251 Swelling and Orientation Phenomena with Nucleoprotein Films. By E. J. Ambrose and J. A. V. Butler . . 261 Hyaluronic Acid, Chondroitin Sulphates and their Protein Complexes. By Karl Meyer . . 271 Composition and Properties of Hyaluronic Acid Complex of Ox Synovial Fluid. By A. G. Ogston and J . E. Stanier . . 275 GENERAL DIscussIoN.-Mr. A. M. Woodin, Mr. J. H. Verhoog, Prof. M. Stacey, Dr. R. Consden, Prof. F. Haurowitz, Prof. W. T. Astbury and Dr. N. N. Saha, Dr. R. D. B. Fraser, Dr. J. T. Edsall, Dr. E. J. Ambrose, Mr. J. E. Stanier, Dr. A. G. Ogston . . 281 Author Tndex . . 288

ISSN:0366-9033    

DOI:10.1039/DF9531300001

出版商:RSC    

年代:1953

数据来源: RSC

ISSN:0366-9033    

DOI:10.1039/DF95313BX003

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

THE PHYSICAL CHEMISTRY OF PROTEINS SIXTH SPIERS MEMORIAL LECTURE THE MOLECULAR SHAPES OF CERTAIN PROTEINS AND SOME OF THEIR INTERACTIONS WITH OTHER SUBSTANCES BY JOHN T. EDSALL" University Laboratory of Physical Chemistry Related to Medicine and Public Health Harvard University Boston Mass. Received 30th September 1952 The Discussions of the Faraday Society have developed in recent years into events of international significance for many scientists. To-day as in a number of the previous Discussions the focus of interest is on the great border area between the provinces of physics chemistry and biology. In those earlier Discussions many ideas of extraordinary fruitfulness were presented and vigorously debated before they had become familiar and respectable.I have often turned back for illumination to the printed record of their proceedings. Three years ago I had for the first time the opportunity to take part in one of these Discussions-that on Lipoproteins at Birmingham. Hence I feel deeply the honour of giving the Sixth Spiers Memorial Lecture. It was never my good fortune to know Mr. Spiers personally but it was he especially who guided the pattern of this Society's development from small beginnings until it grew into an active and significant organization and its discussion meetings became as they are to-day the focal point for investigators from every land where science is free and active. The untimely death of Mr. Spiers occurred in 1926. At that time although the Faraday Society had held many conferences of international importance on the border line of physical chemistry and other sciences it is scarcely likely that it could have contemplated a two-day discussion on the physical chemistry of the proteins.It is doubtful indeed whether in 1926 more than half a dozen laboratories throughout the world could have made effective contributions to such a discussion. L can well remember that year for in the midst of it I returned to Harvard after two years of study here in Cambridge where I had imbibed the broad vision of the scope and the future of biochemistry that was to be found in Cambridge in Hopkins's laboratory. Also I had had the good fortune to know that great pioneer in the physical chemistry of the proteins Sir William Hardy then no longer active in that field but still with a freshness of mind a vitality and zest in life that I have seldom seen matched in other men.In those days we scarcely knew the molecular weight of a single protein. The view was indeed held by some distinguished chemists that proteins were actually very small molecules with molecular weights below 1000. This theory was short- lived being quite demolished late in 1926 by a few well-chosen freezing-point * Fulbright Visiting Lecturer University of Cambridge 1952. 9 A SIXTH SPIERS MEMORIAL LECTURE 10 measurements by Cohn and Conant. Sorensen indeed had carried out a decade earlier his classical investigations on egg albumin and his careful studies of osmotic pressure had given the basic information for the determination of its molecular size.Adair had propounded in 1925 the startling view that the molecular weight of mammalian haemoglobin was near 67,000 and that the molecule therefore contained four iron atoms. I can well remember the profound scepticism with which this report was received by more than one professor of great eminence; and the doubts persisted in high quarters until the ultracentrifugal measurements of Svedberg a year or two later gave complete support to Adair’s conclusions. The ultracentrifuge had indeed been under development since about 1922 but there were few at that time who anticipated its immense and revolutionary influence on protein chemistry. If relatively little was known of the sizes of protein molecules almost nothing was known about their shapes; indeed the problem of molecular shape was scarcely envisaged at that time by protein chemists.The air was still filled with the strife of the conflict between Jacques Loeb and the colloid chemists of the school of Wolfgang Ostwald. Loeb’s dzmonstration that some of the colloidal properties of proteins could be explained on simple chemical principles a demon- stration expounded brilliantly and with a fighting spirit appeared to many besides myself as if it were a new revelation. To-day I still appreciate the value of Loeb’s contribution. but I am also aware that T. B. Osborne in New Haven many years before had been quietly carrying on his work on the plant proteins treating them as chemical substances to be understood on general chemical principles and paying no attention to the clamour of conflicting schools.Also there was one idea expressed by distinguished protein chemists as late as twenty years ago that proteins furnished essentially the inert structural frame- work of the animal organism while the active pattern of biochemical trans- formations was determined by the interplay of other molecules of quite a different sort. Certainly this view was never upheld in the laboratory headed by Edwin Cohn. Nevertheless the fact that such a belief could have prevailed among many biochemists even for a few years seems astonishing to-day; and indeed it represented the denial of a much older tradition going back over a century to Liebig and Mulder which held that proteins were indeed of absolutely prime importance to the living organism.To Mulder in 1840 this must have been largely a matter of faith and intuition ; but now in the last quarter century we have seen that faith abundantly justified by works. I have tried to recall here something of the temper of the times the prevailing atmosphere of thought among the relatively small band of protein chemists in 1926 when I came back from Cambridge to Harvard as a third year medical student and started working in my spare time under the direction of Edwin Cohn on a viscous and peculiar globulin from skeletal muscle continuing work which had been begun by W. T. Salter. Later we called this globulin myosin and still later it was rechristened by Szent-Gyorgyi as a form of actomyosin. It proved a most refractory and difficult substance to work with but in the midst of our struggles Alexander von Muralt arrived from Switzerland and introduced the measurement of double refraction into the laboratory.His brilliant contributions both to experiment and to thinking brought us face to face with the problem of molecular shape; from then on we knew that although many proteins might be more or less globular some-and these indeed of supreme importance-were long and filamentous even in solution. In these casual remarks I shall not try to review the intervening years with the colossal advances that they have seen. Protein chemistry which was a rela- tively unfashionable subject a quarter of a century ago has now become almost embarrassingly fashionable.It does not seem likely to go out of fashion unless mankind decides to turn away from science and repudiate it altogether-a possible but I still hope a relatively improbable event. JOHN T . EDSALL 11 However of all the advances in this quarter century perhaps none will stand out more than two that have cccurred in the last two years-the elucidation of the complete sequence of amino acid residues in the A and B chains of insulin by Sanger Tuppy and Thompson,l and the detailed formulation of spatial con- figurations of the polypeptide chain by Pauling and Corey.2 The aspects of protein chemistry in which these developments are included will not be dealt with by other authors in the present Discussion but these achievements are bound to influence the thinking of all of us so profoundly that I cannot leave them without mention here.We cannot yet write a structural formula for insulin until the arrangement of the disulphide bridges is specified but that the goal has now been so nearly approached is an achievement which would have seemed incredible even five years ago. Protein chemists will now really begin to think for the first time in terms of structural organic chemistry and all of us here who are physical chemists will be permeated more and more by that influence. However in protein chemistry more than anywhere else structural chemistry is of little use without stereochemistry. Proteins with their marvellously specific patterns are the supreme examples of molecillar geometry. The work of Pauling and Corey is neither the beginning nor the end of the deciphering of these patterns; their achievement is built on the work of many and brilliant predecessors and what they have done is only a beginning.Their work still has its sharp critics; but I have been led more and more to the conclusion that their a-helix does quite closely represent the spatial pattern of long chain synthetic polyamino acids and not improbably also that of the fibrous proteins which are found in the cc configuration. Also-though there is less concrete evidence for this view-the pleated sheets which they have proposed appear to be by far the best existing models for the extended pattern of protein chains in the ,B configuration. In several crystals of corpuscular proteins the presence of intramolecular bundles of parallel chains has been inferred from the analysis of X-ray data.In the case of haemoglobin Bragg Howells and Perutz 3 inferred that these chains were polypeptide chains in the cc configuration. Well-defined chains were in- ferred by Kendrew 4 in the myoglobin molecule. The study of crystalline ribo- nuclease 5 has also suggested the presence of chains parallel to the c-axis of the crystal but the configuration of these chains seems still to be a matter for dispute. Some of the most striking evidence for chain structures in a protein crystal comes from the recent work of my colleague Barbara W. Low at Harvard on the ortho- rhombic crystals of acid insulin sulphate,6 originally prepared in our laboratory by Dr. Eric Ellenbogen working with Prof.Oncley. The three-dimensional Patterson diagrams of these crystals in the air-dried form show a most striking pattern of parallel rods with axes parallel to the a-axis of the unit cell which has a length of approximately 45 A. Dr. Low has constructed possible packing models for the polypeptide chains in this crystal taking account of Sanger’s findings on the sequence of amino acid residues. I should like to turn now from these brief remarks on protein structure at the deeper level to some of the problems related to the general size shape and inter- actions of proteins. I shall confine myself to two proteins with which I have become particularly familiar in recent years fibrinogen and serum albumin FIBRINOGEN SIZE AND SHAPE Fibrinogen occupies a place apart among the plasma proteins because of its high molecular asymmetry and its low solubility the latter property rendering it the first major constituent of plasma to be precipitated in almost any system of fractionation.Its high viscosity its marked flow birefringence its high frictional ratio and its significant angular dissymmetry of light scattering all mark it out as a molecule of shape very far from spherical. There are now data available on both human and bovine fibrinogen in solution which are too numerous to 12 S I X T H SPIERS MEMORIAL LECTURE discuss here in detail. They are summarized in table 1 and appear to indicate that human and bovine fibrinogen are remarkably similar in size and shape. The molecular weight indeed appears at present more uncertain than the absolute dimensions-many workers have accepted a value of 450,000-500,000 but the TABLE 1 .-PHYSICAL CONSTANTS OF HUMAN AND BOVINE FIBRINOGEN sedimentation constant S ~ O ~ (Svedberg units) partial specific volume V20 intrinsic viscosity Eio rotary diffusion constant 0 2 0 (sec-1) (from double refraction of flow) molecular weight (in thousands) osmotic pressure (Mo) sedimentation and viscosity (Mg) light scattering molecular length (A) double refraction of flow osmotic pressure and viscosity sedimentation and viscosity light scattering molecular diameter (A) H = Holmberg (Arkiv.Kemi Min. Geol. A 1944 17 no. 28). NoTEs.-T~~s table is also published in BZood Cells and Plasma Proteins ( J .L. Tullis ed. Academic Press New York 1953 in press) G. S. Adair obtained a molecular weight from osmotic pressure near 500,000 (species not recorded) as reported by Bailey Advances in Protein Chem. 1 308 (1944). Holmberg reports a translational diffusion constant for human fibrinogen which gives a molecular weight near 700,000 if taken with sedimentation constants given above. A new investigation of the diffusion constant would be desirable. ABMH = Armstrong Budka Morrison and Hasson (J. Amer. Ch~m. SOC. 1947,69,1747). EFS = Edsall Foster and Scheinberg (J. Amer. Chem. Soc. 1947 69 2731). HLS = Hocking Laskowski and Scheraga (J. Amer. Chem. SOC. 1952 74 775). OSB OSB - K = Koenig (Arch. Biochem. 1950 25 241). KGSF = Katz Gutfreund Shulman Ferry J.Amer. Chem. SOC. 1952 74 5706. KP = Koenig and Pedersen (Arch. Biochem. 1950 25 97). ML = Morrison and Lontie (unpublished measurements from this laboratory). N = Nanninga (Arch. neerland. physiol. 1946 28 241). OSB = Oncley Scatchard and Brown (J. Physic. Chem. 1947 51 184). SF = Shulman and Ferry (unpublished). SL = Steiner and Laki (Arch. Biochem. Biophys. 1951 34 24). recent work of Hocking Laskowski and Scheraga 7 points to a value near 400,000 ; and very recently Katz Gutfreund Shulman and Ferry,8 in what appears to be a particularly careful study of light scattering have obtained a value of only 340,000 for bovine fibrinogen. From the angular dissymmetry of the scattering they also calculated a length of 520A for the molecule if it were assumed to be a rod or of 650A if it be taken as an ellipsoid of revolution.This marked difference shows how sensitive the calculated absolute dimensions are to the particular * Hocking Laskowski and Scheraga believe this value to be too high because of the form of the angular dissymmetry curves obtained. references numerical values bovine human 8.5-9 9 human OSB 0.725 bovine 8.4-8.6 7.9-8.2 0-706 0.25 0.25 H ABMH OSB EFS KP SF K N HLS 39,400 35,000 580 400 440 OSB - ML - - 440 - 540" 407 340 EFS - - N SL HLS KGSF HLS - N 700 - 700 670 725 - 840" - 38 SL HLS 38 JOHN T . EDSALL FIBRINOGEN AND FIBRIN INTERACTIONS 13 type of model assumed-a fact which if carefully borne in mind would help to obviate some of the disputes about molecular dimensions that now and then arise.Using the ellipsoidal model Foster Scheinberg and I 9 calculated a length near 700w for human fibrinogen and Hocking Laskowski and Scheraga7 more recently obtained a value of 670w for bovine fibrinogen. Both of these values obtained from flow birefringence measurements are in very satisfactory agree- ment with the light scattering value. Moreover the angular dissymmetry of scattering taken in conjunction with the order of magnitude of the molecular weight seems to be compatible only with a prolate or rod-shaped molecule; an oblate type of structure showing such high dissymmetry should have a much higher molecular weight.The dimensions of the fibrinogen molecule have aIso been studied by the electron microscopists. Hall 10 observed rod-shaped structures of varying length the most commonly found length being near 700A as might have been expected from the measurements in solution. The rods appeared to be built up of sub- units so that they looked a little like a string of beads tightly bound together. Except for the marked heterogeneity of the size of the molecules this was quite compatible with the studies on fibrinogen in solution. However to make the problem more complicated other electron microscopists have reported quite different findings. Porter and Hawn 11 reported particles which they believed to be disc shaped about 200w across from bovine fibrinogen; and Mitchell12 found only globular units about 50 in diameter and short filaments 100-300 A long in preparations from human fibrinogen.How to interpret these conflicting findings I do not know ; I must confess to a strong prejudice that Hall’s findings are closest to the truth and in any case the high asymmetry and rodlike shape of fibrinogen in solution seem well established whatever may happen to it in the drying process that is necessary before it can be examined in the electron microscope. The supremely characteristic property of fibrinogen is its interaction with thrombin to give an altered molecule which is capable of association with other like molecules to give the fibrin clot. Bailey and Bettelheim in Cambridge,l3 and Lorand and Middlebrook in Leeds,l4 have done much to eludicate the process.The transformation certainly involves the splitting-off of a peptide (fibrinopeptide) from the fibrinogen molecule and the unmasking of free terminal a-amino groups of glycine in the resulting reactive fibrinogen derivative from which the peptide has been dissociated. The subsequent association processes culminating in the fully developed fibrin clot have been studied in many ways. Here I shall only speak briefly of some influences that are exerted on the proccss by the very strong interactions of certain ions and neutral molecules with fibrinogen or fibrin or both. These effects were observed by Dr. Walter Lever and myseIf,ls studying the rate of clot formation from fibrinogen and thrombin by direct weighing of the fibrin formed and examining the general structure of the clot by measuring its turbidity.Ferry and Morrison,ls in the fundamental studies they had carried out in our laboratory during the war had already defined two extreme types of fibrin clots; the coarse type formed at low pH and low ionic strength which is opaque and synerizes readily under pressure and the fine type relatively transparent and very friable which forms at high pH or high ionic strength. Lever and I carried out our studies for the most part at constant pH and at a fixed ionic strength of 0.15. Even when these factors were fixed many ions and neutral molecules were found to exert profound and specific effects upon the process. Notable among these were urea and guanidine hydrochloride long of course well known as denatur- ing agents for proteins.In this system however these reagents were studied at 14 concentrations far below those ever reported to produce denaturation. Fig. 1 shows the effects of urea on the rate of fibrin formation. It will be seen that even at a concentration of 0.1 M the rate of fibrin formation in urea solution is distinctly lower than in the control solution without urea. At 0.5 M the decrease in the rate of fibrin formation is profound. Moreover the presence of urea affected the structure of the clots as well as the rate of their formation ; invariably the clots formed in the presence of urea were less turbid and more friable than the controls. It has become well known from the work of Laki and L0rand~17.18 that urea at much higher concentrations (above 3 M) actually dissolves fibrin clots formed from purified fibrinogen.It seems probable that this is due to its strong capacity for hydrogen-bond formation so that the hydrogen bonds between urea and the protein displace the protein-protein bonds that normally link the protein molecules together into the fibrin clot. Our measurements at very low urea concentrations which were carried out in 1946-47 before the work of Laki and Lorand was SIXTH SPIERS MEMORIAL LECTURE I 2 0 4 24 3 Time in Hours FIG. 1.-Effect of urea on the rate of fibrin formation from human fibrinogen (fraction I) and thrombin. Thrombin concentration 0.1 unitlml. All solutions contained 0.1 5 M sodium chloride at pH 6.3.The top curve is for a solution containing no urea; the others are for solutjons containing the amounts indicated on the diagram. From Edsall and Lever.15 published show the same effect in incipient form. The formation of highly turbid clots of the coarse type is the result of extensive lateral aggregation of the first thin strands developing in the early stages of clot formation.16 Increase of pH increases the negative net charge on the fibrin molecules and thus tends to decrease lateral association by electrostatic repulsion. Addition of urea blocks lateral association by hydrogen-bond formation between urea and protein leading to the same result. In either case the clot becomes more translucent and friable. The effect of guanidine hydrochloride is very different.Like urea indeed it greatly retards the rate of formation of fibrin even at 0.05 M there is a very marked retardation and at 0.075 M the effect is profound (fig. 2). However the effect on the structure of the clot is in the opposite direction from that of urea; as the structure becomes fully developed it becomes markedly more coarse and turbid than the control clot; indeed the pattern of development of the clot both as regards increase of turbidity and rate of fibrin formation is nearly the same for a control clot at pH 6-3 and for a clot at pH 7.3 which contains guanidine hydro- chloride at a concentration of 0-075 M at least in the earlier stages of the clotting process (fig. 2 and 3). Adding guanidine hydrochloride at this concentration thus has nearly the same effect as lowering the pH by one unit ; the initial rate of fibrin 15 0 0.135 M NaCl; pH 6.3.0.8 0-6 0.4 0.2 JOHN T . EDSALL formation is greatly retarded and the final turbidity of the fully developed clot is greatly increased. The results suggest that the positively charged guanidinium ion FIG. 2.-Effect of guanidine hydrochloride on rate of fibrin formation at pH 6.3 and 7-3. Total ionic strength in all solutions was 0.15. pH was controlled by imidazole buffers the concentration of imidazole hydrochloride being 0.01 5 in ail experiments except in the top curve where it was 0.03. 0 sodium chloride (0.12 M) plus imidazole buffer at pH 7.3. @ 0.075 M guanidine hydrochloride (C(NH&+Cl-) ; 0.06 M sodium chloride ; pH 7.3.0 0.05 M C(NH&+CI- ; 0.085 M NaCl ; pH 6.3. 0 0-075 M C(NH&+Cl- ; 0-06 M NaCl ; pH 6.3 (From measurements of Walter F. Lever.) Time in Hours FIG. 3.-Effect of guanidine hydrochloride on turbidity of fibrin clots at pH 6.3 and 7.3. The solutions had the same composition as those described in fig. 2 and corresponding curves are denoted by the same symbols in both figures. Note the high turbidity of the solution containing 0-075 M C(NH&+C1- at pH 7.3 as compared to the extremely low turbidity of the control solution containing only sodium chloride at the same pH. (From measurements of Walter F. Lever.) combines preferentially with the protein decreasing its negative net charge just as hydrogen ion would do.Quantitative studies of the binding of guanidinium ion to 16 fibrinogen and fibrin have not been carried out but these results suggest that the binding would be strong. K'I- pH 6*50,,+ .' :-- 0 / -0 "0 I / ,4 / */o/@--o- K+SCN- pH 6 - 2 5 ' 1 P / / I Time in Hours * FIG. 4.-Effect of iodide and thiocyanate on rate of fibrin formation from human 2 4 NaC1; middle curve 0.135 M KI + 0.015 M NaCI; bottom curve 0.135 M KSCN + fibrinogen and thrombin. Ionic strength in all solutions was 0.15. Top curve 0.15 M 0.015 M NaCl. Note that the pH in the iodide solution was significantly higher than in the other two. This increased pH accelerated the reaction; thus the rate of fibrin formation in the iodide solution would have been lower at lower pH.Control experi- ments showed that the effect of potassium salts was indistinguishable from that of sodium salts with the same anion with respect to rate of fibrin formation and clot turbidity. 5 (From measurements of Walter F. Lever.) -0.8 Y A7 0 SIXTH S P I E R S MEMORlAL LECTUKE I 9 J 3 2 Time in Hours FIG. 5.-Effect of iodide and thiocyanate on turbidity of fibrin clots as a function of time. Molar concentrations of salts in these experiments were identical with those denoted by corresponding symbols in fig. 4. (From measurements by Walter F. Lever.) * Acknowledgments are made to the Josiah Macy Jr. Foundation for permission to publish fig. 4 and 5 which are slightly modified versions of those appearing in Blood Clotting rmd Allied Psohltwis.Trmsnctions of' the Forirtk CorlJ;.renre ( I95 I) pp. 241-2. JOHN T . EDSALL SERUM ALBUMINS SIZE AND SHAPE 17 Certain anions such as iodide and thiocyanate-also well known as denaturing agents for many proteins at much higher concentrations-act in the opposite direction on the structure of the clot greatly decreasing its turbidity as compared with that of the control at the same pH and ionic strength (fig. 4 and 5). In these cases therefore there is suggestive evidence of selective anion binding. It should be noted that in all cases these reagents diminish the initial rate of fibrin formation as shown in fig. 2 and 4 whatever their effect on the structure of the clot. In no case however was there any destruction of thrombin by the added reagent as control experiments showed so that the decreased rate of reaction cannot be laid to this.Closely related to these studies are the extensive and important studies of J. D. Ferry and Sidney Shulman on the effects of many reagents in inhibiting the conversion of fibrinogen to fibrin. These have been presented by Dr. Shulman 19 in a paper to be discussed at this conference so that I shall not treat them further here. The serum albumins have been among the most intensively studied of all pro- teins ; yet even the size and general shape of the albumin molecule are still a matter of dispute. Let us consider some of the evidence. (i) By the use of dinitrofluorobenzene van Vunakis and Brand 20 could find only one free terminal a-amino group in human horse or bovine serum albumin per molecule of molecular weight approximately 70,000.In all three cases this amino group belonged to an aspartic acid (or asparagine) residue. Thus it would appear that the molecule consists of a single peptide chain. Yet Weber,21 in the paper he is presenting to this meeting has concluded from polarization fluorescence studies that serum albumin dissociates into smaller units in both acid and alkaline solution. There is no necessary incompatibility between these findings but inevitably they raise certain questions. Does the splitting of the albumin molecule involve the breaking of some exceptionally labile peptide linkages? If so what brings about the reassociation that Weber has found to occur on neutralizing the acid albumin solution? Or on the other hand are there actually two or more peptide chains within the albumin molecule only one of which-presumably for steric reasons-reacts with dinitrofluorobenzene? Or is it possible that Weber's findings can be explained in terms of strong electro- static interactions between molecules carrying a high net charge without actually having to assume molecular dissociation? The latter possibility seems to me relatively unlikely but it should not be altogether disregarded.(ii) The molecular weight of human and bovine serum albumin has generally been taken in recent years as 69,000 chiefly on the basis of the very careful osmotic pressure studies of Scatchard Batchelder and Brown.22 The recent X-ray measurements on albumin crystals in our laboratory by Barbara LOW,^^ combined with accurate determinations of density and composition definitely indicate a lower molecular weight near 65,500.Quite independently Creeth 24 has deduced an almost identical value for bovine serum albumin from very careful sedimentation and diffusion measurements. There is converging evidence indeed from several laboratories both in England and the United States that some of the generally accepted measurements of sedimentation constants are in fact too high and require some downward revision-a process which inevitably involves a downward re- vision of molecular weights as well. (iii) In 1947 Oncley Scatchard and Brown25 gave a detailed discussion of sedimentation viscosity and other data for serum albumin and-assuming 20 % hydration-described the molecule approximately as a prolate ellipsoid about I50 A long and 38 A in diameter at the middle.Their choicc of a prolate rather 18 t C SIXTH SPIERS MEMORIAL LECTURE than an oblate shape to describe the molecule was determined largely by Oncley’s dielectric dispersion data which indicated two distinctly different relaxation times -a finding incompatible with an oblate shape if the preparation consists of molecules which are all alike. Recently Oncley Dintzis and Hollies 25a have obtained dielectric measurements of much higher precision on carefully purified human mercaptalbumin and other serum albumin preparations freed from fatty acid and other impurities by treatment with ion exchange resins.These prepara- tions gave considerably higher dipole moments than earlier less pure samples. The form of the dielectric dispersion curve was quite sensitive to the protein con- centration ; but at sufficiently low concentrations the curve was entirely consistent with that to be expected for a prolate ellipsoid of molar volume 60 I. and an axial ratio of 4 to 1. No oblate ellipsoidal mode1 could be chosen to fit the data. The studies of Weber 26 on the polarization of fluorescence from albumin derivatives also give important evidence on this point and on the whole Weber’s findings appear to confirm those of Oncley. l 0 -1 0 2 0 L FIG. 6.-Model for possible modes of packing of human serum albumin in the crystal of mercaptalbumin mercury dimer (a wet crystal; b and c air dried crystals).All drawings represent the c plane projection. Two different modifications of Oncley’s model for the serum albumin molecule are indicated. From Low.23 (iv) As was discovered by Hughes,27 two molecules of human mercaptalbumin are readily linked by means of their sulphydryl groups through a single mercury atom to form a dimer. Oncley was led to reconsider his earlier model for the albumin molecule in order to account for the steric relations involved in the forma- tion of this dimer. This revised model has been employed by Low 23 to describe the packing of serum albumin molecules in the crystal of mercaptalbumin mercury dimer. The model consists of a prism approximately 145 A long 45A in dia- meter at the middle and 22A thick.Such a model can fit well into the unit cell of the crystal as determined by Low; and the indications of a layer structure which are given by the X-ray data are well accounted for by the piling into layers of such long thin molecules (fig. 6). It should be clearly understood that the X-ray data do not prove the structure but they are apparently compatible with it and the model itself-to be taken of course only as a very rough approximation -seems reasonably plausible on other grounds. (v) As was found by Foster and myself,2* human serum albumin shows flow birefringence although only at very high gradients in solvents of high viscosity. From our measurements we estimated a length at least as great as that of l50& inferred by Oncley on other grounds.However these measurements on so relatively short a molecule were exceedingly difficult and subject to considerable JOHN T . EDSALL 19 error ; they should not be taken as having precise quantitative significance. They do point however to a somewhat asymmetrical shape. (vi) In contrast to all these conclusions Riley and Oster 29 have inferred from low angle X-ray diffraction studies on very concentrated albumin solutions that the shape of the molecules cannot be very far from spherical. The theory under- lying this deduction is one which I do not feel competent to criticize ; but I feel very reluctant to accept the conclusion itself in view of the other evidence summarized above. (vii) Very recently Anderegg Beeman and Shulman30 have carried out low angle X-ray scattering studies on relatively dilute albumin solutions.Here the interpretation of the data is more straightforward than for the very concentrated systems studied by Riley and Oster. At vzry low angles the scattering curves indicate that the radius of gyration is definitely greater than for a sphere of the same molecular weight while the scattering at somewhat higher angles which is more characteristic of shape appears to be in better accord with that expected for an oblate than for a prolate ellipsoid of revolution. Assuming an oblate ellipsoid the ratio of the molecular axes is found to be near 3-5 to 1. These data must I think be taken very seriously; we cannot evade the attempt to find some explanation that will make them understandable in conjunction with all the other findings that seem to point to an elongated shape.Dr. Beeman has remarked in a recent letter to me on the subject “ We can say from our data that we are much more convinced that the molecule is not a prolate ellipsoid of revolution than that it is an oblate ellipsoid of revolution.” Indeed the major moral of all this may be that we should not take too seriously calculations based upon simple shapes. They are useful for representing the results of particular researches and for correlating the properties of proteins derived from hydrodynamic measure- ments but in detail the description of the shapes of protein molecules must involve something more complex than the very simple geometrical figures that are con- veniently used for preliminary descriptions.It would be a feasible though certainly a very tedious task to calculate the scattering function for the prismatic model of serum albumin previously discussed; and the curve so derived might be quite different from that of a prolate ellipsoid ; but the case seems a little too special at present to warrant the work involved. It does seem to me that the weight of the evidence does rather favour a general model of this sort and that an oblate ellipsoidal shape for albumin or anything very close to such a shape is unlikely. However I do not wish to be dogmatic on the subject ; what I hope to do in this lecture is to ask more questions than I can answer and I trust that others will come forward with the answers or will set to work to find them.ON THE POSSIBLE FLEXIBILITY OF PROTEIN MOLECULES In contrast to the arrangement of most synthetic polymers which coil about in random fashion in solution proteins have a highly ordered pattern. The evidence for this order is clearest from X-ray studies on protein crystals. Such order also implies a high degree of molecular rigidity which is shown clearly for proteins in solution by the dielectric studies of Oncley.31 Protein molecules orient as entire units in an electric field not as a series of more or less independent segments as do the polar polymers. All this is true and important ; yet we should certainly not assume that this rigidity is strict and absolute. Indeed Klotz and Ayers,32 in a paper presented at this meeting conclude that the molecule of serum albumin must be able to change its configuration with pH ; that it may swell or unfold somewhat as its net charge increases and that these efTects are reversible.Others I think have made similar suggestions before. Likewise the remarkable interactions involved in the combination of haemoglobin with oxygen have led Pauling,33 and also Wyman and Allen,34 to suggest that the addition of oxygen SIXTH SPIERS MEMORIAL LECTURE SERUM ALBUMINS INTERACTlONS WITH CERTAIN CATIONS 20 actually leads to a rather fundamental change in the configuration of the mole- cule as a whole. Pauling and Wyman hold rather different views as to the nature of this change and we certainly need more proof before we assume that the change really occurs but the suggestive evidence is strong.Even more striking I think are some recent unpublished studies here in Cambridge on the enzyme fumarase from pig heart recently crystallized by Vincent Massey 35 working with Dr. Malcolm Dixon. Combined observations by Massey on enzyme activity and by Weber using polarization of fluorescence suggest alterations of molecular size or shape or both with varying pH and ionic strength ; and these variations appear closely correlated with changes in enzyme activity and the formation of the enzyme-substrate complex. We have long been accustomed to change of shape on the part of the fibrous proteins as in the a-/3 transformation of keratin. Indeed the myosin system of muscle could not do its job unless it changed its shape radically and rapidly.Alterations of shape among the corpuscular proteins are presumably much less drastic-that is if they occur under conditions in which the protein still remains undenatured-but they may be of profound importance both chemically and biologically. The fluorescence polarization technique of Dr. Weber may be of great value in such studies for unlike the dielectric dispersion method it can be applied over a very wide range of pH and salt concentration to give information about the relaxation times of proteins. The serum albumins are famous for the strength and diversity of their inter- actions with anions; the story is a long and remarkable one and I have nothing to add to it to-day.Dr. Klotz and Ayers 32 are reporting some new developments in this area and I shall confine myself to a discussion of the interactions of serum albumin with two cations both from group 2 B of the periodic table namely zinc and mercury. The use of these two cations in the fractionation of blood plasma proteins is discussed at this meeting by Cohn Surgenor Schmid Batchelor and Isliker.36 The far-reaching developments which they are presenting speak for themselves; here I shall discuss only some recent work from our laboratory which bears on the chemical nature of the interactions involved. The tendency of metallic ions toward complex formation has been admirably surveyed by Jannik Bjerrum,37 who himself has done so much for the advance- ment of knowledge in this field.From his data it is apparent that the tendency of zinc ion to complex formation with the carboxylate ion is relatively weak (log k is approximately zero for zinc and acetate ion where k is the association constant). The association tendency with ammonia is much stronger (log k = 2-37) and this should give an approximate measure of the affinity of zinc for amino groups. However the affinity of amino groups for hydrogen ion is so strong that the zinc binding capacity at pH 7 or below is relatively small. One very important group found in proteins the irnidazole group of histidine had not been previously investigated; Gurd and Goodman undertook to study the combination of zinc ion and imidazole while Felsenfeld and I were studying its combination with cupric ion.33 Both these ions were found to bind four molecules of imidazole the successive values of log k being tentatively estimated (at 4.5") for copper as 4.58 3-76 3.14 and 2.18.For Zn2+ they were 2.76 2.38 2.40 and 2-22. These values of k for zinc ion are defined by the relation In these equations Im denotes uncharged imidazole. On correcting for the statistical factors involved it is interesting to note that although the first Cu2+ ion is bound much more strongly than the first Zn2' the binding of one imidazole JOHN T . EDSALL 21 by copper decreases thc probability of binding the next whereas the binding of the first imidazole by zinc increases the affinity for more imidazoles until saturation is achieved. While this work was proceeding Gurd and Goodman 39 also made a most careful study of the binding of zinc ion by serum albumin employing the dialysis FIG.7.-Effect of zinc ion concentration on zinc binding to human serum albumin. From Gurd and Goodman.39 Temp.= 0°C 0.15M NaNO -2.5% Albumin Q 7.5 o 5.8 9 14.0 10.0 OH- Added I / Albumin 4 I FIG. &-Dependence of n on v (see text) in the binding of zinc ions by human serum albumin. From Gurd and Goodman.33 equilibrium technique. This work required great care in setting up the experi- ments and the analysis of the data involved a quite elaborate examination of the competition between zinc and hydrogen ions for the various sites on the protein and of the influence of electrostatic forces due to the charge on the protein on the affinity constants.Some of their results are shown in fig. 7 and 8. The former SIXTH SPIERS MEMORIAL LEC'TIJRE 22 illustrates zinc binding as a function of free zinc ion concentration for various amounts of OH- ion added to the isoionic protein; while the latter shows the number n o€ imidazole groups available to combine with hydrogen ions as a function of C the number of Zn2+ ions bound per molecule of albumin. The conclusion from these studies was clear cut ; the binding of Zn2+ by albumin in the pH range 5.5 to 7.5 is due almost entirely to the 16 imidazole groups of the histidine residues. At more acid pH values very little zinc is bound and at more alkaline values the precipitation of zinc hydroxide renders experimental measurement impracticable.Moreover the association constant involved is defined by the relation log k" = 2-82 where k" denotes the association constant corrected for the influence of electro- static forces. This value agrees extraordinarily well with the first association constant in the relation between the zinc ion and the imidazole molecule (log kl = 2.76). Indeed such very close agreement could hardly be expected and is perhaps accidental. Nevertheless this value certainly adds strong confirmatory evidence for the significance of the imidazole groups in the binding of zinc by albumin. The carboxylate groups in spite of their much greater number-106 per albumin molecule-show no significant binding at least in aqueous solutions. However in the presence of concentrated urea as Cohn et al.36 are reporting at this meeting there is evidence from recent work of Dr.Gurd that carboxylate groups become available for binding. The amino groups would certainly combine at more alkaline pH values but their relatively strong basicity renders them unreactive with zinc at neutral pH. Quite independently of Gurd and Goodman Tanford 40 studied polaro- graphically the binding of zinc and several other ions by serum albumin and has come to identical conclusions concerning the significance of the imidazole groups for the binding. The mercuric ion in spite of its electronic relationship to that of zinc differs profoundly in its interactions with the groups found in proteins. Its affinity constant for amino groups is more than a million times as great as that of the Zn2+ ion; and its affinity for imidazole groups although the exact figure is not known to me is certainly far greater also.However its well-known affinity for sulphydryl groups is the dominating feature in its interactions with proteins as long as the protein has any free sulphydryl groups left to react. My colleague Dr. W. L. Hughes Jr. whose work in this area has been basic for all that the rest of us have done estimates that log k for the association of Hg2+ with a sulp- hydryl group is of the order of 17 to 19. On the other hand we must remember that the amount of free Hg2+ ion present in most aqueous solutions of a mercuric salt is extremely small because of its ex- tremely strong affinity for the hydroxyl ion and for all halogen ions except fluoride.The reversible combination of mercury with mercaptal bumin was discovered by Hughes,27 who formulated the essential features of the reaction and crystallized the mercury dimer of mercaptalbumin. Oncley by means of ultracentrifuge measurements showed the simultaneous presence of dimer and monomer in solutions derived from these crystals and obtained indications that they existed in a state of reversible equilibrium. Over a period of several years we have continued the study of the process by light-scattering measurements and have characterized in a more quantitative manner both the kinetics of the formation and dissociation of the dimer and the position of the equilibrium as a function of pH ionic strength the concentration of certain specific ions in the solution temperature and other variables.It was also discovered that certain organic mercurials containing two or more mercury atoms were also capable of linking two albumin molecules together to form a dimer. The one most thoroughly studied was the dioxane derivative introduced into the laboratory by Straessle 41 CH2-0 \CH . CH2. Hg+ +Hg . CH2. HC/ \O-CHz/ JOHN T. EDSALL ASH + HgC12 + ASHgCl + H+ + C1-. ASHgCl + ASH + ASHgSA + Hf + C1-. Kcl/RhO = M-1 + 2Bc1 111. ASHgSA + HgC12 + 2ASHgCl. The exact formulation of these reactions is open to some discussion. In the solution before reaction starts and at the concentrations used in our studies most of the mercury is in the form of undissociated HgC12.Some however must be present in combination with hydroxyl or acetate ion; some presumably as HgCl+ ion. The amount of free Hg2+ is certainly minute at equilibrium. Probably however the reactions I I1 and 111 represent the most convenient simple way of formulating the process. Dr. Hughes has demonstrated the release of hydrogen ions accompanying reactions I and 11. and for the dimer of molecular weight 2M Kc~/R$~ = (2M)-1 + 2B~2. 23 which for brevity will be denoted as HgRHg. This compound as will be seen induces dimer formation far more rapidly than mercuric chloride itself. Other organic mercurials prepared by Dr. Howard Dintzis are now being studied. This work has been carried on by several investigators in our laboratory; its progress is due to the thinking and the experimental skill of Harold Edelhoch R.B. Simpson Rudolf Straessle Ephraim Katchalski and R. H. Maybury. The insight of Dr. Hughes has been invaluable to us throughout the progress of the work. The reaction may be described as proceeding in three stages. First is the combination of mercury with mercaptalbumin (denoted here as ASH) through the sulphydryl group of the latter (1) This reaction involves no significant change of the molecular weight of the protein ; it therefore cannot be measured by light scattering and it is in any case very rapid-probably too fast to measure by most techniques. The product of the first reaction then reacts with a second molecule of mercaptalbumin to form the dimer (11) This is the reaction which is directly observed by light scattering; it proceeds very slowly as compared to reaction I and is readily followed.Finally if an excess of mercuric chloride is added the dimer rapidly dissociates according to reaction (111) The interpretation of the light-scattering measurements in order to derive weight average molecular weights also raises certain problems. For the monomer of molecular weight M we can employ the usual equation for light scattering in dilute solutions (W (V) Here c1 and c2 denote the weight concentrations of monomer and dimer respectively expressed in g/ml RJO and R30 denotes the corresponding values for the reduced intensities of scattering at 90" from monomer and dimer and K denotes the factor K = 2~2n2(dn/dc)2/NoA-f where n denotes refractive index N is Avogadro's number and ho is the wave- length of the light in vacuo (Doty and Edsall42).The factor B is the interaction constant which is determined in large measure by the net charge on the protein and the ionic strength of the solution (Edsall Edelhoch Lontie and Morrison 43). (VI) If the protein is isoelectric B is zero or nearly zero for both monomer and dimer and the weight average molecular weight can be inferred directly from the reduced intensity. However it was of great interest to study the system under conditions of varying net charge on the protein and under these conditions B is SIXTH SPIERS MEMORIAL LECTURE (VW (VIII) Using this approach we have been able to obtain extensive information con- cerning the velocity of the reaction and the proportion of dimer which is present at equilibrium.Repeated experiments have shown that true equilibrium is actually attained. In a given medium one may start either from pure monomer with an appropriate amount of mercury added or from a solution which is nearly pure dimer ; and on adjusting to the same weight concentration of protein in each case one attains the same final equilibrium value. If the activity of hydrogen and of chloride ions is fixed this equilibrium may conveniently be defined in terms of an apparent equilibrium constant (ASHg SA) 24 in generai different from zero. Fortunately it proved in practice that in a given medium the value of B for the pure mononier and the pure dimer is the same within experimental error.We have made the assumption-and it can be justified by theoretical arguments developed by Dr. Katchalski which I need not discuss here-that the same value of B can then be used for any mixture of monomer and dimer in the same solution and we can then obtain the molecular weight by extra- polation. For the mixture of the two species then we may write in fig. 9. Kc/RgO = X-1 -t ~ B c where c = c1 + c2 and X = M(c1 -+ 2c~)/c. Since c and R90 are measured experi- mentally X may then be calculated from (VIl) provided that the value of B has been determined for the pure monomer or dimer in the same solvent in which the reaction is taking place. The weight fraction of dimer is then given by the equation (ASHgCI)(ASH) Kapp == and the variation of this apparent constant with change in pH and ionic strength may then be studied.To our surprise Kapp was found to vary little over the pH range 4-25-6 the mean value being 2.6 (& 0-4) x lo4 over the whole range. The measurements were carried out in phosphate buffers at pH 6 and in acetate buffers at the lower pH values. In simple terms this value of Kapp means that in a system containing 0.5 mole Hg per mole albumin if the total weight con- centration of protein is 10 g/l. then the weight fraction of dimer at equilibrium is near 0.5. Preliminary studies of the temperature coefficient of equilibrium indi- cate that rise of temperature favours dimer formation and therefore that the process is presumably endothermic.For a given protein concentration in a given medium the weight fraction of dimer at equilibrium varies in a characteristic fashion with the amount of mercury added. It is of course zero in the absence of added mercury when all mercapt- albumin is present as ASH and falls again to zero when one mole of mercury has been added per mole of albumin all of the mercaptalbumin being then in the form ASHgCI. The maximum amount of dimer formation always occurs when the molar ratio of mercury to albumin is 0.5. The value of the weight fraction of dimer at this point increases with the value Kapp. The form of the whole curve indeed can be calculated from the value of the equilibrium constant. The curve is symmetrical about its midpoint and is similar in charactcr to the curves theoret- ically deduced by Michaelis 44 for the fraction of semiquinone formed in the re- versible titration of an oxidation-reduction system as a function of the total of oxidant or reductant added and of the semiquinone formation constant.A family of some of the curves to be expected for different values of Kapp is shown We have also made extensive determinations of the velocity constant kf of the combination reaction between ASHgCl and ASH. This constant expressed in 1. mole-1 min-1 is equal about to 40 from pH 4.25-4.75. As the pH increases JOHN T. EDSALL 25 further however the value decreases progressively to 0.69 at pH 6 . I t seems probable that this variation at pH values above 4.7 can be at least partly explained by the electrostatic effect of the increasing negative net charge on the albumin molecules above pH 5.The treatment involved here would be essentially that developed by Bronsted N. Bjerrum Scatchard and others for simpler i0ns.~5 However since the equilibrium constant is essentially independent of pH the results indicate clearly that the dissociation of dimer into monomer is far slower at pH 6 or above than below pH 5. This has indeed been confirmed experi- mentally. A concentrated solution of the dimer may be prepared and adjusted to a pH somewhat above 6 in phosphate buffer. Even if the solution is then greatly diluted the dissociation of the dimer is found to be extremely slow as 100 80 L (Y E *6 60 .c. c 0 e 0 ) 2 40 20 0 Molar Ratio HgCI,/(ASH) at t= 0 FIG.9.-Percent of mercaptalbumin-dimer formed at equilibrium as a function of HgC12/ mercaptalbumin ratio. Curves are shown for various values of the parameter Kappa (apparent association constant multiplied by the molar concentration of albumin). compared to the rate below pH 5. In this way dilute solutions containing nearly pure dimer with very little monomer can be obtained for study over a considerable period of time as for instance in ultracentrifuge runs. Studies of interaction of mercaptalbumin with the organic dioxane derivative discussed above (HgRHg) indicate a striking difference from the interactions with mercuric chloride. The equilibrium at all the pH values studied seems to lie almost completely in the direction of dimer formation.At low pH values below 4.8 the velocity of the process is so great that no accurate velocity constants for dimer formation could be determined by our methods. Between pH 5 and 6 the value of kf decreases with rising pH in much the same manner as for the re- action with mercuric chloride. However the absolute values of kf at any pH in this range are very much greater-indeed several thousand times as high as those for the reaction involving mercuric chloride. A plot of the two sets of kfvalues is given in fig. 10. The great contrast between the rates and the equilibrium constants found in these two different types of reaction with mercury compounds is presumably to 26 be explained largely on steric grounds. When two albumin molecules are linked through their sulphydryl groups by a single mercury atom the juxtaposition of the adjoining surfaces of the two protein molecules must be extraordinarily close.It is indeed remarkable that the surfaces are so shaped that they can be fitted together at all. It will be of great interest to see whether chemical modification of other groups in the albumin will lead to alterations in the tendency to undergo this reaction. In any case the relative slowness and incompleteness of the reaction are not surprising. On the other hand for the dioxane derivative HgRHg in which there is a mercury atom at each end there is an effective distance of the order of lOA between the two mercury atoms. Thus the coupling of the two albumin molecules which are linked by these atoms is far looser than when a single mercury serves as the link.The conditions for the combination are much less critical and pre- sumably the reaction is possible in a much greater variety of relative orientations t PH SIXTH SPIERS MEMORIAL LECTURE 4; 6 A S Hq RHq SA \ A5 Hq SA - 5;8 5 4 log K f 10 I :2 5 0 FIG. IO.-Logarithm of the velocity constant kf for dimer formation of human Serum mercaptalbumin with mercurials. Lower curve reaction with mercuric chloride ; upper curve reaction with the dioxane derivative containing two mercury atoms. Phosphate buffers were employed near pH 6 acetate buffers at lower pH values; ionic strength 0.05 in acetate buffers; 0.05-0.15 in phosphate; temp. 25". (From studies of E.Katchalski H. Edelhoch and R. B. Simpson.) of the two albumin molecules than would be the case if the fitting were closer. Thus the reaction goes relatively rapidly and it goes largely to completion. One may suspect although this is not proved that the two halves of the dirner ASHgRHgSA may be much more loosely coupled than the halves of the dimer ASHgSA. Thus in the former case a much greater degree of free rotation of one-half of the dimer relative to the other should perhaps be expected than in the latter; a possibility which may perhaps be explored by such methods as dielectric dispersion. It should be added that in spite of the presumably very close fitting of the two halves of the dimer ASHgSA the mercury atom does not appear to be buried within the protein in such a way as to be inaccessible to the solvent.Addition of other ions such as bromide iodide or cyanide which have a strong affinity for mercury leads to quite rapid dissociation of the dimer. Evidently these ions can penetrate readily to the mercury atom which is then released from its com- bination from one of the sulphydryl groups to form the monomer ASHgBr ASHgI or ASHgCN. The reaction has been studied by us particularly with JOHN T. EDSALL 282 729. 3 Bragg Howells and Perutz Acta Cryst. 1952 5 136. 4Kendrew Proc. Roy. SOC. A 1950 201 62. 5 Carlisle and Scouloudi Proc. Roy. SOC. A 1951 207 496. 6 Low Nature 1952 169 955. 7 Hocking Laskowski and Scheraga J. .4mer. Chem. SOC. 1952 74 775. 8 Katz Gutfreund Shulman and Ferry J.Amer. Chem. SOC. 1952 74 5706. Bettelheim and Bailey ibid. 9 578. 14 Lorand Nature 1951 167 992. 15 Edsall and Lever J. Biol. Chem. 1951 191 735. 16 Ferry and Morrison J. Amer. Chem. SOC. 1947 69 388. 17 Laki and Lorand Science 1948 108 280. 18 Lorand Nature 1950 166 694. 19 Shulman this Discussion. 27 the iodide ion. The addition of even a single mole of iodide per mole of mercury in the dimer leads to a relatively rapid and very marked dissociation of the dimer. Bromide is inuch less effective and chloride far less than bromide. Indeed these studies appear to be significant as a method of evaluating the relative affinity for mercury of various inorganic ions and thus obtaining data which should be of interest for inorganic chemistry.This is of course not our primary concern; we are interested in these reactions chiefly because of what we can learn from them concerning the external form the surface properties and re- activity and perhaps something of the finer pattern of the configuration of the albumin molecule. The extensive studies carried out by Lewin 46 in our laboratory demonstrated that mercaptalbumin mercury dimer could be crystallized in association with many other metallic cations and also with anions containing atoms of high atomic number. Dimer formation of the type first observed by Hughes27 with serum albumin has not yet apparently been observed with other proteins. Several years earlier however the successful use of mercury in the crystallization of enolase was reported by Warburg and Christian.47 In this case the crystallized mercury derivative contained just one mole of mercury per mole of enolase and other derivatives have not been found.The interaction of mercury with proteins is of course not confined to a single group. However the affinity of mercury for the sulphydryl group is so much greater than for any other groups found in proteins that no other reaction will occur until all such groups are saturated with mercury. Beyond this point however important interactions begin to occur with imidazole amino and other groups. These interactions between mercury and proteins have recently been exploited with great success by Dr. Cohn and his associates in fractionation processes .36 1 Sanger and Tuppy Biochem.J. 1951,49,463,481. Sanger Thompson and Tuppy Symposiiiin sur les Hormones Proteines et Derivtes des Proteines (Paris 1952). 2 Pauling and Corey Proc. Nut. Acad. Sci. 1951 37 205 235 241 251 261 272 9 Edsall Foster and Scheinberg J. Amer. Chem. SOC. 1947 69 273. 10 Hall J. Biol. Chem. 1949 179 857. 11 Porter and Hawn J. Expt. Meci. 1949 90 225. 12 Mitchell Biochem. Bioplzys. Acta 1952 9 430. 13 Bailey Bettelheim Lorand and Middlebrook Nature 1951 167 233 ; Bettelheim and Bailey ResumL des Communications I P Congres Internationale de Biochemie 1952 411; Lorand and Middlebrook Biochem. Biophys. Acta 1952 9 581; 20 van Vunakis and Brand Abstr. 119th meeting Amer. Chem. SOC. April 1951 p. 28c. 21 Weber this Discussion. 22 Scatchard Batchelder and Brown J.Amer. Chem. SOC. 1946 68 2320. 23 Low J. Amer. Chem. SOC. 1952 74 4830. 24 Creeth Biochem. J. 1952 51 10. 25 Oncley Scatchard and Brown J. Physic. Chem. 1947 51 184. 28 SIXTH SPIERS MEMORIAL LECTURE 25a Oncley Dintzis and Hollies Abstr. 122nd meetirrg Amer. Chem. SOC. Sept. 1952 p. 12p; Dintzis Thesis Harvard University 1952. 26 Weber Biochenr. J . 1952 51 145 155. Biol. 1950 14 79. 27 Hughes J. Amer. Chem. Sac. 1947 69 1836 ; CoM Spring Harbor Symp. Quant. 28 Edsall and Foster J. Amer. Chem. SOC. 1948 70 1860. 29 Riley and Oster Furuday SOC. Discussions 1951 11 107. 30 Anderegg Beeman and Shulman paper presented to Amer. Physic. SOC. April 1952 ; and unpublished work. 31 Oncley Chem. Rev. 1942 30 433. 32 Klotz and Ayers this Discussion 33 St.George and Pauling Science 1951 114 629. 34 Wyman and Allen J. Polymer Sci. 1951 7 499. 35 Massey Biochem. J. 1952 51 490. 36 Cohn Surgenor Schmid Batchelor Isliker and Alameri this Discussion. 37 Bjerrum Chem. Rev. 1950 46 38 1. 38 Gurd Edsall Felsenfeld and Goodman Fed. Proc. 1952 11 224. 39 Gurd and Goodman J. Amer. Chem. SOC. 1952,74 670. 40 Tanford J. Amer. Chem. SOC. 1952 74 21 1. 41 Straessle J. Amer. Chem. SOC. 1951 73 504. 42 Doty and Edsall Advances in Protein Chem. 1951 6 35. 43 Edsall Edelhoch Lontie and Morrison J. Amer. Chem. SOC. 1950 72 4641. 44 Michaelis Chem. Rev. 1935 16 243 ; see especially fig. 4. 45 see for instance the discussion by Laidler Chemical Kinetics (McGraw Hill Pub- lishing Co. New York 1950) p.123. 46 Lewin J. Amer. Chem. SOC. 1951 73 3906. 47 Warburg and Christian Biochem. Z. 1941 310 384. THE PHYSICAL CHEMISTRY OF PROTEINS SIXTH SPIERS MEMORIAL LECTURE THE MOLECULAR SHAPES OF CERTAIN PROTEINS AND SOME OF THEIR INTERACTIONS WITH OTHER SUBSTANCES BY JOHN T. EDSALL" University Laboratory of Physical Chemistry Related to Medicine and Public Health Harvard University Boston Mass. Received 30th September 1952 The Discussions of the Faraday Society have developed in recent years into events of international significance for many scientists. To-day as in a number of the previous Discussions the focus of interest is on the great border area between the provinces of physics chemistry and biology. In those earlier Discussions many ideas of extraordinary fruitfulness were presented and vigorously debated before they had become familiar and respectable.I have often turned back for illumination to the printed record of their proceedings. Three years ago I had for the first time the opportunity to take part in one of these Discussions-that on Lipoproteins at Birmingham. Hence I feel deeply the honour of giving the Sixth Spiers Memorial Lecture. It was never my good fortune to know Mr. Spiers personally but it was he especially who guided the pattern of this Society's development from small beginnings until it grew into an active and significant organization and its discussion meetings became as they are to-day the focal point for investigators from every land where science is free and active. At that time although the Faraday Society had held many conferences of international importance on the border line of physical chemistry and other sciences it is scarcely likely that it could have contemplated a two-day discussion on the physical chemistry of the proteins.It is doubtful indeed whether in 1926 more than half a dozen laboratories throughout the world could have made effective contributions to such a discussion. L can well remember that year for in the midst of it I returned to Harvard after two years of study here in Cambridge where I had imbibed the broad vision of the scope and the future of biochemistry that was to be found in Cambridge in Hopkins's laboratory. Also I had had the good fortune to know that great pioneer in the physical chemistry of the proteins Sir William Hardy then no longer active in that field but still with a freshness of mind a vitality and zest in life that I have seldom seen matched in other men.The view was indeed held by some distinguished chemists that proteins were actually very small molecules with molecular weights below 1000. This theory was short-lived being quite demolished late in 1926 by a few well-chosen freezing-point * Fulbright Visiting Lecturer University of Cambridge 1952. The untimely death of Mr. Spiers occurred in 1926. In those days we scarcely knew the molecular weight of a single protein. A 10 SIXTH SPIERS MEMORIAL LECTURE measurements by Cohn and Conant. Sorensen indeed had carried out a decade earlier his classical investigations on egg albumin and his careful studies of osmotic pressure had given the basic information for the determination of its molecular size.Adair had propounded in 1925 the startling view that the molecular weight of mammalian haemoglobin was near 67,000 and that the molecule therefore contained four iron atoms. I can well remember the profound scepticism with which this report was received by more than one professor of great eminence; and the doubts persisted in high quarters until the ultracentrifugal measurements of Svedberg a year or two later gave complete support to Adair’s conclusions. The ultracentrifuge had indeed been under development since about 1922 but there were few at that time who anticipated its immense and revolutionary influence on protein chemistry. If relatively little was known of the sizes of protein molecules almost nothing was known about their shapes; indeed the problem of molecular shape was scarcely envisaged at that time by protein chemists.The air was still filled with the strife of the conflict between Jacques Loeb and the colloid chemists of the school of Wolfgang Ostwald. Loeb’s dzmonstration that some of the colloidal properties of proteins could be explained on simple chemical principles a demon-stration expounded brilliantly and with a fighting spirit appeared to many besides myself as if it were a new revelation. To-day I still appreciate the value of Loeb’s contribution. but I am also aware that T. B. Osborne in New Haven, many years before had been quietly carrying on his work on the plant proteins, treating them as chemical substances to be understood on general chemical principles and paying no attention to the clamour of conflicting schools.Also there was one idea expressed by distinguished protein chemists as late as twenty years ago that proteins furnished essentially the inert structural frame-work of the animal organism while the active pattern of biochemical trans-formations was determined by the interplay of other molecules of quite a different sort. Certainly this view was never upheld in the laboratory headed by Edwin Cohn. Nevertheless the fact that such a belief could have prevailed among many biochemists even for a few years seems astonishing to-day; and indeed it represented the denial of a much older tradition going back over a century to Liebig and Mulder which held that proteins were indeed of absolutely prime importance to the living organism.To Mulder in 1840 this must have been largely a matter of faith and intuition ; but now in the last quarter century we have seen that faith abundantly justified by works. I have tried to recall here something of the temper of the times the prevailing atmosphere of thought among the relatively small band of protein chemists in 1926 when I came back from Cambridge to Harvard as a third year medical student and started working in my spare time under the direction of Edwin Cohn on a viscous and peculiar globulin from skeletal muscle continuing work which had been begun by W. T. Salter. Later we called this globulin myosin and still later it was rechristened by Szent-Gyorgyi as a form of actomyosin.It proved a most refractory and difficult substance to work with but in the midst of our struggles Alexander von Muralt arrived from Switzerland and introduced the measurement of double refraction into the laboratory. His brilliant contributions, both to experiment and to thinking brought us face to face with the problem of molecular shape; from then on we knew that although many proteins might be more or less globular some-and these indeed of supreme importance-were long and filamentous even in solution. In these casual remarks I shall not try to review the intervening years with the colossal advances that they have seen. Protein chemistry which was a rela-tively unfashionable subject a quarter of a century ago has now become almost embarrassingly fashionable.It does not seem likely to go out of fashion unless mankind decides to turn away from science and repudiate it altogether-a possible but I still hope a relatively improbable event JOHN T . EDSALL 11 However of all the advances in this quarter century perhaps none will stand out more than two that have cccurred in the last two years-the elucidation of the complete sequence of amino acid residues in the A and B chains of insulin by Sanger Tuppy and Thompson,l and the detailed formulation of spatial con-figurations of the polypeptide chain by Pauling and Corey.2 The aspects of protein chemistry in which these developments are included will not be dealt with by other authors in the present Discussion but these achievements are bound to influence the thinking of all of us so profoundly that I cannot leave them without mention here.We cannot yet write a structural formula for insulin until the arrangement of the disulphide bridges is specified but that the goal has now been so nearly approached is an achievement which would have seemed incredible even five years ago. Protein chemists will now really begin to think for the first time in terms of structural organic chemistry and all of us here who are physical chemists will be permeated more and more by that influence. However in protein chemistry more than anywhere else structural chemistry is of little use without stereochemistry. Proteins with their marvellously specific patterns are the supreme examples of molecillar geometry. The work of Pauling and Corey is neither the beginning nor the end of the deciphering of these patterns; their achievement is built on the work of many and brilliant predecessors and what they have done is only a beginning.Their work still has its sharp critics; but I have been led more and more to the conclusion that their a-helix does quite closely represent the spatial pattern of long chain synthetic polyamino acids and not improbably also that of the fibrous proteins which are found in the cc configuration. Also-though there is less concrete evidence for this view-the pleated sheets which they have proposed appear to be by far the best existing models for the extended pattern of protein chains in the ,B configuration. In several crystals of corpuscular proteins the presence of intramolecular bundles of parallel chains has been inferred from the analysis of X-ray data.In the case of haemoglobin Bragg Howells and Perutz 3 inferred that these chains were polypeptide chains in the cc configuration. Well-defined chains were in-ferred by Kendrew 4 in the myoglobin molecule. The study of crystalline ribo-nuclease 5 has also suggested the presence of chains parallel to the c-axis of the crystal but the configuration of these chains seems still to be a matter for dispute. Some of the most striking evidence for chain structures in a protein crystal comes from the recent work of my colleague Barbara W. Low at Harvard on the ortho-rhombic crystals of acid insulin sulphate,6 originally prepared in our laboratory by Dr. Eric Ellenbogen working with Prof.Oncley. The three-dimensional Patterson diagrams of these crystals in the air-dried form show a most striking pattern of parallel rods with axes parallel to the a-axis of the unit cell which has a length of approximately 45 A. Dr. Low has constructed possible packing models for the polypeptide chains in this crystal taking account of Sanger’s findings on the sequence of amino acid residues. I should like to turn now from these brief remarks on protein structure at the deeper level to some of the problems related to the general size shape and inter-actions of proteins. I shall confine myself to two proteins with which I have become particularly familiar in recent years fibrinogen and serum albumin, FIBRINOGEN SIZE AND SHAPE Fibrinogen occupies a place apart among the plasma proteins because of its high molecular asymmetry and its low solubility the latter property rendering it the first major constituent of plasma to be precipitated in almost any system of fractionation.Its high viscosity its marked flow birefringence its high frictional ratio and its significant angular dissymmetry of light scattering all mark it out as a molecule of shape very far from spherical. There are now data available on both human and bovine fibrinogen in solution which are too numerous t 12 S I X T H SPIERS MEMORIAL LECTURE discuss here in detail. They are summarized in table 1 and appear to indicate that human and bovine fibrinogen are remarkably similar in size and shape. The molecular weight indeed appears at present more uncertain than the absolute dimensions-many workers have accepted a value of 450,000-500,000 but the TABLE 1 .-PHYSICAL CONSTANTS OF HUMAN AND BOVINE FIBRINOGEN numerical values references sedimentation constant S ~ O ~ (Svedberg partial specific volume V20 intrinsic viscosity Eio rotary diffusion constant 0 2 0 (sec-1) (from molecular weight (in thousands) units) double refraction of flow) osmotic pressure (Mo) sedimentation and viscosity (Mg) light scattering molecular length (A) double refraction of flow osmotic pressure and viscosity sedimentation and viscosity light scattering molecular diameter (A) human 8.5-9 9 0.725 0.25 35,000 580 400 440 700 700 38 --bovine 8.4-8.6 7.9-8.2 0-706 0.25 39,400 440 540" 407 340 670 725 840" 38 --human H OSB ABMH OSB EFS OSB ML ---EFS OSB OSB --bovine KP SF K N HLS N SL HLS KGSF HLS N SL HLS --NoTEs.-T~~s table is also published in BZood Cells and Plasma Proteins ( J .L. Tullis ed. Academic Press New York 1953 in press), G. S. Adair obtained a molecular weight from osmotic pressure near 500,000 (species not recorded) as reported by Bailey Advances in Protein Chem. 1 308 (1944). Holmberg reports a translational diffusion constant for human fibrinogen which gives a molecular weight near 700,000 if taken with sedimentation constants given above. A new investigation of the diffusion constant would be desirable. ABMH = Armstrong Budka Morrison and Hasson (J.Amer. Ch~m. SOC. 1947,69,1747). EFS = Edsall Foster and Scheinberg (J. Amer. Chem. Soc. 1947 69 2731). HLS = Hocking Laskowski and Scheraga (J. Amer. Chem. SOC. 1952 74 775). KGSF = Katz Gutfreund Shulman Ferry J. Amer. Chem. SOC. 1952 74 5706. KP = Koenig and Pedersen (Arch. Biochem. 1950 25 97). ML = Morrison and Lontie (unpublished measurements from this laboratory). OSB = Oncley Scatchard and Brown (J. Physic. Chem. 1947 51 184). H = Holmberg (Arkiv. Kemi Min. Geol. A 1944 17 no. 28). K = Koenig (Arch. Biochem. 1950 25 241). N = Nanninga (Arch. neerland. physiol. 1946 28 241). SF = Shulman and Ferry (unpublished). SL = Steiner and Laki (Arch. Biochem. Biophys. 1951 34 24). recent work of Hocking Laskowski and Scheraga 7 points to a value near 400,000 ; and very recently Katz Gutfreund Shulman and Ferry,8 in what appears to be a particularly careful study of light scattering have obtained a value of only 340,000 for bovine fibrinogen.From the angular dissymmetry of the scattering they also calculated a length of 520A for the molecule if it were assumed to be a rod, or of 650A if it be taken as an ellipsoid of revolution. This marked difference shows how sensitive the calculated absolute dimensions are to the particular * Hocking Laskowski and Scheraga believe this value to be too high because of the form of the angular dissymmetry curves obtained JOHN T . EDSALL 13 type of model assumed-a fact which if carefully borne in mind would help to obviate some of the disputes about molecular dimensions that now and then arise.Using the ellipsoidal model Foster Scheinberg and I 9 calculated a length near 700w for human fibrinogen and Hocking Laskowski and Scheraga7 more recently obtained a value of 670w for bovine fibrinogen. Both of these values, obtained from flow birefringence measurements are in very satisfactory agree-ment with the light scattering value. Moreover the angular dissymmetry of scattering taken in conjunction with the order of magnitude of the molecular weight seems to be compatible only with a prolate or rod-shaped molecule; an oblate type of structure showing such high dissymmetry should have a much higher molecular weight. The dimensions of the fibrinogen molecule have aIso been studied by the electron microscopists. Hall 10 observed rod-shaped structures of varying length, the most commonly found length being near 700A as might have been expected from the measurements in solution.The rods appeared to be built up of sub-units so that they looked a little like a string of beads tightly bound together. Except for the marked heterogeneity of the size of the molecules this was quite compatible with the studies on fibrinogen in solution. However to make the problem more complicated other electron microscopists have reported quite different findings. Porter and Hawn 11 reported particles which they believed to be disc shaped about 200w across from bovine fibrinogen; and Mitchell12 found only globular units about 50 in diameter and short filaments 100-300 A long in preparations from human fibrinogen. How to interpret these conflicting findings I do not know ; I must confess to a strong prejudice that Hall’s findings are closest to the truth and in any case the high asymmetry and rodlike shape of fibrinogen in solution seem well established whatever may happen to it in the drying process that is necessary before it can be examined in the electron microscope.FIBRINOGEN AND FIBRIN INTERACTIONS The supremely characteristic property of fibrinogen is its interaction with thrombin to give an altered molecule which is capable of association with other like molecules to give the fibrin clot. Bailey and Bettelheim in Cambridge,l3 and Lorand and Middlebrook in Leeds,l4 have done much to eludicate the process. The transformation certainly involves the splitting-off of a peptide (fibrinopeptide) from the fibrinogen molecule and the unmasking of free terminal a-amino groups of glycine in the resulting reactive fibrinogen derivative from which the peptide has been dissociated.The subsequent association processes culminating in the fully developed fibrin clot have been studied in many ways. Here I shall only speak briefly of some influences that are exerted on the proccss by the very strong interactions of certain ions and neutral molecules with fibrinogen or fibrin or both. These effects were observed by Dr. Walter Lever and myseIf,ls studying the rate of clot formation from fibrinogen and thrombin by direct weighing of the fibrin formed, and examining the general structure of the clot by measuring its turbidity. Ferry and Morrison,ls in the fundamental studies they had carried out in our laboratory during the war had already defined two extreme types of fibrin clots; the coarse type formed at low pH and low ionic strength which is opaque and synerizes readily under pressure and the fine type relatively transparent and very friable, which forms at high pH or high ionic strength.Lever and I carried out our studies, for the most part at constant pH and at a fixed ionic strength of 0.15. Even when these factors were fixed many ions and neutral molecules were found to exert profound and specific effects upon the process. Notable among these were urea and guanidine hydrochloride long of course well known as denatur-ing agents for proteins. In this system however these reagents were studied a 14 SIXTH SPIERS MEMORIAL LECTURE concentrations far below those ever reported to produce denaturation.Fig. 1 shows the effects of urea on the rate of fibrin formation. It will be seen that even at a concentration of 0.1 M the rate of fibrin formation in urea solution is distinctly lower than in the control solution without urea. At 0.5 M the decrease in the rate of fibrin formation is profound. Moreover the presence of urea affected the structure of the clots as well as the rate of their formation ; invariably the clots formed in the presence of urea were less turbid and more friable than the controls. It has become well known from the work of Laki and L0rand~17.18 that urea at much higher concentrations (above 3 M) actually dissolves fibrin clots formed from purified fibrinogen.It seems probable that this is due to its strong capacity for hydrogen-bond formation so that the hydrogen bonds between urea and the protein displace the protein-protein bonds that normally link the protein molecules together into the fibrin clot. Our measurements at very low urea concentrations, which were carried out in 1946-47 before the work of Laki and Lorand was 0 I 2 3 4 24 Time in Hours FIG. 1.-Effect of urea on the rate of fibrin formation from human fibrinogen (fraction I) and thrombin. Thrombin concentration 0.1 unitlml. All solutions contained 0.1 5 M sodium chloride at pH 6.3. The top curve is for a solution containing no urea; the others are for solutjons containing the amounts indicated on the diagram. From Edsall and Lever.15 published show the same effect in incipient form.The formation of highly turbid clots of the coarse type is the result of extensive lateral aggregation of the first thin strands developing in the early stages of clot formation.16 Increase of pH increases the negative net charge on the fibrin molecules and thus tends to decrease lateral association by electrostatic repulsion. Addition of urea blocks lateral association by hydrogen-bond formation between urea and protein leading to the same result. In either case the clot becomes more translucent and friable. Like urea indeed it greatly retards the rate of formation of fibrin even at 0.05 M there is a very marked retardation and at 0.075 M the effect is profound (fig. 2). However the effect on the structure of the clot is in the opposite direction from that of urea; as the structure becomes fully developed it becomes markedly more coarse and turbid than the control clot; indeed the pattern of development of the clot both as regards increase of turbidity and rate of fibrin formation is nearly the same for a control clot at pH 6-3 and for a clot at pH 7.3 which contains guanidine hydro-chloride at a concentration of 0-075 M at least in the earlier stages of the clotting process (fig.2 and 3). Adding guanidine hydrochloride at this concentration thus has nearly the same effect as lowering the pH by one unit ; the initial rate of fibrin The effect of guanidine hydrochloride is very different JOHN T . EDSALL 15 formation is greatly retarded and the final turbidity of the fully developed clot is greatly increased.The results suggest that the positively charged guanidinium ion FIG. 2.-Effect of guanidine hydrochloride on rate of fibrin formation at pH 6.3 and 7-3. Total ionic strength in all solutions was 0.15. pH was controlled by imidazole buffers the concentration of imidazole hydrochloride being 0.01 5 in ail experiments, except in the top curve where it was 0.03. 0 sodium chloride (0.12 M) plus imidazole buffer at pH 7.3. @ 0.075 M guanidine hydrochloride (C(NH&+Cl-) ; 0.06 M sodium chloride ; pH 7.3. 0 0.135 M NaCl; pH 6.3. 0 0-075 M C(NH&+Cl- ; 0-06 M NaCl ; pH 6.3, 0 0.05 M C(NH&+CI- ; 0.085 M NaCl ; pH 6.3. (From measurements of Walter F. Lever.) 0.8 0-6 0.4 0.2 Time in Hours FIG. 3.-Effect of guanidine hydrochloride on turbidity of fibrin clots at pH 6.3 and 7.3.The solutions had the same composition as those described in fig. 2 and corresponding curves are denoted by the same symbols in both figures. Note the high turbidity of the solution containing 0-075 M C(NH&+C1- at pH 7.3 as compared to the extremely low turbidity of the control solution containing only sodium chloride at the same pH. (From measurements of Walter F. Lever.) combines preferentially with the protein decreasing its negative net charge just as hydrogen ion would do. Quantitative studies of the binding of guanidinium ion t 16 SIXTH S P I E R S MEMORlAL LECTUKE fibrinogen and fibrin have not been carried out but these results suggest that the binding would be strong. .' K'I- pH 6*50,,+ :--0 P / / / 1 K+SCN- pH 6 - 2 5 ' */o/@--o- / -0 Time in Hours I / "0 I ,4 5 2 4, * FIG.4.-Effect of iodide and thiocyanate on rate of fibrin formation from human fibrinogen and thrombin. Ionic strength in all solutions was 0.15. Top curve 0.15 M NaC1; middle curve 0.135 M KI + 0.015 M NaCI; bottom curve 0.135 M KSCN + 0.015 M NaCl. Note that the pH in the iodide solution was significantly higher than in the other two. This increased pH accelerated the reaction; thus the rate of fibrin formation in the iodide solution would have been lower at lower pH. Control experi-ments showed that the effect of potassium salts was indistinguishable from that of sodium salts with the same anion with respect to rate of fibrin formation and clot turbidity.(From measurements of Walter F. Lever.) -0.8 Y A7 0 J I 2 3 9 Time in Hours FIG. 5.-Effect of iodide and thiocyanate on turbidity of fibrin clots as a function of time. Molar concentrations of salts in these experiments were identical with those denoted by corresponding symbols in fig. 4. (From measurements by Walter F. Lever.) * Acknowledgments are made to the Josiah Macy Jr. Foundation for permission to publish fig. 4 and 5 which are slightly modified versions of those appearing in Blood Clotting rmd Allied Psohltwis. Trmsnctions of' the Forirtk CorlJ;.renre ( I95 I) pp. 241-2 JOHN T . EDSALL 17 Certain anions such as iodide and thiocyanate-also well known as denaturing agents for many proteins at much higher concentrations-act in the opposite direction on the structure of the clot greatly decreasing its turbidity as compared with that of the control at the same pH and ionic strength (fig.4 and 5). In these cases therefore there is suggestive evidence of selective anion binding. It should be noted that in all cases these reagents diminish the initial rate of fibrin formation, as shown in fig. 2 and 4 whatever their effect on the structure of the clot. In no case however was there any destruction of thrombin by the added reagent as control experiments showed so that the decreased rate of reaction cannot be laid to this. Closely related to these studies are the extensive and important studies of J. D. Ferry and Sidney Shulman on the effects of many reagents in inhibiting the conversion of fibrinogen to fibrin.These have been presented by Dr. Shulman 19 in a paper to be discussed at this conference so that I shall not treat them further here. SERUM ALBUMINS SIZE AND SHAPE The serum albumins have been among the most intensively studied of all pro-teins ; yet even the size and general shape of the albumin molecule are still a matter of dispute. Let us consider some of the evidence. (i) By the use of dinitrofluorobenzene van Vunakis and Brand 20 could find only one free terminal a-amino group in human horse or bovine serum albumin per molecule of molecular weight approximately 70,000. In all three cases this amino group belonged to an aspartic acid (or asparagine) residue. Thus it would appear that the molecule consists of a single peptide chain. Yet Weber,21 in the paper he is presenting to this meeting has concluded from polarization fluorescence studies that serum albumin dissociates into smaller units in both acid and alkaline solution.There is no necessary incompatibility between these findings but inevitably they raise certain questions. Does the splitting of the albumin molecule involve the breaking of some exceptionally labile peptide linkages? If so what brings about the reassociation that Weber has found to occur on neutralizing the acid albumin solution? Or on the other hand are there actually two or more peptide chains within the albumin molecule only one of which-presumably for steric reasons-reacts with dinitrofluorobenzene? Or is it possible that Weber's findings can be explained in terms of strong electro-static interactions between molecules carrying a high net charge without actually having to assume molecular dissociation? The latter possibility seems to me relatively unlikely but it should not be altogether disregarded.(ii) The molecular weight of human and bovine serum albumin has generally been taken in recent years as 69,000 chiefly on the basis of the very careful osmotic pressure studies of Scatchard Batchelder and Brown.22 The recent X-ray measurements on albumin crystals in our laboratory by Barbara LOW,^^ combined with accurate determinations of density and composition definitely indicate a lower molecular weight near 65,500. Quite independently Creeth 24 has deduced an almost identical value for bovine serum albumin from very careful sedimentation and diffusion measurements.There is converging evidence indeed from several laboratories both in England and the United States that some of the generally accepted measurements of sedimentation constants are in fact too high and require some downward revision-a process which inevitably involves a downward re-vision of molecular weights as well. (iii) In 1947 Oncley Scatchard and Brown25 gave a detailed discussion of sedimentation viscosity and other data for serum albumin and-assuming 20 % hydration-described the molecule approximately as a prolate ellipsoid about I50 A long and 38 A in diameter at the middle. Their choicc of a prolate rathe 18 SIXTH SPIERS MEMORIAL LECTURE than an oblate shape to describe the molecule was determined largely by Oncley’s dielectric dispersion data which indicated two distinctly different relaxation times -a finding incompatible with an oblate shape if the preparation consists of molecules which are all alike.Recently Oncley Dintzis and Hollies 25a have obtained dielectric measurements of much higher precision on carefully purified human mercaptalbumin and other serum albumin preparations freed from fatty acid and other impurities by treatment with ion exchange resins. These prepara-tions gave considerably higher dipole moments than earlier less pure samples. The form of the dielectric dispersion curve was quite sensitive to the protein con-centration ; but at sufficiently low concentrations the curve was entirely consistent with that to be expected for a prolate ellipsoid of molar volume 60 I.and an axial ratio of 4 to 1. The studies of Weber 26 on the polarization of fluorescence from albumin derivatives, also give important evidence on this point and on the whole Weber’s findings appear to confirm those of Oncley. No oblate ellipsoidal mode1 could be chosen to fit the data. t l C 0 0 2 0 -1 L FIG. 6.-Model for possible modes of packing of human serum albumin in the crystal of mercaptalbumin mercury dimer (a wet crystal; b and c air dried crystals). All drawings represent the c plane projection. Two different modifications of Oncley’s model for the serum albumin molecule are indicated. From Low.23 (iv) As was discovered by Hughes,27 two molecules of human mercaptalbumin are readily linked by means of their sulphydryl groups through a single mercury atom to form a dimer.Oncley was led to reconsider his earlier model for the albumin molecule in order to account for the steric relations involved in the forma-tion of this dimer. This revised model has been employed by Low 23 to describe the packing of serum albumin molecules in the crystal of mercaptalbumin mercury dimer. The model consists of a prism approximately 145 A long 45A in dia-meter at the middle and 22A thick. Such a model can fit well into the unit cell of the crystal as determined by Low; and the indications of a layer structure which are given by the X-ray data are well accounted for by the piling into layers of such long thin molecules (fig. 6). It should be clearly understood that the X-ray data do not prove the structure but they are apparently compatible with it and the model itself-to be taken of course only as a very rough approximation -seems reasonably plausible on other grounds.(v) As was found by Foster and myself,2* human serum albumin shows flow birefringence although only at very high gradients in solvents of high viscosity. From our measurements we estimated a length at least as great as that of l50& inferred by Oncley on other grounds. However these measurements on so relatively short a molecule were exceedingly difficult and subject to considerabl JOHN T . EDSALL 19 error ; they should not be taken as having precise quantitative significance. They do point however to a somewhat asymmetrical shape. (vi) In contrast to all these conclusions Riley and Oster 29 have inferred from low angle X-ray diffraction studies on very concentrated albumin solutions that the shape of the molecules cannot be very far from spherical.The theory under-lying this deduction is one which I do not feel competent to criticize ; but I feel very reluctant to accept the conclusion itself in view of the other evidence summarized above. (vii) Very recently Anderegg Beeman and Shulman30 have carried out low angle X-ray scattering studies on relatively dilute albumin solutions. Here the interpretation of the data is more straightforward than for the very concentrated systems studied by Riley and Oster. At vzry low angles the scattering curves indicate that the radius of gyration is definitely greater than for a sphere of the same molecular weight while the scattering at somewhat higher angles which is more characteristic of shape appears to be in better accord with that expected for an oblate than for a prolate ellipsoid of revolution.Assuming an oblate ellipsoid the ratio of the molecular axes is found to be near 3-5 to 1. These data must I think be taken very seriously; we cannot evade the attempt to find some explanation that will make them understandable in conjunction with all the other findings that seem to point to an elongated shape. Dr. Beeman has remarked, in a recent letter to me on the subject “ We can say from our data that we are much more convinced that the molecule is not a prolate ellipsoid of revolution than that it is an oblate ellipsoid of revolution.” Indeed the major moral of all this may be that we should not take too seriously calculations based upon simple shapes.They are useful for representing the results of particular researches, and for correlating the properties of proteins derived from hydrodynamic measure-ments but in detail the description of the shapes of protein molecules must involve something more complex than the very simple geometrical figures that are con-veniently used for preliminary descriptions. It would be a feasible though certainly a very tedious task to calculate the scattering function for the prismatic model of serum albumin previously discussed; and the curve so derived might be quite different from that of a prolate ellipsoid ; but the case seems a little too special at present to warrant the work involved. It does seem to me that the weight of the evidence does rather favour a general model of this sort and that an oblate ellipsoidal shape for albumin or anything very close to such a shape is unlikely.However I do not wish to be dogmatic on the subject ; what I hope to do in this lecture is to ask more questions than I can answer and I trust that others will come forward with the answers or will set to work to find them. ON THE POSSIBLE FLEXIBILITY OF PROTEIN MOLECULES In contrast to the arrangement of most synthetic polymers which coil about in random fashion in solution proteins have a highly ordered pattern. The evidence for this order is clearest from X-ray studies on protein crystals. Such order also implies a high degree of molecular rigidity which is shown clearly for proteins in solution by the dielectric studies of Oncley.31 Protein molecules orient as entire units in an electric field not as a series of more or less independent segments as do the polar polymers.All this is true and important ; yet we should certainly not assume that this rigidity is strict and absolute. Indeed Klotz and Ayers,32 in a paper presented at this meeting conclude that the molecule of serum albumin must be able to change its configuration with pH ; that it may swell or unfold somewhat as its net charge increases and that these efTects are reversible. Others I think have made similar suggestions before. Likewise the remarkable interactions involved in the combination of haemoglobin with oxygen have led Pauling,33 and also Wyman and Allen,34 to suggest that the addition of oxyge 20 SIXTH SPIERS MEMORIAL LECTURE actually leads to a rather fundamental change in the configuration of the mole-cule as a whole.Pauling and Wyman hold rather different views as to the nature of this change and we certainly need more proof before we assume that the change really occurs but the suggestive evidence is strong. Even more striking I think are some recent unpublished studies here in Cambridge on the enzyme fumarase from pig heart recently crystallized by Vincent Massey 35 working with Dr. Malcolm Dixon. Combined observations by Massey on enzyme activity and by Weber using polarization of fluorescence, suggest alterations of molecular size or shape or both with varying pH and ionic strength ; and these variations appear closely correlated with changes in enzyme activity and the formation of the enzyme-substrate complex.We have long been accustomed to change of shape on the part of the fibrous proteins as in the a-/3 transformation of keratin. Indeed the myosin system of muscle could not do its job unless it changed its shape radically and rapidly. Alterations of shape among the corpuscular proteins are presumably much less drastic-that is if they occur under conditions in which the protein still remains undenatured-but they may be of profound importance both chemically and biologically. The fluorescence polarization technique of Dr. Weber may be of great value in such studies for unlike the dielectric dispersion method it can be applied over a very wide range of pH and salt concentration to give information about the relaxation times of proteins.SERUM ALBUMINS INTERACTlONS WITH CERTAIN CATIONS The serum albumins are famous for the strength and diversity of their inter-actions with anions; the story is a long and remarkable one and I have nothing to add to it to-day. Dr. Klotz and Ayers 32 are reporting some new developments in this area and I shall confine myself to a discussion of the interactions of serum albumin with two cations both from group 2 B of the periodic table namely, zinc and mercury. The use of these two cations in the fractionation of blood plasma proteins is discussed at this meeting by Cohn Surgenor Schmid Batchelor, and Isliker.36 The far-reaching developments which they are presenting speak for themselves; here I shall discuss only some recent work from our laboratory which bears on the chemical nature of the interactions involved.The tendency of metallic ions toward complex formation has been admirably surveyed by Jannik Bjerrum,37 who himself has done so much for the advance-ment of knowledge in this field. From his data it is apparent that the tendency of zinc ion to complex formation with the carboxylate ion is relatively weak (log k is approximately zero for zinc and acetate ion where k is the association constant). The association tendency with ammonia is much stronger (log k = 2-37) and this should give an approximate measure of the affinity of zinc for amino groups. However the affinity of amino groups for hydrogen ion is so strong that the zinc binding capacity at pH 7 or below is relatively small.One very important group found in proteins the irnidazole group of histidine, had not been previously investigated; Gurd and Goodman undertook to study the combination of zinc ion and imidazole while Felsenfeld and I were studying its combination with cupric ion.33 Both these ions were found to bind four molecules of imidazole the successive values of log k being tentatively estimated (at 4.5") for copper as 4.58 3-76 3.14 and 2.18. For Zn2+ they were 2.76 2.38, 2.40 and 2-22. These values of k for zinc ion are defined by the relation : In these equations Im denotes uncharged imidazole. On correcting for the statistical factors involved it is interesting to note that although the first Cu2+ ion is bound much more strongly than the first Zn2' the binding of one imidazol JOHN T .EDSALL 21 by copper decreases thc probability of binding the next whereas the binding of the first imidazole by zinc increases the affinity for more imidazoles until saturation is achieved. While this work was proceeding Gurd and Goodman 39 also made a most careful study of the binding of zinc ion by serum albumin employing the dialysis FIG. 7.-Effect of zinc ion concentration on zinc binding to human serum albumin. From Gurd and Goodman.39 OH- Added Albumin Temp.= 0°C 0.15M NaNO, -2.5% Albumin 9 14.0 I / 10.0 o 5.8 Q 7.5 I 4 FIG. &-Dependence of n on v (see text) in the binding of zinc ions by human serum albumin. From Gurd and Goodman.33 equilibrium technique. This work required great care in setting up the experi-ments and the analysis of the data involved a quite elaborate examination of the competition between zinc and hydrogen ions for the various sites on the protein, and of the influence of electrostatic forces due to the charge on the protein on the affinity constants.The former Some of their results are shown in fig. 7 and 8 22 SIXTH SPIERS MEMORIAL LEC'TIJRE illustrates zinc binding as a function of free zinc ion concentration for various amounts of OH- ion added to the isoionic protein; while the latter shows the number n o€ imidazole groups available to combine with hydrogen ions as a function of C the number of Zn2+ ions bound per molecule of albumin. The conclusion from these studies was clear cut ; the binding of Zn2+ by albumin in the pH range 5.5 to 7.5 is due almost entirely to the 16 imidazole groups of the histidine residues.At more acid pH values very little zinc is bound and at more alkaline values the precipitation of zinc hydroxide renders experimental measurement impracticable. Moreover the association constant involved is defined by the relation log k" = 2-82, where k" denotes the association constant corrected for the influence of electro-static forces. This value agrees extraordinarily well with the first association constant in the relation between the zinc ion and the imidazole molecule (log kl = 2.76). Indeed such very close agreement could hardly be expected and is perhaps accidental. Nevertheless this value certainly adds strong confirmatory evidence for the significance of the imidazole groups in the binding of zinc by albumin.The carboxylate groups in spite of their much greater number-106 per albumin molecule-show no significant binding at least in aqueous solutions. However, in the presence of concentrated urea as Cohn et al.36 are reporting at this meeting there is evidence from recent work of Dr. Gurd that carboxylate groups become available for binding. The amino groups would certainly combine at more alkaline pH values but their relatively strong basicity renders them unreactive with zinc at neutral pH. Quite independently of Gurd and Goodman Tanford 40 studied polaro-graphically the binding of zinc and several other ions by serum albumin and has come to identical conclusions concerning the significance of the imidazole groups for the binding.The mercuric ion in spite of its electronic relationship to that of zinc differs profoundly in its interactions with the groups found in proteins. Its affinity constant for amino groups is more than a million times as great as that of the Zn2+ ion; and its affinity for imidazole groups although the exact figure is not known to me is certainly far greater also. However its well-known affinity for sulphydryl groups is the dominating feature in its interactions with proteins as long as the protein has any free sulphydryl groups left to react. My colleague, Dr. W. L. Hughes Jr. whose work in this area has been basic for all that the rest of us have done estimates that log k for the association of Hg2+ with a sulp-hydryl group is of the order of 17 to 19.On the other hand we must remember that the amount of free Hg2+ ion present in most aqueous solutions of a mercuric salt is extremely small because of its ex-tremely strong affinity for the hydroxyl ion and for all halogen ions except fluoride. The reversible combination of mercury with mercaptal bumin was discovered by Hughes,27 who formulated the essential features of the reaction and crystallized the mercury dimer of mercaptalbumin. Oncley by means of ultracentrifuge measurements showed the simultaneous presence of dimer and monomer in solutions derived from these crystals and obtained indications that they existed in a state of reversible equilibrium. Over a period of several years we have continued the study of the process by light-scattering measurements and have characterized in a more quantitative manner both the kinetics of the formation and dissociation of the dimer and the position of the equilibrium as a function of pH ionic strength the concentration of certain specific ions in the solution, temperature and other variables.It was also discovered that certain organic mercurials containing two or more mercury atoms were also capable of linking two albumin molecules together to form a dimer. The one most thoroughly studied was the dioxane derivative introduced into the laboratory by Straessle 41 CH2-0 \O-CHz/ +Hg . CH2. HC/ \CH . CH2. Hg JOHN T. EDSALL 23 which for brevity will be denoted as HgRHg. This compound as will be seen, induces dimer formation far more rapidly than mercuric chloride itself.Other organic mercurials prepared by Dr. Howard Dintzis are now being studied. This work has been carried on by several investigators in our laboratory; its progress is due to the thinking and the experimental skill of Harold Edelhoch, R. B. Simpson Rudolf Straessle Ephraim Katchalski and R. H. Maybury. The insight of Dr. Hughes has been invaluable to us throughout the progress of the work. First is the combination of mercury with mercaptalbumin (denoted here as ASH) through the sulphydryl group of the latter : The reaction may be described as proceeding in three stages. ASH + HgC12 + ASHgCl + H+ + C1-. (1) This reaction involves no significant change of the molecular weight of the protein ; it therefore cannot be measured by light scattering and it is in any case very rapid-probably too fast to measure by most techniques.The product of the first reaction then reacts with a second molecule of mercaptalbumin to form the dimer : (11) This is the reaction which is directly observed by light scattering; it proceeds very slowly as compared to reaction I and is readily followed. Finally if an excess of mercuric chloride is added the dimer rapidly dissociates according to reaction 111. ASHgCl + ASH + ASHgSA + Hf + C1-. ASHgSA + HgC12 + 2ASHgCl. (111) The exact formulation of these reactions is open to some discussion. In the solution before reaction starts and at the concentrations used in our studies, most of the mercury is in the form of undissociated HgC12. Some however must be present in combination with hydroxyl or acetate ion; some presumably as HgCl+ ion.The amount of free Hg2+ is certainly minute at equilibrium. Probably, however the reactions I I1 and 111 represent the most convenient simple way of formulating the process. Dr. Hughes has demonstrated the release of hydrogen ions accompanying reactions I and 11. The interpretation of the light-scattering measurements in order to derive weight average molecular weights also raises certain problems. For the monomer, of molecular weight M we can employ the usual equation for light scattering in dilute solutions : Kcl/RhO = M-1 + 2Bc1, Kc~/R$~ = (2M)-1 + 2B~2. (W (V) and for the dimer of molecular weight 2M: Here c1 and c2 denote the weight concentrations of monomer and dimer respectively expressed in g/ml RJO and R30 denotes the corresponding values for the reduced intensities of scattering at 90" from monomer and dimer and K denotes the factor : K = 2~2n2(dn/dc)2/NoA-f (VI) where n denotes refractive index N is Avogadro's number and ho is the wave-length of the light in vacuo (Doty and Edsall42).The factor B is the interaction constant which is determined in large measure by the net charge on the protein and the ionic strength of the solution (Edsall Edelhoch Lontie and Morrison 43). If the protein is isoelectric B is zero or nearly zero for both monomer and dimer and the weight average molecular weight can be inferred directly from the reduced intensity. However it was of great interest to study the system under conditions of varying net charge on the protein and under these conditions B i 24 SIXTH SPIERS MEMORIAL LECTURE in generai different from zero.Fortunately it proved in practice that in a given medium the value of B for the pure mononier and the pure dimer is the same within experimental error. We have made the assumption-and it can be justified by theoretical arguments developed by Dr. Katchalski which I need not discuss here-that the same value of B can then be used for any mixture of monomer and dimer in the same solution and we can then obtain the molecular weight by extra-polation. For the mixture of the two species then we may write (VW where c = c1 + c2 and X = M(c1 -+ 2c~)/c. Since c and R90 are measured experi-mentally X may then be calculated from (VIl) provided that the value of B has been determined for the pure monomer or dimer in the same solvent in which the reaction is taking place.The weight fraction of dimer is then given by the equation Kc/RgO = X-1 -t ~ B c , (VIII) Using this approach we have been able to obtain extensive information con-cerning the velocity of the reaction and the proportion of dimer which is present at equilibrium. Repeated experiments have shown that true equilibrium is actually attained. In a given medium one may start either from pure monomer with an appropriate amount of mercury added or from a solution which is nearly pure dimer ; and on adjusting to the same weight concentration of protein in each case one attains the same final equilibrium value. If the activity of hydrogen and of chloride ions is fixed this equilibrium may conveniently be defined in terms of an apparent equilibrium constant (ASHg SA) (ASHgCI)(ASH) Kapp == and the variation of this apparent constant with change in pH and ionic strength may then be studied.To our surprise Kapp was found to vary little over the pH range 4-25-6 the mean value being 2.6 (& 0-4) x lo4 over the whole range. The measurements were carried out in phosphate buffers at pH 6 and in acetate buffers at the lower pH values. In simple terms this value of Kapp means that in a system containing 0.5 mole Hg per mole albumin if the total weight con-centration of protein is 10 g/l. then the weight fraction of dimer at equilibrium is near 0.5. Preliminary studies of the temperature coefficient of equilibrium indi-cate that rise of temperature favours dimer formation and therefore that the process is presumably endothermic.For a given protein concentration in a given medium the weight fraction of dimer at equilibrium varies in a characteristic fashion with the amount of mercury added. It is of course zero in the absence of added mercury when all mercapt-albumin is present as ASH and falls again to zero when one mole of mercury has been added per mole of albumin all of the mercaptalbumin being then in the form ASHgCI. The maximum amount of dimer formation always occurs when the molar ratio of mercury to albumin is 0.5. The value of the weight fraction of dimer at this point increases with the value Kapp. The form of the whole curve indeed can be calculated from the value of the equilibrium constant.The curve is symmetrical about its midpoint and is similar in charactcr to the curves theoret-ically deduced by Michaelis 44 for the fraction of semiquinone formed in the re-versible titration of an oxidation-reduction system as a function of the total of oxidant or reductant added and of the semiquinone formation constant. A family of some of the curves to be expected for different values of Kapp is shown in fig. 9. We have also made extensive determinations of the velocity constant kf of the combination reaction between ASHgCl and ASH. This constant expressed in 1. mole-1 min-1 is equal about to 40 from pH 4.25-4.75. As the pH increase JOHN T. EDSALL 25 further however the value decreases progressively to 0.69 at pH 6 .I t seems probable that this variation at pH values above 4.7 can be at least partly explained by the electrostatic effect of the increasing negative net charge on the albumin molecules above pH 5. The treatment involved here would be essentially that developed by Bronsted N. Bjerrum Scatchard and others for simpler i0ns.~5 However since the equilibrium constant is essentially independent of pH the results indicate clearly that the dissociation of dimer into monomer is far slower at pH 6 or above than below pH 5. This has indeed been confirmed experi-mentally. A concentrated solution of the dimer may be prepared and adjusted to a pH somewhat above 6 in phosphate buffer. Even if the solution is then greatly diluted the dissociation of the dimer is found to be extremely slow as 100 80 L (Y E *6 60 .c.0 0 ) c e 40 2 20 0 Molar Ratio HgCI,/(ASH) at t= 0 FIG. 9.-Percent of mercaptalbumin-dimer formed at equilibrium as a function of HgC12/ mercaptalbumin ratio. Curves are shown for various values of the parameter Kappa (apparent association constant multiplied by the molar concentration of albumin). compared to the rate below pH 5. In this way dilute solutions containing nearly pure dimer with very little monomer can be obtained for study over a considerable period of time as for instance in ultracentrifuge runs. Studies of interaction of mercaptalbumin with the organic dioxane derivative discussed above (HgRHg) indicate a striking difference from the interactions with mercuric chloride. The equilibrium at all the pH values studied seems to lie almost completely in the direction of dimer formation.At low pH values, below 4.8 the velocity of the process is so great that no accurate velocity constants for dimer formation could be determined by our methods. Between pH 5 and 6, the value of kf decreases with rising pH in much the same manner as for the re-action with mercuric chloride. However the absolute values of kf at any pH in this range are very much greater-indeed several thousand times as high as those for the reaction involving mercuric chloride. A plot of the two sets of kfvalues is given in fig. 10. The great contrast between the rates and the equilibrium constants found in these two different types of reaction with mercury compounds is presumably t 26 SIXTH SPIERS MEMORIAL LECTURE be explained largely on steric grounds.When two albumin molecules are linked through their sulphydryl groups by a single mercury atom the juxtaposition of the adjoining surfaces of the two protein molecules must be extraordinarily close. It is indeed remarkable that the surfaces are so shaped that they can be fitted together at all. It will be of great interest to see whether chemical modification of other groups in the albumin will lead to alterations in the tendency to undergo this reaction. In any case the relative slowness and incompleteness of the reaction are not surprising. On the other hand for the dioxane derivative HgRHg in which there is a mercury atom at each end there is an effective distance of the order of lOA between the two mercury atoms.Thus the coupling of the two albumin molecules which are linked by these atoms is far looser than when a single mercury serves as the link. The conditions for the combination are much less critical and pre-sumably the reaction is possible in a much greater variety of relative orientations A S Hq RHq SA \ log K f t: 10 A5 Hq SA I -PH :2 4; 6 5 0 5 4 5;8 , FIG. IO.-Logarithm of the velocity constant kf for dimer formation of human Serum mercaptalbumin with mercurials. Lower curve reaction with mercuric chloride ; upper curve reaction with the dioxane derivative containing two mercury atoms. Phosphate buffers were employed near pH 6 acetate buffers at lower pH values; ionic strength 0.05 in acetate buffers; 0.05-0.15 in phosphate; temp.25". (From studies of E. Katchalski H. Edelhoch and R. B. Simpson.) of the two albumin molecules than would be the case if the fitting were closer. Thus the reaction goes relatively rapidly and it goes largely to completion. One may suspect although this is not proved that the two halves of the dirner ASHgRHgSA may be much more loosely coupled than the halves of the dimer ASHgSA. Thus in the former case a much greater degree of free rotation of one-half of the dimer relative to the other should perhaps be expected than in the latter; a possibility which may perhaps be explored by such methods as dielectric dispersion. It should be added that in spite of the presumably very close fitting of the two halves of the dimer ASHgSA the mercury atom does not appear to be buried within the protein in such a way as to be inaccessible to the solvent.Addition of other ions such as bromide iodide or cyanide which have a strong affinity for mercury leads to quite rapid dissociation of the dimer. Evidently these ions can penetrate readily to the mercury atom which is then released from its com-bination from one of the sulphydryl groups to form the monomer ASHgBr, ASHgI or ASHgCN. The reaction has been studied by us particularly wit JOHN T. EDSALL 27 the iodide ion. The addition of even a single mole of iodide per mole of mercury in the dimer leads to a relatively rapid and very marked dissociation of the dimer. Bromide is inuch less effective and chloride far less than bromide. Indeed, these studies appear to be significant as a method of evaluating the relative affinity for mercury of various inorganic ions and thus obtaining data which should be of interest for inorganic chemistry.This is of course not our primary concern; we are interested in these reactions chiefly because of what we can learn from them concerning the external form the surface properties and re-activity and perhaps something of the finer pattern of the configuration of the albumin molecule. The extensive studies carried out by Lewin 46 in our laboratory demonstrated that mercaptalbumin mercury dimer could be crystallized in association with many other metallic cations and also with anions containing atoms of high atomic number. Dimer formation of the type first observed by Hughes27 with serum albumin has not yet apparently been observed with other proteins.Several years earlier however the successful use of mercury in the crystallization of enolase was reported by Warburg and Christian.47 In this case the crystallized mercury derivative contained just one mole of mercury per mole of enolase and other derivatives have not been found. The interaction of mercury with proteins is of course not confined to a single group. However the affinity of mercury for the sulphydryl group is so much greater than for any other groups found in proteins that no other reaction will occur until all such groups are saturated with mercury. Beyond this point, however important interactions begin to occur with imidazole amino and other groups. These interactions between mercury and proteins have recently been exploited with great success by Dr.Cohn and his associates in fractionation processes .36 1 Sanger and Tuppy Biochem. J. 1951,49,463,481. Sanger Thompson and Tuppy, 2 Pauling and Corey Proc. Nut. Acad. Sci. 1951 37 205 235 241 251 261 272, 3 Bragg Howells and Perutz Acta Cryst. 1952 5 136. 4Kendrew Proc. Roy. SOC. A 1950 201 62. 5 Carlisle and Scouloudi Proc. Roy. SOC. A 1951 207 496. 6 Low Nature 1952 169 955. 7 Hocking Laskowski and Scheraga J. .4mer. Chem. SOC. 1952 74 775. 8 Katz Gutfreund Shulman and Ferry J. Amer. Chem. SOC. 1952 74 5706. 9 Edsall Foster and Scheinberg J. Amer. Chem. SOC. 1947 69 273. 10 Hall J. Biol. Chem. 1949 179 857. 11 Porter and Hawn J. Expt. Meci. 1949 90 225. 12 Mitchell Biochem. Bioplzys. Acta 1952 9 430. 13 Bailey Bettelheim Lorand and Middlebrook Nature 1951 167 233 ; Bettelheim and Bailey ResumL des Communications I P Congres Internationale de Biochemie, 1952 411; Lorand and Middlebrook Biochem. Biophys. Acta 1952 9 581; Bettelheim and Bailey ibid. 9 578. Symposiiiin sur les Hormones Proteines et Derivtes des Proteines (Paris 1952). 282 729. 14 Lorand Nature 1951 167 992. 15 Edsall and Lever J. Biol. Chem. 1951 191 735. 16 Ferry and Morrison J. Amer. Chem. SOC. 1947 69 388. 17 Laki and Lorand Science 1948 108 280. 18 Lorand Nature 1950 166 694. 19 Shulman this Discussion. 20 van Vunakis and Brand Abstr. 119th meeting Amer. Chem. SOC. April 1951 p. 28c. 21 Weber this Discussion. 22 Scatchard Batchelder and Brown J. Amer. Chem. SOC. 1946 68 2320. 23 Low J. Amer. Chem. SOC. 1952 74 4830. 24 Creeth Biochem. J. 1952 51 10. 25 Oncley Scatchard and Brown J. Physic. Chem. 1947 51 184 28 SIXTH SPIERS MEMORIAL LECTURE 25a Oncley Dintzis and Hollies Abstr. 122nd meetirrg Amer. Chem. SOC. Sept. 1952: 26 Weber Biochenr. J . 1952 51 145 155. 27 Hughes J. Amer. Chem. Sac. 1947 69 1836 ; CoM Spring Harbor Symp. Quant. 28 Edsall and Foster J. Amer. Chem. SOC. 1948 70 1860. 29 Riley and Oster Furuday SOC. Discussions 1951 11 107. 30 Anderegg Beeman and Shulman paper presented to Amer. Physic. SOC. April, 1952 ; and unpublished work. 31 Oncley Chem. Rev. 1942 30 433. 32 Klotz and Ayers this Discussion, 33 St. George and Pauling Science 1951 114 629. 34 Wyman and Allen J. Polymer Sci. 1951 7 499. 35 Massey Biochem. J. 1952 51 490. 36 Cohn Surgenor Schmid Batchelor Isliker and Alameri this Discussion. 37 Bjerrum Chem. Rev. 1950 46 38 1. 38 Gurd Edsall Felsenfeld and Goodman Fed. Proc. 1952 11 224. 39 Gurd and Goodman J. Amer. Chem. SOC. 1952,74 670. 40 Tanford J. Amer. Chem. SOC. 1952 74 21 1. 41 Straessle J. Amer. Chem. SOC. 1951 73 504. 42 Doty and Edsall Advances in Protein Chem. 1951 6 35. 43 Edsall Edelhoch Lontie and Morrison J. Amer. Chem. SOC. 1950 72 4641. 44 Michaelis Chem. Rev. 1935 16 243 ; see especially fig. 4. 45 see for instance the discussion by Laidler Chemical Kinetics (McGraw Hill Pub-46 Lewin J. Amer. Chem. SOC. 1951 73 3906. 47 Warburg and Christian Biochem. Z. 1941 310 384. p. 12p; Dintzis Thesis Harvard University 1952. Biol. 1950 14 79. lishing Co. New York 1950) p. 123

ISSN:0366-9033    

DOI:10.1039/DF9531300009

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

I. EXPERIMENTAL TECHNIQUES ZONE ELECTROPHORESIS IN FILTER PAPER AND OTHER MEDIA BY ARNE TISELIUS Biokemiska Institutionen, Uppsala Universitet, Uppsala, Sweden Received 8th May, 1952 Zone electrophoresis in immobilized media (filter paper strips or powder, glass beads, starch, gels, etc.) has several advantages over boundary electrophoresis as a separation method. Completely separated zones may be obtained, and it is easy to work on a micro- scale and at low concentrations. The method is suitable also for the separation of many low molecular weight substances, like amino acids, peptides, nucleotides, etc. On the other hand electro-osmosis and adsorption phenomena interfere. There are certain ways of minimizing the influence of these and it is shown that, under suitable conditions, zone electrophoresis measurements may be used for determination of mobilities and isoelectric points.Electrophoresis of proteins and other biochemically important substances of large molecular weight is usually performed in free solution in a suitable U-tube apparatus. Observation of electrophoretic migration and separation (electro- phoretic analysis) in this modification of the method is essentially an observation of the migration of boundaries by optical or analytical methods. The separation obtained is a boundary separation of overlapping zones, not a complete separation into zones of different migration. Such a separation generally cannot be achieved in free solution, as convection due to gravity would upset the zones ; this requires some sort of an immobilized medium like glass powder, sand, various gels or filter paper, which were sometimes used in early work on ionic migration (Lodge, Hittorf).Several workers in this field have recently attempted to use such arrangements especially for preparative work, where accurate definition of mobilities is not so essential. Already in 1907 Field and Teague 1 observed the migration of diphtheria toxin and antitoxin in agar jelly. Ionic migration experiments in gels were performed by Kendall et al.2 for the separation of rare earths. Coolidge in 1939 separated serum proteins into zones in an electrophoresis tube med with ground glass ~001.3 Consden, Gordon and Martin in 1946 separated amino acids and peptides by migration through a slab of silica gel (ionophoresis).4 They also introduced an ingenious method of localizing the zones by taking a print of the surface of the gel on a sheet of filter paper which was subsequently stained with ninhydrin.Other modifications have recently been used by Butler and Stephen 5 by Gordon, Keil and Sebesta,6 by Haglund and Tiselius 7 and many others. Separations of isotopic ions have been achieved by Brewer, Madorsky and Westhower in layers of sand or glass powder.* One should also mention attempts to arrange zone electrophor- esis in free solutions by using a large number of U-tubes or other elements, coupled in series. Unless the units are very small and their number quite large, separations in such apparatus will tend to become less distinct, as convection currents are set up within each unit.They have, however, been used with advantage in some in- vestigations.9 Recently filter paper strips have been widely used with notable 2930 ZONE ELECTROPHORESIS success as an immobilizing medium for electrophoresis of proteins and substances of lower molecular weight. This has the advantage of providing a micro-method and requires only a very simple apparatus. Contributions to the development of this modification have been made by, for example, Wieland and Fischer,lo Turba and Enenke1,ll Durrum,l2 Cremer and Tiselius,l3 Kunkel and Tiselius 14 and many others. Obviously the great success of filter paper chromatography has been a source of inspiration in this work. We have proposed the term zone electrophoresis for the type of experiment in which a separation into zones is achieved, whereas the common electrophoresis in U-tubes with free solutions may be called boundary electrophoresis.* It is perhaps not generally realized that the advantages of zone electrophoresis are not only the possibility of complete separation, fairly easy isolation of the fractions and microscale operation ; in addition, much lower concentration can be used than in free boundary electrophoresis, where a certain minimum concen- tration is required for the stability of the boundaries against convections.Con- sequently the so-called " boundary anomalies " can be largely eliminated. These effects, which in boundary electrophoresis usually mark their presence by a pro- nounced difference in the sharpness and rate of migration of the boundaries in the two limbs of the U-tube, may cause grave errors and even make electrophoretical determinations impossible.They depend essentially on that contribution to the total conductivity of the solution which is due to the migrating substances (pro- teins, etc.) studied. With proteins-for a given concentration-the effect is usually small ; with amino acids, for example, it may be so large that it virtually makes the experiment meaningless. Amino acids, peptides and many other low molecular weight substances can, however, easily be studied electrophoretically in zone electrophoresis. If the con- centration is low, the zones are not spread out too much and a complete separation can be achieved. This was first made clearly evident in the work of Consden, Gordon and Martin.3 The advantages of the zone method over the boundary method have, however, to be gained at the cost of less well-defined conditions of migration in other respects, especially with regard to the accurate definition of the mobilities.There are three main factors to be taken into account, all of which depend upon the properties of the immobilizing medium used. First there is the purely geometrical effect of the structure of the medium, which makes the charged particles and the electric current travel in a manner which deviates considerably from the conditions in a homo- genous medium. Secondly, there is the possible interaction (by adsorption or other effects) between the medium and the migrating substances. And thirdly, we always have in media of this kind an electro-osmotic transport of the whole solution, superimposed upon the migration.As the migration is observed, relative to the solid medium (for example, filter paper) and we want to know the mobility relative to the solution, a correction term must be introduced. One also should pay attention to the reaction changes occurring at the interface between the ends of the filIing-medium column (or sheet of filter paper) and the buffer solution, which changes are analogous to membrane polarization. Thus the choice of filling medium is of great importance. Small particle size is favour- able from point of view of stabilizing the zones but will, on the other hand, tend * It may be qiiestionable to extend the use of the term electrophoresis, originally applied to colloids, proteins and other substances of large molecular weight, to the migration of substances of low molecular weight.However, as the experimental arrangements are essentially the same in both cases if a separation is the main purpose, it seems impractical to distinguish between electrophoresis, ionophoresis or electromigration and it would be preferable to use a common term. In any case these terms do not seem suitable for distinguishing between zone and boundary methods. The author is perhaps somewhat conservative in using the term electrophoresis. We notice the same difficulty in chromato- graphy, which nowadays is not limited to coloured substances by far.FIG. I a.-Apparatus for filter paper electrophoresis (Kunkel and Tiselius). (a) coiled platinum electrodes ; (b) glass wool at the juncture between electrode chambers ; (c) highly porous paper carrying liquid from electrode vessels to the filter paper ; (d) glass plates surrounding the filter paper.FIG. Ib.-Separation of a mixture of (left to right) crystalline lysozyme, purified myclorna protein, crystalline lacto to globulin and crystalline human serum albumin by paper electrophoresis ; staining with bromphenol blue. The line across the strip indicates the position of the original spot (Kunkel and Tiselius). [To face page 31ARNE TISELIUS 31 to increase adsorption effects. With gels, which would represent an extreme case, the resistance against the migration of large molecules due to adsorption or mechan- ical resistance may be appreciable. Thus silica gel is not suitable for proteins, whereas dilute agar jelly offers surprisingly small resistance to the migration of proteins of moderately high molecular weight.6 It is, however, difficult to avoid contamination by the agar when one attempts to elute the protein.Most investigators using these methods have been chiefly interested in separa- tion for preparative or analytical purposes or, in some cases, even for clinical diagnostics (blood plasma), It is important to take note of the factors mentioned above in these applications, but it is still more important when one attempts a quantitative interpretation of mobility. This problem is of considerable interest if one wanls to make use of zone electrophoresis for identification of components and determination of their electrophoretic properties in the same way as in ordinary boundary electrophoresis in free solution.Some results, obtained in filter paper electrophoresis by Kunkel and the author,l4 may be quoted as typical. The simple apparatus used, made of Perspex, is shown in fig. 1 . The strip of filter paper (Munktell no. 20 was found particularly useful) is enclosed between two thick plane glass plates, which are firmly clamped together. The surfaces received an anti-wetting treatment (Dow Coming Silicone 200, or a touch of silicon grease) and the edges are sealed with silicon grease. The ends of the strips are pressed against pads of filter paper which are partially immersed in the electrode vessels containing buffer solution. The technique for staining the strips with brom- phenol blue after the conclusion of an experiment is essentially that of Durrum.12 The " hanging strip " used by this author appears less suitable for mobility studies as it is difficult to obtain constant potential gradient along the strip, the quantity of buffer solution taken up by the filter paper being smaller at the top than at the bottom.Analysis of the protein contents of the different zones can be made by cutting the strip into segments and applying a micro-colorimetric method for protein,l4 or by evaluating the colour intensity of the bromphenol blue (or other suitable staining reagents) photometrically. Special photometers have been con- structed for this purpose. As the affinity between the dye and different proteins may vary considerably, great care has to be exercised in such procedures, however, even though they are very tempting to use because of convenience.Table 1 and fig. 2 shows the results from an experiment on normal human serum. Many similar experiments have been published during the last few years. TABLE 1 Comparison between the percentage composition of the various components of a normal serum as calculated from the areas under the curves obtained by paper electrophoresis and free electrophoresis. No attempt was made to correct the dye values for differences in affinity with the different components. (Experiments by Kunkel and Tiselius, J . Gen. PhysioE., 1951, 35, 89.) composition % method - alb. a1 a2 B Y mod. Folin method 52.9 6.3 12.2 10.1 18.5 paper electrophoresis- dye elution method 65.9 3.9 8-0 9.0 13.1 free electrophoresis- ascending 53.33 6.21 10.87 13-79 15-62 descending 52-06 5-28 11-26 14.66 16.74 For mobility determinations it is essential first to correct for the electro-osmotic flow.This can easily be done by introducing into the mixture to be studied a small amount of a sub.-tance of known mobility, preferably one with zero mobility.32 ZONE ELECTROPHORESIS As such an “ index substance ” we have used dextran which has the advantage of being uncharged and is also easily detected with the bromphenol blue reagent. If distances of migration are measured from the index spot, the effect of electro- osmosis is eliminated. There may, however, be disturbances due to this pheno- menon, especially at low buffer concentrations, if electro-osmosis sets in at the glass plate surfaces, and it is necessary to press the glass plates firmly against the strip to avoid trouble of this kind.The geometry of the migration can also be dealt with fairly simply.14 The migrating substances and the current follow the same somewhat crooked path through a porous medium, the length of which I’ is greater than the length of the strip 1. Thus an observed migration of d cm in the strip corresponds to an actual migration of Vd/l cm at a potential gradient of ljl’ . v/l and the apparent mobility U,, as observed on the strip after correction for electro-osmosis, is related to the real mobility U in the pores by the expression u = (l/Z’)2Ua. 0 ? FIG. 2.-Mobility values plotted against pH for human serum albumin on filter paper (O), and in free solution ( x ), The individual points (0) represent dextran mobilities (Kunkel and Tiselius).The factor l/Z’ can be determined by measuring the resistance of the strip when it has taken up a known weight of a standard conductivity solution. If both the electro-osmotic and geometrical corrections are thus applied, mobilities and iso- electric points agree remarkably well with those obtained in free solution (fig. 2). Adsorption phenomena which cause tailing, and sometimes even complele immobilization of the zones, are probably the greatest difficulties in this sort of work. Various qualities of filter paper differ in this respect (it is recommended that electro-dialyzed paper be used). Some proteins show rather low adsorption (e.g.serum proteins) but others, especially those which carry a strongly positive or negative charge, may be tenaciously held. Porath and Flodin in the author’s laboratory have had some success in using for such substances filter paper which by suitable pretreatment has been given a marked positive or negative charge (unpublished experiments). Columns of small, spherical glass beads or of ionic exchange resins (Dowex) have also been found useful in such cases. In the separation of larger quantities, it is convenient to substitute the paper strip in the apparatus fig. 1 with a rectangular trough containing, for example, filter paper powder. Such an arrangement is used in the author’s laboratory forARNE TISELIUS 33 analysis of mixtures of the basic amino acids.For continuous separation of still larger quantities, arrangements based upon a combination of vertical flow and horizontal electrophoresis in a glass powder layer or in a filter paper sheet have been worked 0~t.15~16 If gels are used as immobilizing media it appears possible to make use of the increasing resistance to migration of substances of increasing molecular weight for separation purposes.6917 Electro-osmosis will tend to transport all substances, charged or uncharged, through the gel and a kind of " zone ultrafiltration " will result, which appears to offer interesting applications. Zone electrophoresis in immobilized media is rapidly gaining importance, and has some obvious advantages, and in particular, the possibility of working with small quantities and of obtaining complete separation.Only very simple apparatus is required and yet, under suitable conditions, it is still possible to make quanti- tative measurements. The method is rapidly coming into general use in biochemical and clinical laboratories for the study of proteins, nucleic acids, nucleotides, polypeptides, peptides, amino acids and many other types of substances. Also in enzyme chemistry it offers great promise. As compared to chromatographic procedures its most valuable field of application is for substances of comparatively large molec- ular weight, but there are of course many cases where both methods may be used and where each supplements the other. 1 Field and Teague, J. Expt. Med., 1907, 9, 86, 225. 2 Kendall, Jette and West, J. Amer. Chem. SOC., 1926, 48, 31 14. 3 Coolidge, J. Biol. Chem., 1939, 127, 551. 4 Consden, Gordon and Martin, Biochem. J., 1946, 40, 33 ; 1947,41, 590. 5 Butler and Stephen, Nature, 1947, 160,468. 6 Gordon, Keil and Sebesta, Nature, 1949, 164,498. 7 Haglund and Tiselius, Acta Chem. Scund., 1950, 4, 957. 8 Brewer, Madorsky and Westhover, Science, 1946, 104, 156. 9 Reference is made to a review by Svensson, Advances in Protein Chemistry, voI. 4, 10 Wieland and Fischer, Naturwiss., 1948, 35, 29. 11 Turba and Enenkel, Naturwiss., 1950, 37, 93. IZDurrum, J. Amer. Chem. SOC., 1950, 72, 2943. 13 Cremer and Tiselius, Biochem. Z., 1950, 320, 273. 14 Kunkel and Tiselius, J. Gen. Physiol., 1951, 35, 89. 15 Svensson and Brattsten, Arkiv Kemi, 1949, 1, 401. 16 Grassmann and Hannig, 2. angew. Chem., 1950, 62, 170. 17 Synge and Tiselius, Biochem. J., 1950, 46,41. pp. 252-277 (Academic Press, Inc., New York, 1948).

ISSN:0366-9033    

DOI:10.1039/DF9531300029

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

ARNE TISELIUS 33 POLARIZATION OF THE FLUORESCENCE OF LABELLED PROTEIN MOLECULES BY G. WEBER Sir William Dunn Institute of Biochemistry, Cambridge Received 8th May, 1952 The recent development of a method of determination of the average rotational relaxation time of protein molecules in solution by measurements of the polarization of the fluorescence of protein conjugates is reviewed. A method for the purification of the conjugates by ion exchange is described, together with the use of paper chromatography to distinguish between reversible adsorption and chemical combination of small mole- cules to proteins. The reversible dissociation of bovine serum albumin in acid first detected by observations of the changes in the polarization of the fluorescence with pH34 POLARIZATION OF FLUORESCENCE has been confirmed by measurements of sedimentation and diffusion at pH -1-8.A study of the effect of neutral salts on the dissociated protein shows that reversal of the dissoci- ation depends on the anion and increases in the order C1 < Br < NO3 < SCN < p - toluensulphonate. On this basis it is concluded that the electrostatic repulsion between the subunits is indispensable for the acid dissociation. The independence of the alkaline dissociation of bovine serum albumin from the ionic composition and strength of the medium is discussed. THEORETICAL.-The studies of Perrin 1, 2 have shown that the polarization of the fluorescence emitted by molecules in solution depends only on the relaxation time of the rotation, the lifetime of the excited state of the fluorescent molecules and the relative orientation of the virtual oscillators of absorption and emission of light.For spherical molecules a particularly simple relation obtains between these quantities : where Here p is the partial linear polarization of the fluorescent light emitted at right- angles to the direction of propogztion of the exciting light. The minus signs in l / p correspond to excitation by polarized light vibrating at right-angles to the directions of excitation and observation, the plus signs to ex- citation by natural light. T is the lifetime of the excited state of the fluorescence and p the rotational relaxation time ; po is the polarization obtaining when 3r/p -+ 0, and as 7 is a quantity of the order of 10-8 sec, po does not differ appreciably from the polarization observed at room temperature in glycerol solution for most fluorescent molecules.For ellipsoidal molecules the relations between these quantities are consider- ably more complex since the general equations developed by Perrin2 require a knowledge of the angles that the directions of the oscillators make with the axis of revolution of the ellipsoid. However, as I have shown elsewhere 3 a consider- able simplification is possible if the fluorescent units carrying the oscillators are attached to a much larger molecule with random orientation, that is, if all the angles between the oscillators and the axis of revolution are equally probable. In such cases eqn. (1) becomes 8 and l/po 'f 7 1 1 5 , 9T2 1 -i- - - < - T - PO 2 I n 2 n l ! 8 p2 in which po = 377 Y,/RT is the relaxation time of the rotation of a sphere of volume Ve equal to that of the ellipsoid.If p1 is the rotational relaxation time about the axis of revolution and p 2 the rotational relaxation time about a direction normal to it, then When the experimentally accessible values of T / ~ O are sufficiently small the last equation becomes n1 = pdpo; n2 = P2/PO.FIG. 1 . acetate 1 2 3 -Fluorescence of ascending paper chromatogram. Watman no. 1 ethanol + pH 4.8 ; fluorescence excited by Hg arc with Wood's filter ; camera filter : 2 cm 1, bovine serum albumin conjugate before filtering through resin. 2, same after filtering through resin. 3, 1-dimethylamino-naphthalene-5 sulphonate. layer of saturated NaN02 solution.[To face page 35G . WEBER 35 Therefore in general if a straight line is obtained on plotting l/p against absolute temperature/viscosity, the relaxation time of the rotation calculated from the slope and intercept is ph the harmonic mean of the principal relaxation times : 2 ph = ~ 1 1 ’ - + - P1 P2 (4) When the depolarization is due to more than one relaxation lime the plot of l/p against T/q may yield a curve concave towards the latter axis. If the solution is monodisperse it may safely be concluded that the emitting units are elongated molecules since the relaxation times of the rotation of an oblate ellipsoid never differ by more than 10 % from each other 4 and would in their effects be indis- tinguishable from a sphere even under the most favourable conditions.If the plot shows curvature the elongation may be determined by comparison with the theo- retical curves given by eqn. (2). If the axial ratio of the ellipsoid is greater than 10, po/p1 can be considered effectively 0 and po/p2 = 3. For such particles therefore the dynamic volume may be calculated directly, and for proteins an approximate molecular weight may be obtained if due allowance for hydration is made. A curvature convex towards the T/q axis is only possible if T lengthens as the temperature increases or ifph is not alinear function of q/T. The former is extremely improbable on theoretical grounds. It has been suggested by Perrin5 that the latter obtains in the Brownian motion of solute molecules differing little in size from the molecules of a solvent of low viscosity.For macromolecules this possi- bility may be discarded and a curvature convex towards the T/T axis is only possible if the total number of rotational degrees of freedom increases with the temperature. Therefore either new modes of intramolecular rotation appear that were originally frozen at lower temperatures or dissociation into fragments takes place. In most cases it is possible to distinguish between these alternatives.4 EXPERIMENTAL To study the rotational diffusion of proteins by the depolarization of the fluorescence they must be converted into stable fluorescent conjugates. With this object the protein is reacted with 1 to 2 % of its weight of 1-dimethylamino-naphthalene-5-sulphonyl chloride.6 Part of the acid chloride combines with the protein, mainly or only with the primary amino groups yielding substituted sulphonamides, and the rest hydrolyzes giving the strongly fluorescent sulphonate.The separation of the conjugate from the latter may be accomplished by dialysis against salt solutions or by ethanol precipitation. Lately I have used a more rapid and very effective method : the reaction mixture is dialyzed against M/15 phosphate buffer at pH 7 for 24 h and then filtered slowly through a column of a basic exchange resin (Dowex 2, mesh 200). The strongly acidic sulphonate is retained while the protein conjugate passes through and is quantitatively recovered. Spectro- photometric measurements indicate that 50 to 60 % of the acid chloride reacts with the protein.6 The presence of free sulphonate as opposed to chemically bound sulphonamide may be detected by paper chromatography in a medium where the protein is insoluble while the sulphonate has some solubility.A mixture of 55 parts of ethanol and 45 parts of 0-2 M acetate buffer of pH 4-8 is very convenient. Here the Rf of the proteins exam- ined was effectively 0 and the Rf of the sulphonate nearly 0.9. Fig. 1 is the photograph of such a chromatogram. It shows the elimination of the sulphonate by the ion exchange resin and also the clear separation between the fluorescence of the conjugate and that of the sulphonate. There is little tailing of the fast spot in spite of the fact that serum albumin in solution adsorbs the sulphonate strongly.7 When 1-dimethylamino-naphthal- ene-5 sulphonate or any of several acidic dyes was dissolved together with unlabelled bovine serum albumin and then subjected to chromatography, complete separation of the dye occurred, all the fluorescence and/or the colour appearing in the moving spot.This method is clearly a general one by which reversible adsorption may be distinguished from the actual chemical binding of small molecules to proteins. It allows a study of the stability of the conjugates under a variety of conditions. Thus it is found that heating36 POLARIZATION OF FLUORESCENCE at neutral pH at 60" for 1 h results in hydrolysis of less than 1 % of the sulphonamide and that at pH 2 the same treatment may result in up to 5 % hydrolysis. DISCUSSION Two proteins, ovalbumin and bovine serum albumin have been studied in detail.6 On plotting l/p against T/v, straight lines are obtained for solutions in water or dilute electrolyte at neutral pH between 3" and 50".Moreover the extra- polated values of po agree within the precision of the measurements with the polarization observed in 60 % sucrose (w/v) where 3r/ph is effectively 0 (table 1). Therefore eqn. (3) may be used. If the values of ph obtained from the data of TABLE 1 .-FLUORESCENCE POLARIZATION OF PROTEIN CONJUGATES S is the calculated regression coefficient of l/p upon T/q. po (ext.) is the extrapolated value obtained from this regression coefficient. PO observed, is the polarization recorded in 60 % (w/v) sucrose at 4". ph at 20" has been calculated from eqn. (3) with 7 = 1.4 x lo-gsec. ph/pu is the ratio of the observed value of the rotational relaxation time to the value of a sphere of molecular weight (known or assumed) given in the second column. Notice the difference between pa (anhydrous sphere), and pa (hydrated sphere) appearing in eqn.(2). Therefore ph/po depends only on shape, ph/pu on both shape and hydration and has a significance similar to f/fo in the translational diffusion. The figures given for F- and G-actin are from observations of Dr. T.-C. Tsao. protein molecular weight solvent PH S X 105 po(ext) bovine serum 0.06 M phosphate 7.0 4-26 0-258 0.257 1.42 2.22 bovine serum - distilled water 1.8 11.6 0.245 0.244 0.56 - bovine serum - N/10 NaOH 13.0 11.9 0.233 0.230 0.58 - albumin 69,000 buffer albumin + HC1 albumin ovalbumin 45,000 0.06 M phos- 7.0 7.5 0.236 0.236 0.88 2.12 phate buffer ovalbumin 45,000 N/lONaOH 13-0 7.5 0-236 - 0.88 2.12 ovalbumin heat - 0.06 M phos- 7.0 - - 0.253 2-2 to - F-actin 147,000 0.1 M KCl 7.0 2.21 0.193 0.195 3.6 2.66 denatured phate buffer 4.8 0.06 M phosphate 10-4 M ATP G-actin 71,000 lO-3M Na 7.5 4.84 0.202 0.203 1.6 2.44 G-actin (dimer) 143,000 water, salt-free 7.0 2.30 0.193 0.193 3.5 2.66 (monomer) versinate Oncley8 using the dielectric dispersion method are introduced in eqn.(3) it is found that r has the same value in both conjugates, namely, 1-4 x 10-8 sec. Reproducible results are obtained without difficulty in the range of pH 1-5-14. Between these limits no conspicuous changes in the lifetime of the excited state of the fluorescence seem to occur, so that changes in the observed slope in the plot of l / p against T/y may be attributed to real changes in the mean harmonic relaxation time of the protein particles.It is thus possible to show that bovine serum albumin dissociates into subunits outside a stability region which at room temperature extends between pH 3.9 and 9. Both dissociations in acid and in alkali are rever- sible, a t least as regards the relaxation time of the rotation. They differ consider- ably in certain respects. The dissociation in acid is strongly dependent upon the ionic composition and strength of the solvent, the alkaline dissociation is practically independent of it. The lowest relaxation times in acid are observed in proteinG . WEBER 37 solutions dialyzed against distilled water for a period of 100 to 200 h and subse- quently brought to pH 1-8-2.2 by addition of a small amount of HCl.On addition of neutral salts to these solutions the polarization of the fluorescence increases with ionic strength and with most salts tends asymptotically to the polarization observed in neutral solution. Different salts vary in their ability to induce re-aggregation of the acid protein. Table 2 gives the concentrations of electrolyte in which a rotational relaxation time midway between those of the neutral and salt-free acid TABLE 2 Electrolyte concentration at which ph at 20" = 1.02 x 10-7 sec; 0.2 % bovine serum albumin dialyzed for 100 h against distilled water and subsequently adjusted to pH 2. with HCI. concentration electrolyte (equiv. /I.) electrolyte (equiv./ 1 .) KCI 0.18 KBr 0.075 N-CI 0.18 &So4 0.032 LiCl 0.12 KNO3 0-025 MgC12 0.16 KSCN 0.0082 CaC12 0.17 p-KSO3. C6H4. CH3 0.0059 concentration BaCl2 0.21 protein is observed. This characteristic concentration is independent of the cation and dependent on the anion, increasing for the latter in the series, Cl < Br < NO3 < SCN < p . CH3. C&4. sO3. The series is the same as is given by Scatchard and Black 9 for the relative adsorption of anions by iso-ionic serum albumin. The conclusion may be drawn that re- combination of the subunits takes place when the electrostatic repulsion between them is sufficiently decreased by the adsorption of the ion of opposite sign. The acid dissociation of the protein has been confirmed by sedimentation and diffusion measurements.Preliminary observations made by Dr. R. A. Kekwick on bovine serum albumin dissolved in 0-2 M KCl adjusted with HCl to give pH 1-82 showed in the ultracentrifuge only one component with 320 = 2-7 X 10-13. (For the neutral protein, s20 = 4.3 x 10-13.) Measurements of the free diffusion of the protein in the same solvent yielded a mean value for D20 of 6.85 x 10-7. (For the neutral molecule, D20 = 6.1 x 10-7.) The calculated molecular weight is 36,000 with f l f o = 1-43. Observations of the polarization of the fluorescence in the same medium gave (fig. 2) at 20" ph = 7.9 x 10-8. For the quoted molec- ular weight phipa = 2.4, while for the neutral molecule ph/pa = 2.2 and f / f o = 1.34. Therefore neglecting improbable changes in hydration, both translational and rotational diffusion indicate an increase in assymetry on dissociation.This is best explained by the splitting lengthwise of the neutral molecule into two equal or nearly equal parts. Fig. 2 shows that shorter relaxation times are observed in the absence of salt as compared with the medium in which the sedimentation study was carried out, suggesting a further splitting of the molecule under these conditions. After dialysis against distilled water for 48 h the relaxation time in HC1 solution was (pH 2), ph = 6-5 x 10-8 sec.5 After dialysis for a further 100 h, ph = 5.6 x 10-8sec in the same medium. Longer diaylsis periods did not result in any further change in the relaxation time. In N/10 NaOH the calculated relaxation time at 20" was 5.8 x 10-8 sec which does not differ significantly from 5-6 x 10-8 observed in acid.Apparently both dissociations result in the same products. The polarization of the fluorescence in alkali is not modified by the addition of neutral salts up to molar concentration. This difference with the acid dissociation may give an indication of the nature of the groups that bind the subunits together. If some of these have a pK of 9-11, the binding form being the one present at the lower pH, the disappearance of this38 POLARIZATION OF FLUORESCENCE form would result in dissociation even if the electrostatic repulsion is nzinimized by the addition of salt. On the other hand, in the acid dissociation the bonds would be broken by the electrostatic repulsion when the dissociation of the carboxyls is suppressed but could be restored if the approach of the subunits is made possible by addition of salt since the binding groups are present in the appropriate ionic form.The results just quoted and others obtained by Dr. T.-C. Tsao in his study of muscle proteins indicate that observations of the polarization of the fluorescence of labelled protein molecules is a particularIy useful tool in the investigation of those r A I I / t z 1 3 14 1 5 1 6 I FIG. 2.-Polarization of the fluorescence of 0-2 % bovine serum albumin conjugates. 1, pH 7.0, 0.1 M phosphate buffer. 2, pH 1-5, 0.2 M acetate + HCl buffer. 3. pH 1.82, 0.15 M HCl + KCl. 4, conjugate dialyzed for 150 h against distilled water and brought to pH 1-8 by 2 and 3 are curves convex towards the T/v axis showing thermal dissociation ; 4, where addition of HCl.presumably dissociation is complete, shows no detectable curvature. protein reactions that result in conspicuous changes of the molecuiar size and/or shape. Perhaps the most important feature of this method is that it can be used in the absence or presence of salt, and also over a wide range of both pH (1.5-14) and temperature (0-60"). Also the study of protein interactions is considerably facilitated because it is possible to label one molecular species and study its behaviour in a complex protein system provided this is non-fluorescent. Within wide limits the polarization of the fluorescence is independent of the concentration so that the protein concentration may be ignored in all cases where interactions between the labelled protein molecules is negligible. As indicated by Perrin 4 the molecular rotations are independent of the simultaneous translations although the converse is not true.It may therefore be expected that interactions which appear as changes in the translational diffusion with concentration may not be observed in a studyG . WEBER 39 of the rotational diffusion alone. This is exemplified by the successful use of solutions of high protein concentration in the study of dielectric dispersions.8 Conjugates of native bovine serum albunlin and ovalbumin showed polarizations which were independent of the protein concentration at all the concentrations investigated (2 % to 0.05 %). When p1/7 is greater than 50 an increase in temperature over the experimental range cannot be expected to increase materially the depolarization.The range of molecular sizes that can be studied depends upon the lifetime of the excited state of the fluorescence of the label. With l-dimethylamino-naphthalene-5-sulphonyl chloride the upper limit corresponds to globular molecules of molecular weight 300,000. It is possible that by the use of other labels with longer lifetime of the excited state molecules of 2 to 3 times this size may be studied. Fluorescent conjugates have been used by Coons 10 to trace the fate of anti- bodies in animal tissues. Besides the general use of labelled proteins as tracers other applications may ultimately be of importance. A study of the absorption spectra of the conjugates may give indications regard- ing the nature of the protein environment that surrounds the attached groups, and its changes under varying conditions.Such changes have been observed following the denaturation of ovalbumin conjugates by heat.6 Parallel observations on the fluorescent spectrum and yield may give much important information on the same matter. The accessibility of the protein surface to certain ions and molecules that are quenchers of the fluorescence may also be explored. For example, H ions which quench the fluorescence of the naphthalene sulphonates and its sulphonamido derivatives at pH 4 fail to affect the fluorescence of the conjugates even at a much lower pH, but do so on addition of neutral salts. A quantitative study of this effect will no doubt give valuable evidence relating to the ionic atmosphere surround- ing the protein. The use of the polarization measurements in the study of the reversible adsorp- tion of fluorescent dyes to macromolecules has been discussed recently by Laurence. By combining the polarization measurements with observations of the absorption spectrum and the yield of the fluorescence Laurence was able to characterize the binding sites in bovine serum albumin as basic groups imbedded in a lipophilic environment. 1 Perrin, J . Physique Rad., 1926, 7, 390. 2 Perrin, J. Physique Rad., 1936, 7, 1. 3 Weber, Biochem. J . , 1952, 51, 145. 4 Perrin, J. Physique Rad., 1934, 5, 497. 5 Perrin, Acta Physic. Polon., 1936, 5, 335. 6 Weber, Biochem. J., i952, 51, 155. 7 Laurence, Bioclzem. J., 1952, 51, 168. 8 Oncley, Chem. Rew., i942, 30, 433. 9 Scatchard and Black, J. Physic. Chein., 1949, 53, 88. 10 Coons and Kaplan, J . Expt. Met/., 1650, 91, 1.

ISSN:0366-9033    

DOI:10.1039/DF9531300033

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

THE APPLICATION OF SOLUBILITY MEASUREMENTS TO THE STUDY OF COMPLEX PROTEIN SOLUTIONS AND TO THE ISOLATION OF INDIVIDUAL PROTEINS BY J. S. FALCONER, D. J. JENDEN,* AND D. B. TAYLOR -f Division of Pharmacology and Experimental Therapeutics, University of California Medical Centre, San Francisco, and Physiology Department, Medical School, King’s College, Newcastle-upon-Tyne Received 2nd May, 1952 The influence of total protein concentration on the separability of two proteins is discussed theoretically and rules governing the use of this variable in protein separation deduced. The possible types of specific property solubility test are given, and their use in conjunction with the above rules stated. A new method for the fractionation of rat liver based on the insolubility of proteins in strong salt solutions is described, with some preliminary results of the application of solubility methods to one of these fractions.Solubility experiments on complex protein mixtures can provide valuable information about their composition, and as a result prove useful in separating individual proteins. Moreover, they can provide us with some of the most critical and sensitive criteria of purity which it is possible to obtain. While the meaning of the term “ pure ”, as it is applied to a protein is funda- mental, it has been discussed previously 1 and will not be enlarged upon. From the standpoint of this paper, it seems more useful to examine theoretically some less explored portions of the problem of protein separation and of the analysis of complex protein mixtures.It has long been known that many intracellular proteins and enzymes are not in free solution in the cell but are attached to and form a part of organized cell structures. Before a homogeneous true solution of cytoplasmic or nuclear proteins can be obtained a series of problems relating to tissue disintegration and handling have to be solved. Unlike solubility measurements, these problems cannot be covered by a general theory; rather the methods used should be based on the observance of relevant biological principles, and it will be part of the object of the second section of this paper to comment on some recent developments in this field. In general, the variables which we must recognize in determining a protein solubility are pH, temperature, total protein concentration and concentration of reagent used to alter the solubility of the protein.Experimentally the solubility may be measured as a function of any one of these variables, while the others are held constant ; studies relating solubility either to total protein concentration or to salt concentration have been most extensively used. The former, in which the protein solubility is measured as a function of total protein concentration at con- stant pH and temperature in a solvent of constant composition, can be referred to as a constant solvent test 2 , 3 and is beautifully illustrated by the classical work of Northrop, Kunitz and Herriott.4 If the composition of the solvent is used as the independent variable while pH, temperature and total protein concentration are * United States Public Health Service Fellow.t Introductory section by J. S. Falconer, D. J. Jenden and D. B. Taylor ; experimental section by D. J. Jenden and D. B. Taylor. 40J . S. FALCONER, L). J . JENDEN AND D . B . TAYLOR 41 held constant, we have the variable solvent test. The use of this is demonstrated by the very accurate measurements of Roche and Derrien.5 We cannot expect a variable solvent solubility test to do much more than indicate a lower limit to the number of proteins in solution and give some very approximate idea of the concentration of some of these. The presence of an inflection on the salting-out curve indicates the appearance of a new solid phase, but proteins present in low concentration will not necessarily be detected by gross methods such as nitrogen analyses.Moreover the adsorption of a soluble protein onto, or solution in, a solid phase already precipitated may render detection impossible. The relative positions of inflections do not provide us with a means of identifying a protein, for change in individual protein concen- tration may reverse the order of appearance of saturation points of two proteins. The specific property test was introduced because of the desirability of giving full weight to the important activities and characteristics of individual proteins. These characteristic properties are often extremely specific, and for enzymes suffi- ciently marked to enable detection and measurement to be made in very dilute solution. tion may sometimes be reduced by the sacrifice of active material, the consequences of this should be clearly recognized.If the losses during an isolation are extensive the final product will be a highly selected sample. If the molecules of the original protein are all identical this does not matter, but in many cases, we may be isolating one of a group of proteins all of which have some activity. For example, crystalline catalase 6 has been shown by Brown 7 3 8 to be a mixture of proteins with varying enzymatic activity in attacking hydrogen peroxide, and he has pointed out that some of the disagreement in the literature may have been due to different workers isolating selected samples of the original material. There is evidence that more than one pig liver esterase exists, but the activities of the various enzymes isolated seem to be very similar.2 A clearer understanding of the factors influencing yield in fractionation procedures therefore seems desirable.This problem is bound up with the influence of total protein concentration on the separability of saturation points of individual proteins in a variable solvent test and will be considered in the next section. property solubility test enables the optimum pH, temperature, salt and protein concentration for purification to be estimated. The methods for estimating the first three have already been discussed,2s 3 but the influence of total protein con- centration requires further quantitative calculation. In general successive steps in an isolation tend to increase in difficulty because the remaining impurities are those whose solubility characteristics resemble the protein to be isolated.At the same time, however, the system becomes simpler and therefore more susceptible to analysis. Ultimately we shall be concerned with the separation of two proteins, and we can therefore simplify our calculations to the influence of total protein concentration on the separability of two proteins. The relation between the solubilities of the great majority of proteins and the salt concentration is exponential or nearly so; it follows that in salting-out, the precipitation range of every protein in solution overlaps that of every other protein present. In separating proteins by salting-out, therefore, the problem to be solved is to determine the experimental conditions which reduce precipitation range over- lap to a minimum. Moreover, if two proteins occur in such concentrations that their saturation points occur at the same salt strength, no single fractionation procedure, without alteration in protein concentration, can yield any pure protein.To obtain the maximum yield of pure protein the conditions for maximum separa- tion of saturation points should be determined. The following calculation is useful in that it provides information on the influence of total protein concentration on the ratio of the amounts of the two proteins present as a function of salt strength. THE PROBLEM OF YIELD IN PROTEIN ISOLATlON.-~hile the difficulties Of a11 iSOla- THE USE OF THE SPECIFIC PROPERTY TEST IN PROTEIN ISoLATION.-The SpeCifiC €342 S o L u B I L i r y MEASUKEMENI s Cohn's equation relates solubility S to salt strength I when /3 and k are con- stants as follows : S = exp (p - - Ad).From this the ratio R of impurity to enzyme in a solution saturated with respect where the suffixes E and I refer to enzyme and impurity respectively. On differentiation, dR/dI =;: (kE - kj)R, so that if ki > kE, dR/dl is negative and R decreases with increase in salt strength; on the other hand, if k E > ki, R must decrease with increase in salt strength. For given amounts of two proteins alteration in R must result in separation of saturation points which occurred at the same salt strength, reduced overlap of precipitation ranges and absolute separ- ability of the two proteins. Even when the saturation points are already separate, they may be further parted by altering the total protein concentration in the direction indicated by the relative values of the constants k .It is clear, therefore, that alteration in total protein concentration can be used in separating proteins. It to both is given by R - exp (pi - ,BE -- I(ki -- k ~ } ) , FIG. 1 .-Possible types of specific property solubility test. Vertical axes represent protein in solution ; horizontal axes represent activities. also follows that if ki = kE there is nothing to be gained by altering the total pro- tein concentration, and also that the conditions which make the difference between ki and k~ a maximum enable the greatest benefit to be derived from alterations in protein concentration. Since there will always be some overlap of precipitation ranges the yield of pure protein in the final steps can never be theoretical; its actual value will depend on the limits of the range of protein concentration over which we can work and on the inevitable losses which occur during fractionation.The experimental limits to the range of protein concentration we can use are worth considering. It is usually not practicable to work with protein solutions more concentrated than can be prepared by dissolving a well-pressed moist filter cake in an equal weight of water; such solutions contain about 10-30 % protein. On the other hand, the general procedures of protein purification such as fractionation, etc., are not par- ticularly satisfactory when the total protein concentration goes below 0.5 mg protein per ml.A several hundredfold concentration difference is therefore about the maximum range available for use in fractionation procedures. proteins we can reduce the possible types of solubility tests to four which are illustrated in fig. 1. The linear parts of curves I1 and IV represent salting out of PO§§IBLE VARIATIONS OF SPECIFIC PROPERTY TEST.-Again considering Only twoJ . S. FALCONER, I ) . J . JENDEN A N D D. B. 'TAYLOR 43 pure enzyme and the slopes of these lines provide an estimate of its specific activity, while in I and 111, the linear portions are due to the precipitation of impurity with- out any concomitant precipitation of enzyme. As the curved portions of I and I11 approach thc origin, the ratio of enzyme to impurity steadily increases, the constant k of Cohn's equation for the impurity being greater than the constant k for the enzyme.In I11 and IV the reverse is true and ke > k'. The slope of the tangents to I and 11 at the origin also provide an estimate of the specific activity of the enzyme. No such estimate of the specific activity can be deduced directly from 111. In this case, removal of non-overlapping impurity followed by dilution should give a mixture from which pure enzyme should salt out first as in IV. EXPERIMENTAL It is clearly desirable for studies on the fractionation of cell proteins to start with the separation of the particulate cell components. Each fraction can then be extracted separ- ately and the soluble proteins produced subjected to the available analytical procedures.The methods available for the initial particulate fractionation are broadly divisible into two classes : (i) those employing aqueous medias-14 all of which will dissolve proteins, and cannot be relied upon to leave all proteins in their natural cell components ; (ii) those employing non-aqueous media, viz., the Behrens procedure and its modi- fications.15-17 Mixtures of carbon tetrachloride and cyciohexane or other organic solvents are used to suspend the material for differential centrifugation, after preliminary lyophilization and homogenization in a ball-mill. These procedures are lengthy (1-2 days), and again leave room for doubt as to possible protein losses ; for example, lipo- proteins will almost certainly be dissociated, and catalase has been shown to lose much of its activity after freeze-drying.18 Before a cell fractionation process can be accepted as completely adequate for the systematic examination of cell proteins it must satisfy at least four criteria : (i) Each cell fraction must contain all the proteins in the proportion in which they occur in association with it in the living cell.(ii) The yield must be high. In addition to the desirability of a high yield from the standpoint of reducing initial bulk, it is essential in order to avoid the possibility of selection.19 (iii) Each fraction should be pure as regards freedom from extraneous particulate matter and dissolved or adsorbed proteins from plasma or other cell fractions. (iv) Proteins must be stable in the medium and under the conditions used.This point has already been commented upon in connection with the Behrens procedure, but pre- vention of autolysis and degradation resulting from natural instability must be prevented. This may be facilitated by speed and by working at low temperatures. THE AMMONIUM SWLPHATE TECHNIQUE FOR THE SEPARATION OF RAT LIVER NUCLEI.-- Method-A rat which has been fasted overnight is killed and bled. The liver is removed as fast as possible and forced through a perforated steel plate (holes, 0-015 in. diam.) by means of a press into three times its volume of saturated ammonium sulphate at 0" C . The suspension is then homogenized for 3-4 min in a precision bore glass tube (int. diam. 1 in.) with a Plexiglas rotor (diam. 0-998 in.) at a speed of 780 rev/min.Cooling is effected by immersing the tube in a sodium chloride+ice mixture. After dilution of the homogenate with about half its volume of 0.8 saturated ammonium sulphate it is filtered through one layer of Irish linen (63 threads to 1 in.) and transferred to a refriger- ated centrifuge to be spun at 20,000 g for 10 min. This procedure separates the homo- genate into nuclei, which are packed in the bottom of the tube, cells, mitochondria and precipitated proteins which are packed at the top, and a brownish fluid of varying clarity between. The exact concentration of ammonium sulphate necessary to effect this separa- tion is almost always between 0.8 and 0.9 saturated provided that a fasted rat was used, but must be adjusted to suit each batch if necessary.If the rat has not been fasted, it has been found that the entire homogenate generally sink, even in saturated ammonium sulphate, so that fractionation is impossible. If the two fractions obtained are resuspended and centrifuged under the same con- ditions, the sediments and floating layers separately recombined and the procedure re- peated, further cleaning of the nuclei can be achieved. The rapidity and completion of this process can be facilitated at some sacrifice in yield by increasing or decreasing the44 salt concentration in cleaning the lower or upper fractions respectively, and by reducing the time of centrifugation. If the number of intact cells in the upper fraction is large, this is removed and rehomogenized ; the number of nuclei destroyed in this procedure is small since most of them have already been separated.The entire process as described can be completed in about 1 h. Preliminary experiments show that other salting out agents, e.g. potassium phosphate, 3-5-45 M, containing equal molar proportions of the monobasic and dibasic salts, may also be used. DISCUSSION OF METHoD.-Although it is not claimed by any means that the above technique satisfies all the criteria mentioned earlier, it has some advantages over previously described methods. Differential floating on a dense medium has been used in Behrens type procedures, and achieves a very high degree of separation, while the present method is shorter. The stability of proteins in strong ammonium sulphate solutions is well known,4 and filter cakes prepared from such suspensions can be kept for long periods of so L u B I L 1.r Y M E A s u R EM E N T s FIG.2.Variable solvent test on extract of some soluble rat liver proteins. = optical density at 280 mp, + = optical density at 405 mp, and 0 = pg nitrogen per ml. time at low temperatures. The rapidity with which the tissue is brought into intimate contact with saturated ammonium sulphate ensures that protein loss from all fractions is minimized, since practically all proteins are insoluble in the 0.8 saturated salt. The proportion of three volumes of saturated salt solution to one of liver was chosen to give a final concentration of 0.8 or larger after dilution with the liver fluid. A further advantage of this method is that it allows temperatures below 0" C to be maintained during the pro- cessing owing to the depression of freezing point by the strong salt ; in this way autolysis may be considerably slowed or stopped, especially since it is probable that the enzymes responsible for it have been precipitated.Preliminary perfusion of the liver with saline has been used by several groups of investigators in order to remove red blood cells and plasma proteins ; this has not been used in the present method, partly because the red cells are lysed by the strong salt solution, and partly because perfusion almost certainly removes some protein from the liver cells, while at the same time it increases autolysis owing to the delay.J . S. FALCONER, D. J . JENDEN A N D D . B . TAYLOR 45 Certain disadvantages must be admitted ; adsorption of the soluble cytoplasmic pro- teins on to particulate fractions almost certainly occurs, and some plasma protein remains to contaminate the fractions.PROTEIN FRACTIONATION ExPEmMmTs.-In a protein isolation the repeated fractiona- tions used tend both to reduce the complexity of the system and to leave the impurities behind which are harder to separate. The increasing difficulty of eliminating residual impurities is offset by the fact that specific property test analysis is greatly facilitated by the increasing simplicity of the mixture. For complex solutions, however, different methods will have to be used and the present work was designed to study methods by which useful information about such solutions might be obtained. The existence of well-defined points of inflection on salting- out curves, corresponding to the appearance of new solid phase, has been demonstrated.5 If in conjunction with the determination of such a curve, any characteristics of a protein or group of proteins were measured we could obtain information about the distribution FIG.3.-Relation between protein in solution expressed in pg nitrogen per ml and absorption at 405 mp. or pattern of these characteristics with respect to the relative solubilities of the various proteins. In the present study a variable solvent test was carried out on an aqueous extract of the filter cake prepared from the floating layer described in the previous section. The salting-out was studied by estimating the protein nitrogen and absorption at 265, 280 and 405 mp.The curves show a few well-defined inflections and it is possible that increase in the number of points would provide evidence for many more. Those well-defined inflections which occur provide suitable points for fractionation and it seems reasonable that separation and concentration of a fraction followed by further solubility tests would be a better method of study than simply increasing the number of points. Comparison of the material absorbing at 405 mp with nitrogen left in solution shows some of the complexity of the mixture. Catalase will clearly be responsible for part of the absorption at 405 mp but it is probable that adsorbed bile pigments and other haem proteins also contribute. That catalase itself is not a single protein seems to be estab- lished 20 and its salting-out curve has been experimentally investigated.7, 8 Comparison of protein nitrogen and of material absorbing at 405 mp indicates that the latter is chiefly associated with protein salted-out at the beginning and end of the curve.Investigations of this type, designed to develop analytical methods for complex protein solutions, are proceeding.46 GENERAL DISCUSSION This work was supported by a grant from the Medical Research Committee of the University of California. 1 Eyering, Anal. Chem., 1948, 20, 96. 2 Falconer and Taylor, Biochem. J., 1946, 40, 855. 3 Falconer and Taylor, Proc. llth Int. Cong. Pure Appl. Chem., 1947. 4 Northrop, Kunitz and Herriott, Crystalline Enzymes (Columbia University Press, 5 Roche and Derrien, Proc. llth Int. Congr. Pure Appl. Chem., 1947. 6 Sumner and Dounce, J. Biol. Chem., 1937, 121,417. 7 Brown, Ph. D. Thesis (University of London, 1950). 8 Randall, Proc. Roy. SOC. B, 1951, 138, 301. 9 Douuce, J. Biol. Chem., 1943, 147, 685. 10 Claude, J. Expt. Med., 1944, 80, 19. 11 Schneider, J. Biol. Chem., 1946, 165, 585. 12 Mirsky and Pollister, J. Gen. Physiol., 1947, 30, 117. 13 Schneider and Peterman, Cancer Res., 1950, 10, 751. 14 Wilbur and Anderson, Expf. Cell. Res., 1951, 2, 47. 15 Behrens, 2. Physiol. Chem., 1932, 209, 59. 16 Mayer and Gulick, J. Biol. Chem., 1942, 146, 433. 17 Allfrey, Stern, Mirsky and Saetren, J. Gen. Physiol., 1952, 35, 529. 18 Dounce and Howland, Science, 1943,97,21. 19 Dounce, Ann. N. Y. Acad. Sci., 1950, 50, 982. 20 Sumner, Adv. Eng., 1941, 1, 163. New York, 1948), 2nd ed.

ISSN:0366-9033    

DOI:10.1039/DF9531300040

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

46 GENERAL DISCUSSION GENERAL DISCUSSION Dr. P. Flodin (Inst. Biochemistry, Upsala) said: During work on the elimina- tion of adsorption in fdter paper performed at the Institute of Biochemistry, Upsala, attempts have been made to give the surface of the paper the same charge as the substances to be investigated. By repulsion the substances would then be prevented from coming into contact with the surface at which secondary forces may cause adsorption. Hoffpauir and Guthrie 1 have treated cotton with 2-aminoethylsulphuric acid to give it anion-exchanging properties. When filter paper was treated in the same manner it was found to keep its wet strength and its water-absorptive property and was generally suitable for filter paper electrophoresis in the same way as ordinary paper.The presence of free amino groups was demonstrated by its strong reaction with ninhydrin. To make the paper suitable for experiments with positively charged substances it had to be converted to its -NH3+ form with e.g. hydrochloric acid. Positively charged proteins, e.g. serum albumin, P-lactoglobulin and legumin at low pH, have been run without detectable adsorption although these proteins are strongly adsorbed on untreated paper. Surprisingly it was found that even negatively charged proteins could be run without irreversible adsorption. Blood serum has been separated into components at pH 8-6 in a few experiments on pre-treated paper. The possibilities of this modified paper for use in zone electrophoresis and chromatography will be further investigated.Dr. R. Consden (Canadian Red Cross Memorial Hospital, Maidenhead) said : A simple paper electrophoresis apparatus, which is in regular use for routine investigations of pathological sera and for ionophoresis of amino-acids, peptides and carbohydrates, consists of a rectangular glass tray 35 x 20 cm* and 5 cm in depth, in which is placed a close-fitting rectangular glass frame. This frame is constructed of two rectangles of glass rod mounted one over the other, and 1 Hoffpauir and Guthrie, J . Biol. Chern., 1949, 178, 207.GENERAL DISCUSSION 47 separated from each other by about 1.5 cm by sealed-on pieces of glass, or by pieces of cork. The frame stands on short legs, and it carries the paper sheet and keeps it stretched horizontally. The free ends of the paper pass over the edges of the tray and dip into Perspex boxes containing electrolyte and electrodes.After the paper is wetted with electrolyte, and the solution to be analyzed is applied, an organic liquid (e.g. chlorobenzene) is poured into the tray until the paper is covered. Glass pieces are placed in the electrolyte boxes to keep the paper ends away from the electrodes, and the whole tray may be covered with a glass lid. Electrophoresis may be carried out either across the shorter width of the tray or across the longer. Voltage gradients are 10-1 1 V/cm in the former method, and 6-7 V/cm in the latter. The organic liquid prevents evaporation of electrolyte from the paper, and acts as a cooler. The frame is convenient for dealing with wet paper, after the electrophoresis, and eliminates undesirable contamination by contact with the hands, when chromatography is to be subsequently carried out.For a given voltage gradient and temperature, movement is linear with time, and good repro- ducibility for a given substance is obtained. Dr. E. L. Durrum (Army Medical Service Grad. School, Washington, D.C.) said : Professor Tiselius has provided a very complete list of references. However, a paper by v. Klobusitzky and Konig 1 has recently come to our attention in which a paper strip electrophoresis technique developed in 1937 by Konig was used in 1939 to separate a yellow pigment fraction in snake venom. It is interesting to note that this work was done before paper chromatography became so widely used. Also, in connection with the work of Svensson and Brattsten and the work of Grassmann and Hannig on continuous electrophoresis it may be in order to call attention to work of Haugaard and Kroner, who in 1948 applied for a United States patent 2 which has been issued only recently and which clearly discloses the principle of this type of separation independently arrived at by these workers.I should like to comment on one other point which Prof. Tiselius has mentioned. That is the difficulties due to tailing caused by adsorption phenomena. Except in a few specific cases, in our own experience with the free hanging strip technique, tailing attributable to adsorption is less commonly encountered than that type due to paper overloading. Yet another type of tailing appears to be related to the following : when paper is supported on one or both sides, even when the supporting surface is siliconed, a thin film of electrolyte lies at the interface between the paper and supporting medium in which convection may not be efficiently prevented and in which film the mobility may be different from that in the body of the paper.In our opinion, this is often the reason for the so-called tailing encountered in closed strip techniques. On the other hand, when the strip hangs free, this con- dition is not present, and we believe this to be the explanation why in general zones are more sharply defined in this variation. Of course, if it is desired to measure mobilities, the free-strip technique (where evaporation is permitted) can be used with confidence only if reference substances of known mobility and similar adsorption properties are studied simultaneously, and it is probable that even under the best conditions measurements of this type are not so satisfactorily made as in the method where the strip is totally enclosed and evaporation thus prevented, using, of course, corrections for electroendosmosis and those for migration path length proposed by Kunkel and Tiselius.Dr. D. L. Mould (Rowett Research Institute) said: Prof. Tiselius has men- tioned the application of electro-osmosis for " zone ultrafiltration ". A micro- method has been developed for the fractionation of uncharged molecules such as enzyme-synthesized polysaccharide preparations.3 Electro-osmotic streaming 1 von Klobusitzky and Konig, Arch. exper.Path. Pharmakol, 1939, 192, 271. 2 U.S. Pat. 2,555,487 issued to G. Haugaard and T. D. Kroner, filed on 27th February, 3 Mould and Synge, Biochem. J., 1951, 50, 11 ; Analyst, 1952 (in press). 1948.48 GENERAL DISCUSSION forces a solution through a strip of collodion ultrafiltration membrane and ad- sorption and the molecular sieve effect of the porous membrane structure both tend to retard the larger molecules. Migration zones are formed related to the DP of the synthesized polysaccharide. Dr. E. Barbu (Paris) said: We are interested in the possibility of paper zone electrophoresis giving some indication of the size and shape polydispersity of protein aggregates. Protein aggregates of different sizes can show the same mobilities in boundary electrophoresis but by paper zone electrophoresis we can differentiate the larger aggregates which migrate more slowly than the smaller.An example is shown in fig. 1, 2 and 3 of the paper by Barbu and Joly. (For further details, see Barbu, Macheboeuf and Rebeyrotte, Bull. Soc. Chim. Biol., 1952 (in press)). Dr. L. Robert (Facufte' de Medecine de Paris) said : The Maillard reaction may be used with advantage for the qualitative and quantitative estimation of proteins or amino acids in paper electrophoresis. On exposing the dried paper strips to acetaldehyde vapour in a closed system, the well-known reddish-brown, highly fluorescent, pigment is formed and this may be eluted and quantitatively estim- ated by fluorimetry. This method can be made more sensitive by treating the paper strip with a dilute solution of morpholine, before exposing it to acetaldehyde.The method is carried out in our laboratory as follows : (i) after electrophoretic migration, the filter paper strips are dried, followed by (ii) immersion in a 4 % solution of morpholin in methylal, for 5 min, (iii) immersion in pure methylal to remove the excess of morpholine for 4 min, (iv) immersion in ether for 4 min, (v) drying in air, (vi) exposure to acetaldehyde vapours. For this purpose a 20-30 % solution of pure acetaldehyde in methylal is recommended in a wide Petri dish. The paper strips are placed on glass frames and covered with glass jars. About 15-20 min exposure to acetaldehyde vapours is usually sufficient to give a fairly intense pigment formation. If not sufficiently rinsed, the paper turns brown, because morpholine alone gives with acetaldehyde (in absence of proteins) a reddish-brown pigment.Dioxane, however, removes this pigment without extracting the protein pigment. It may be mentioned that paper strips when treated with the acetaldehyde+ morpholine pigment (prepared by distilling acetaldehyde into a morpholine + ether solution when the pigment separates out and may be used in a butanol solution) a somewhat lighter coloration of the protein is obtained. This modification has the advantage that the pigment bound by the protein is much more easily eluted than the pigment formed with the protein. Elution may in fact sometimes present a problem which may be overcome only by a careful choice of solvents. Pigment formed with aminoacids, casein, and some other proteins are readily soluble in butanol t- acetic acid + water (2/1/2) mixture at 100' C or in butanol + piperidine + water (2/1.5/2).Form- amide is also a suitable solvent, Serum-protein pigments are much more difficult to obtain in solution. We are trying a special fluorimeter based on the same principles as the ordinary densitometers used in paper electrophoresis. Quanti- tative data and other details will be published elsewhere. It should be mentioned that this principle may also be applied to paper chromatography. The use of fluorescence in the quantitative estimation may possibly widen the present con- centration limits in paper electrophoresis. Dr. A. G. Ogston (Oxford University) said: Dr. Weber has attempted to ex- plain the lack of effect of dilution upon the apparent degree of dissociation of albumin by assumptions about the kinetic orders of the association and dissoci- ation reactions.Whatever such assumptions are made do not affect the require-GENERAL DISCUSSION 49 ment that a dissociation equilibrium must obey thermodynamic laws. These demand that, if dilution does not affect the equilibrium, the reaction does not affect the total number of solute particles. It would, therefore, be necessary to assume that some other substance, which is also diluted as the albumin is diluted, takes part in the dissociation reaction; for examples, if A2, A are the associated and dissociated a1 bumin, A~ + BJAB + A or A2 -r B2 7 2AB. Dr. K. 0. Pedersen (Lrpsala) said: I wonder if bovine serum albumin really dissociates in acid solutions under the conditions given by Dr.Weber in his paper. In some studies I made some years ago on the sedimentation, diffusion and viscosity of bovine serum albumin in acid solutions I found that a pronounced change in the protein molecule takes place in the region pH 2.3 to pH 3 (temperature 10" to 35" C). Contrary to my expectation I had to assume that the molecular weight of the serum albumin remained constant. I had to explain the decrease in the sedimentation and diffusion coefficients, and the increase in the relative viscosity by assuming a partly reversible unfolding of the molecule. Increase of the ionic strength of the solution from 0.1 to 0.2, or from 0.2 to 0-3, diminished the effect thus indicating the presence of electrostatic charge effects even in solutions having an ionic strength of 0.1 and 0.2.I think one must be very careful in drawing conclusions from single ultra- centrifugal, diffusion or osmotic pressure experiments in this pH region without knowing more about the magnitude of the electrostatic charge effect. Dr. B. Robert (Institut Pasteur, Paris) said: Barbu and Macheboeuf have demonstrated that most proteins change their native configuration at pH values above 9. Their physical and chemical properties alter instantaneously in alkaline media. We were able to show 1 that this configurational modification rendered accessible the -SH groups of serum albumin, which could reduce ferricyanide at pH 8.5 and were oxidizable by air above pH 10. Kinetic analysis of the oxidation of alkaline protein solutions allow some conclusions to be drawn concerning the mechanism of the structural change occurring in proteins at these high pH values.We found, for example, that the dissociation of a proton (with a pK value of about 13) is essential for autoxidation. We concluded that guanidine groups of arginine and cystine S-S bridges are concerned in maintaining the stability of serum albumin molecules in agreement with the finding of Klotz et al. Structural changes occurring above pH 9 are irreversible because of the above- mentioned oxidation of the molecule. It is therefore possible to obtain this struc- tural change in a reversible manner when such side reactions are avoided. Dr. G. S. Adair (Low Temperature Statiorz, Cambridge) said : Measurements of solubilities and specific properties described by Falconer, Jenden and Taylor give one of the most sensitive tests for the purity of a protein.I should like to amplify one point. In experiments made with varying amounts of protein and a constant solvent, two procedures are possible. (i) The molality or the ratio of salt to water can be kept constant. (ii) The activity or effective concentration of the salt may be kept constant. A close approximation to the second procedure might be obtained by dialyzing a mixture of crystals and mother liquor against a salt solution, and using mixtures containing crystals and dialysate for solubility tests. The difference between methods (i) and (ii) may be insignificant in experiments made with small quantities of protein, as used in specific property tests. The second method may prove useful for work on more concentrated solutions of proteins. Dr. L. Nanninga (Leiden) said: From fig. 1, curves I and 11, of Falconer, Jenden and Taylor's paper, it follows that Se/Si increases when cprot. decreases. 1 Robert and Macheboeuf, Bull. Sur. Citeni. Biol., 1953, 35 (in press).50 GENERAL DISCUSSION However, I do not agree that it follows that ki > ke, since the values j3i and in the solubility formula are not accounted for. As I remains constant with varying cprot. there can be no differentiation with respect to 1 here. Dr . D . B . Taylor (California University) said : The differentiation referred to by Dr. Nanninga was of the equation relating the ratio of the solubilities of the enzyme and of the impurity to the ionic strength. These solubilities are ex- ponential functions of ionic strength in accordance with Cohn’s equation. The differentiation would therefore appear to be in order. The /3 constants do not appear in the equation since we are concerned with the rates of salting out of the proteins as a function of salt strength.

ISSN:0366-9033    

DOI:10.1039/DF9531300046

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

11. LOW MOLECULAR WEIGHT PROTEINS THERMODYNAMICS OF THE ASSOCIATION OF INSULIN MOLECULES BY PAUL DOTY AND GEORGE E. MYERS* Gi bbs Laboratory, Harvard University, Cambridge, Mass. Received 19th May, 1952 Light scattering studies of insulin solutions at pH 1.9 and 2.6 in 0.1 M NaCl and NaHzP04 permit the evaluation of equilibrium constants governing the equilibria among monomers, dimers, and trimers. The variation of the constants appears to be explicable in terms of changes in the electrostatic repulsion. Measurements in the range of 20" to 40" C lead to the determination of the corresponding heats and entropies. These are found to be surprisingly low and suggest that increased solvation of the monomer with respect to the dimer is taking place. In a previous publication 1 it has been shown that the variable particle weight exhibited by insulin in acidic solutions is due to the existence of simultaneous equilibria involving monomer, dimer, trimer, tetramer and possibly higher polymers.The monomer is taken as the 12,000 molecular weight unit. Below a pH of 2.2 it was found that dissociation of the polymers was virtually complete except for dimer. Thus in this region the monomer-dimer equilibrium was isolated and its behaviour could be characterized by a single equilibrium constant. In this paper we report more precise determinations of this equilibrium constant at an ionic strength of 0.1 in NaCl and NaH2P04 and at pH values of 1.9 and 2-6 in order to define quantitatively the influence of anion type and pH on the equilibrium. Under these same conditions we have also determined the temper- ature dependence of the equilibrium constant providing values of the heat and entropy of dissociation of the dimer.At the higher pH in NaCl solution, some trimer appeared and consequently information on the equilibrium involving the trimer was obtained. This investigation, like the one just summarized, has been carried out using the measurement of the reduced intensity of scattered light R90 for the deter- mination of the weight average molecular weight. The relation between these two quantities is given by the basic light scattering equation : Kc/R90 = 1/M + ~ B c , (1) where K = 2712n02 (dn/dc)2/Nh4, no being the refractive index of the solvent, dn/dc the refractive index increment of the solution, N Avogardo's number, and h the wave length of light.The concentration in g/ml is given by c ; B represents the second virial coefficient which characterizes the extent of deviation from ideal solution behaviour. Although reference must be made to reviews 2 for detailed discussion of this method two points deserve emphasis here. First is recognition that the insulin particles met with in this investigation are sufficiently small to scatter symmetrically about 90" thereby requiring only measurements at this one angle to characterize the scattering. The second point is that with the average particle weight being a function of concentration it can only be determined from *Public Health Service Research Fellow of the National Tnstitutes of Health, 195 1-2. 5152 ASSOCIATION OF INSULIN eqn (1) within the limits within which B is known to approach zero.If there were no attractive forces operating among insulin molecules in acid solution, the upper limit of B could be estimated from the well-known Donnan term. How- ever, the limited solubility of insulin as well as the observation that the mono- meric units attract sufficiently to form aggregates indicates that B must be quite small and perhaps slightly negative. If this assumption is not correct, it will show up by the slope at high concentrations taking on a value that is incom- patible with the equilibrium constants deduced from the data at lower con- centrations. EXPERIMENTAL INSuLIN.-The insulin used in these experiments was pancreatic beef insulin 5 times recrystallized (Lot T-2344) obtained from the Eli Lilly Co., Indianapolis.This is the same material as that reported on previously.1 Its zinc content, as reported by the Lilly Co., is 0.59 % ; it was not removed. EXTINCTION COEFFICIENT.-The value of the extinction coefficient determined on the basis of dry weight was found to be 10.9 at the maximum of the 280 mp absorption band. Our concentration measurements are based on this value rather than the previous value 1 of 11.3 since the latter was measured on a different insulin sample. REFRACTIVE INDEX 1NCREMENT.The refractive index increment at A = 436 m p was determined with a Phoenix differential refractometer. The concentration is based on dry weight determinations at 80" C in vacuo. The values reported here are for an approxim- ately 0.8 % insulin solution which was dialyzed against NaCl, ionic strength 0.1, pH 2.6 to remove zinc and possible impurities.Such a procedure necessitated a correction for the refraction of the chloride ions, which are present in excess of those in the salt solution in order to compensate for the positive charge of the protein. Using a value of 9-04 for the molar refraction of chloride ion, the correction applied to the measured refractive index increment is about 4 %. The refractive index increment of undialzyed insulin in NaCl and in NaHzP04 at the same pH and ionic strength were found to be the same. The evaluation of the constant K in eqn. (1) at different temperatures requires know- ledge of the values of the refractive index increment at those temperatures.Consequently, the temperature dependence of the refractive index increment was measured for the dialyzed solutions in sodium chloride and the results are given in table 1. The tem- perature variation is only slightly greater than the probable experimental error and is about the same as has been found for other proteins.3 The values of dn/dc for tem- peratures other than 25" C were taken from the best straight line through the points in table 1 . The value of 0.188 at 25" differs considerably from 0.202 used previously,l but the present value is believed more reliable from the point of view of experimental technique and instrumental calibration and from the fact that the solutions were not dialyzed for the earlier determination. TABLE 1 temperature ("C) 16-0 & 0.3 25.0 0.1 30.0 & 0-3 dn/dc (A = 436 mp) 0-1891 0.1879 0.1874 PREPARATION OF soLuTIoN.-Salt solutions were prepared by dissolving the appro- priate quantity of recrystallized salt in doubly glass-distilled water and adjusting the pH to the desired value.To avoid contact of the protein with concentrated acid or base, sufficient additional acid was added to part of the solvent to compensate for the acid bind- ing of the protein and any slight pH adjustments were then made with dilute acid or base. Both solution and solvent were then centrifuged at 80,000 g for 1 h. LIGHT SCATTERING PHo-roMErER.-Scattering measurements were made on a slightly modified Brice-Speiser light scattering photometer 4 using the blue mercury line ( A = 436 mp). The modi- fications were those involved in thermostating the instrument and in the use of a narrow slit system for the special cells employed. Centrifuged solvent was added to the optically clean photometer cell and the reduced intensity at 90°, R90, was determined.For the dilute (0-0.3 %) range, the centrifuged solution of about 1 % concentration was added stepwise to the solvent, the concentration being determined The calibration of the instrument has been reported previ0usly.4~ 5 Measurements were made in the following manner.PAUL DOTY A N D GEORGE E . MYEKS 53 gravimetrically, the concentration of the centrifuged solution having been determined by extinction measurements. In the more concentrated range the reverse procedure was followed, the solvent being added stepwise to the solution.In every case, stirring was accomplished magnetically using a small glass-encased piece of iron within the cell and the solution in the cell was examined critically at low angles to a strong light beam and rejected if significant optical impurities were present. DESIGN AND OPERATION OF CELLS.-TWO light scattering cells were employed. The first was an optically polished square cell of height 10 cm and internal width 13 mm, obtained from Fisher and Porter Co., Hatboro, Pa. This was modified by sealing an inner ground glass joint to the top, the outer joint being then used as a cap to prevent evaporation and the entrance of dust. The cell was cemented to a square metal base which fitted tightly into the central table of the light scattering photometer.The centring of the cell was checked by measuring the 90" light intensity of a fluorescein solution from all four 90" orientations of the cell. This cell was used for measurements at a fixed temperature ; in this case the photometer and the room were thermostated at 25.0f0-3" C . The second cell permitted relatively rapid measurements at several temperatures. It consisted basically of two cells, one within the other. The inner cell was one of the 13 mm cells described above without the metal base. The outer cell was one of the standard square cells for the Brice-Speiser photometers, 70 mm in height and 30 mm internal width. The smaller cell was placed in the centre of the larger, with the walls of the two parallel and held firmly in position by a Lucite plate at the base and by a Lucite top which fitted tightly into the top of the large cell and around the small cell.All plastic to glass surfaces were cemented by plasticizing the Lucite with a little organic solvent. Two 3 mm copper tubes were screwed through the back two corners of the Lucite top, allowing for the circulation of thermostated water through the space between the inner and outer cells. With the optical slit system employed it was found that the 90" light intensity was inde- pendent of the rate of flow of thermostat water through the cell and of reasonable amounts of dust in that water ; excessive dust and bubbles were removed by passage through a sintered glass filter. Scattering measurements were made on insulin solutions with this cell between 20" and 40" C and they appeared to be completely reversible and reproducible. Prolonged exposure to high temperatures was not necessary since the equilibrium shifts almost instantaneously.Employment of these small cells necessitated the use of a 3 mm slit system rather than the 12 mm system standard with this photometer. Consequently, in order to deter- mine reduced intensities on an absolute scale, it was necessary to calibrate the cells against the standard 30 mm square cells and 12 mm slit system. This is accomplished simply by comparing the 90" scattering of the same solution in both the small and the standard cell ; the ratio of the excess turbidities gives the cell calibration constant. RESULTS EQUILIBRIUM coNsTANTs.-Proceeding in the manner just outlined, we have determined the value of Kc/R90 as a function of concentration at 25" C under the four sets of con- ditions listed in table 2.The data for pH 1.9 and 2.6 in NaH2P04 solution are shown in fig. 1. The data for pH 2.6 in NaCl are shown as the lower points in fig. 2. The lengths of the vertical lines through these points are a measure of the probably experimental error. Although some allowance has been made for the possible contribution of dust and other randomly occurring impurities, in assigning the probable error we must essenti- ally rely upon the reproducibility of our data and the visual inspection of the solutions at very low angles to the incident beam to insure that dust is not contributing significantly. The data can be fitted fairly well by trial arid error assignment of the equilibrium constant as was done previously.However, a more objective treatment is possible.6 If we let x equal the mole fraction of monomer existing as monomer and M be the molecular weight of the monomer (12,000) one can show that the weight average particle weight Mw is related to the concentration by the relation M d In x ---=1+- MW d In c' Consequent I y , In x = Ji (g - 1) d In c. (3)54 ASSUCIATiON OF INSULIN Thus, by nieans of graphical integration x may be determined from the experimental data as a function of c. With the data in this form, it may be used to implement the irdsuLiN T - 2344 SODIUM PHOSPHPiTE +=OI TEMP = 25'C - PH = 1.9 ; K~~ = 7 2 xto-4 MOLESIL 8 0 t - PH = 2.6; K Z I =40 XtO-4 MOLES/L 1 0 5 ~ g ~ - 90 70 6.0 50 0 2 4 10 12 FIG.1. following relation in which K21 is the dissociation constant of the dimer and K31 is the dissociation constant of the trimer into the monomer. 105 x 1 INSULIN T-2344 SODIUM CHLORIDE p = O . l PI{ =2.6 1 - T = 25°C 0 - T = 33°C : _ I Y \ \ 0 - T = 4 0 ° C I I I I I I I I I I - 2 4 1 0 ~ c i 9 / r n l ? 3.5 1 10 FIG. 2. Thus plotting the quotient on the left against x(c/M) should prcduce a line whose inter- cept is 4/&1 and whose initial slope is 9/K31. The appearance of curvature would indicate the existence of polymers higher than the trimer and, providing the precision of the dataP A U L DOTY A N D GEORGE E . MYERS 55 warrant, the equilibrium constants for higher species could be deduced by evaluating the coefficients of higher terms in the series shown in eqn.(4). Treating the data in this manner we find that for the first two cases listed in table 2 a horizontal straight line is obtained confirming our view that only monomer and dimer are present and are in dynamic equilibrium. The values of K21 so obtained are listed. TABLE 2.THE EQUILIBRIUM CONSTANTS FOR THE DISSOCIATION OF INSULIN DIMERS AND TRIMERS AT 25" C (Ic = 0.1) moles/l. cal/mole K321 G l 1-90 NaH2P04 7.2 x 10-4 4320 i 100 - - 2.60 NaH2P04 4.0 x 10-4 4690 & 100 - - 2.00 NaCl 2.5 x 10-4 4930 f 100 10.7 X 10-4 4050 f 100 2.60 NaCl 1.7 X 10-4 5190 k 100 3.4 X 10-4 4770 f 100 moles/l. cal/mole salt KZl Wl PH In the last two cases pH 2.0 and 2.6 in NaCl, the plot of eqn. (4) yields a straight line with a pronounced positive slope.The value of K31 is determined therefrom but in table 2 we list the value of K321 = for use in the discussion. The values for NaCl, pH 2.0, supersede those previously reported,l the difference being due to more stringent pre- cautions in removing dust. The full lines in fig. 1 and the lower curve in fig. 2 are drawn using the values of K21 and K321 from table 2. THE HEATS AND ENTROPIES OF DISSOCIATION.-BY means of the thermostated cell de- scribed above, we have at several temperatures measured R90 at different concentrations for each of the four conditions listed in table 2. When the same solution is measured at various temperatures each term in the quantity Kc/&, varies. Consequently, we have listed these terms separately in table 3 which summarizes the data in NaH2P04 solution.Since it was known from the treatment of the 25" C data in the previous section that only the monomer-dimer equilibrium data were involved here the equilibrium constant for each temperature was obtained in the more direct fashion of calculating a value of the constant for each concentration and averaging the values so obtained at each temperature to provide a mean equilibrium constant for that temperature. This is the way in which the constants in the last column of table 3 have been obtained. A plot of the logarithm TABLE 3.-DATA ON THE TEMPERATURE DEPENDENCE OF THE INSULIN DISSOCIATION pH 1.90 ; NaHzP04 ; p = 0.1 T O C c (moles/l.) x 103 20" 2-07 (K= 5-80 x 10-7) 3.62 6.50 10.09 30" 2-07 ( K = 5.73 x 10-7) 3.60 6.48 10.07 40" 2-07 6-45 10.01 ( K = 5.67 x 10-7) 3.59 Rgo X 105 1-947 3-40 6.57 10.72 1.82 3-40 6.28 10-51 1.731 3.23 5.98 9-86 20" 25" 30" 40" pH 2-60 ; NaH2P04 ; p = 0.1 1-41 1-310 2.70 2.74 1-41 1 -276 2-70 2.63 1-41 1.230 2.70 2.60 1 -40 1-180 2-69 2-50 Kc/Rgo x 105 K21 x 104 6-16 - 5.84 1 5.74 - 5-40 5.82 6.5 1 - 6-05 - 5.90 - 5-50 7.54 6-75 - 6.30 - 6.12 - 5.76 10.6 4-25 - 5-92 3.01 6-40 - 5-92 3-82 6.56 - 5.95 4.28 6.74 - 6-10 5.4356 ASSOCIATlON OF INSULIN of these constants against reciprocal absolute temperature leads to the values of the standard heats of dissociation listed in table 4.TABLE 4.-HEATS AND ENTROPIES OF DISSOCIATION AS,.,, 1.90 NaH2P04 5200 400 3.0 3.0 - - 2.60 NaH2P04 4900 k 500 0.7 + 2.0 - - cal/mole deg. AHL cal/mole salt -4% AS,O, pH (p =0.1) cal/mole cal/mole deg.2.00 NaCl 8100 & 600 12.1 1 2 . 4 8700 & 1000 15.4 + 5 2.60 NaCl 7700 _+- 1000 9.0 i 5.0 8100 -I 1100 19.2 f 14 The corresponding data for NaCl solutions at pH 2.6 are shown directly in fig. 2. Due to the presence of trimer the analytical procedure of the previous section was required. The values of K21 and K321 are plotted as logarithms in fig. 3 and the values of these constants have been used to draw the lines through the constant temperature points in fig. 2. The points in fig. 3 scatter somewhat more than other corresponding plots, partly because a smaller temperature range was employed and because two constants rather than one had to be determined. FIG. 3. The values of the standard heats of dissociation obtained from plots such as that shown in fig.3 and the corresponding values of the entropies obtained from the heats and the free energies listed in table 2 are listed in table 4 together with the precision measures we have estimated for each value. DISCUSSION If we consider first the values of the equilibrium constants listed in table 2, we note that in the presence of both salts the constant is decreased by half when the pH is increased to 2.6 and that at the same pH the constant is twice as great in NaH2P04 as in NaCl solution. These two shifts in the equilibria seem lo be most simply explained in terms of the alteration of the electrostatic repulsion between the like-charged monomers, a point first suggested by Oncley and Ellen- bogen.7 Thus, if we employ the Debye-Hiickel expression for the electricalPAUL DOTY AND GEORGE E.MYERS 57 free energy or the nearly equivalent tabulation provided by Verwey and Overbeek,* we find that for two spheres 20 A in radius carrying 12 charges each (the maximum charge for a monomer unit) the contribution to AGO is about - 4800 cal. Reducing the charge to 11 changes this contribution to - 4100 cal. Thus we estimate that AGO would be increased by about 700 cal if one charge is removed from each monomer unit. The titration curve of insulin at this ionic strength 7 shows that there is a difference of about 0.8 charges between pH values of 1.9 and 2.6. Thus one predicts an increase of 560 cal between the first and second and the third and fourth AGO values in table 2. The agree- ment between these two figures is as good as can be expected taking into account the probable experimental error and the errors inherent in the calculation.The errors in the calculation arise from the failure of the Debye-Huckel limiting law at this ionic strength, the assumption that the charge distribution is spherical and the use of the dielectric constant of water. The differences in dissociation in the two salt solutions at the same pH values can be tentatively assigned to greater chloride ion binding9 In view of the bind- ing of chloride ion observed with other proteins, it would not be unexpected lo find that at this ionic strength about one chloride ion per monomer unit is bound, there by explaining the observed difference. In view of the electrostatic repulsion the trimer would be expected to be less stable relative to the dimer and monomer than the dimer with respect to the monomer.This is borne out by finding that the former dissociation constant is about twice as great as the latter. Although the various geometrical forms which the trimer could take would give rise to different electrostatic contributions, the uncertainties mentioned above in calculating such contributions are so great that an attempt to discriminate among various possible trimer structures does not appear to be justified. Turning next to the heats and entropies listed in table 4, we note at once that the heat of dissociation is remarkably low for an association which persists to such low concentrations. However, further consideration shows that it is the values of the entropies of dissociation that are unexpected.As a basis of com- parison we can calculate the entropy of dissociation of a structureless dimer based upon the gain of translational and rotational freedom. Using the Sackur-Tetrode equation the translational contribution to AS' is found to be 77 cal/mole deg. and taking as a model for the monomer a cylinder 30A in height and 30A in diameter, a contribution of 47 is calculated for the rotational contribution. Upon comparing the sum of these, 122, with the values shown in table 4, it is at once apparent that the dissociation of the insulin dimer is very much different than the simple breaking apart of two monomer units. Although the explanation of thermodynamic data in terms of molecular models is usually hazardous this unusually large discrepancy may possibly justify the speculation that such a large loss of entropy upon dissociation could only come from the simultaneous immobilization of water molecules.The entropy loss due to water being bound can be estimated either directly from the entropy change in the freezing of water or indirectly from the entropy changes found upon charge neutralization by ion binding which is considered to set free bound water.10 By either route one finds that the entropy loss per mole of water bound is about 4.8 cal/mole deg. Thus the observed entropy change could be accounted for by assuming the dissociation reaction to be 12 -1 24 H20 = 2 I x 12 H20. In other words we suggest that upon dissoci- ation the areas of the insulin monomer which had previously been in contact become solvated with about 12 water molecules each. Recognizing that a mono- valent anion can be solvated by about four water molecules,ll about three anionic groups would be required for each monomer. Since 12 such charges exist on the insulin monomer in acidic solution, this requirement could be easily met. The observed difference is about 300.58 ‘TRY PSIN AND RELATED PROTEINS 1 Doty, Gellert and Rabinovitch, J. Amer. Cherrr. SOC., 1952, 74, 2065. 2 Doty and Edsall, Adv. in Protein Chem., 1951, 6, 35. 3 Perlmann and Longsworth, J. Amer. Chem. Soc., 1948, 70, 2719. 4 Brice, Halwer and Speiser, J. Opt. SOC., 1950, 40, 768. 5 Doty and Steiner, J. Chem. Physics, 1950, 18, 121 1. 6 Steiner, private communication. 7 Oncley and Ellenbogen, J, Physic. Chem., 1952, 56, 85. 8 Verwey and Overbeek, Theory of the Stability of Lyophobic Colloids (Amsterdam, 9 Fredericq and Neurath, J. Amer. Chem. SOC., 1950, 72,2684. 10 Klotz, Cold Spring Harbor Symp. on Quantitative Biology, 1949, 14, 97. 11 Bockris, Quart. Rev., 1949, 3, 173. Elsevier, 1948).

ISSN:0366-9033    

DOI:10.1039/DF9531300051

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

58 ‘TRY PSIN AND RELATED PROTEINS MOLECULAR-KINETIC PROPERTIES OF TRYPSPN AND RELATED PROTEINS BY LEON W. CUNNINGHAM, JR., FRANK TIETZE, N. MICHAEL GREEN * AND HANS NEURATH Department of Biochemistry, University of Washington, Seattle, Washington, U.S.A. Received 5th May, 1952 In pH regions favourable for enzymatic activity, the sedimentation behaviour of trypsin is dependent on the age and pH of the protein solution and is indicative of a concentra- tion-dependent monomer-polymer equilibrium. The change of sedimentation pattern and constant with time is coincident with a decay of enzymatic activity and can be ac- counted for in terms of autolysis. Crystalline DFP-trypsin and recrystallized trypsinogen do not show such sedimentation anomalies, nor does trypsin at pH 3 where it is enzym- atically inactive and stable.The enzymatically inactive proteins reveal sedimentation behaviour independent of time and pH and characteristic of a monodisperse solute. In conjunction with preliminary diffusion measurements, a molecular weight of about 24,000 is calculated for these inactive proteins. The preparation and properties of crystalline DFP-trypsin are described. DFP- trypsin does not react with soybean or pancreatic inhibitor. Use was also made of DFP for the preparation of recrystallized trypsinogen. In the system comprised by the proteolytic enzymes of the pancreas, trypsin occupies a central position. It activates trypsinogen by an autocatalytic process, it mediates the activation of chymotrypsinogen and of procarboxypeptidase by catalytic processes, and it is inhibited mole for mole by the pancreatic trypsin inhibitor.1 The time-honoured role which trypsin has occupied for decades as the prototype of proteolytic enzymes is due in part only to its physiological pro- perties; historically, it was of equal importance that trypsin was the first proteo- lytic enzyme of the pancreas which lent itself to crystallization, that it revealed the property of reversible denaturation and inactivation and that it was enzymatically inactive toward certain protein substrates prior to their denaturation.The quantitative kinetic studies by Kuntz 2 of the processes leading to the activation of trypsinogen on one hand, and to the inactivation of trypsin by soybean inhibitor 3 on the other, have added further interest to the properties of this enzyme, its pre- cursor and inhibitors.Of more recent interest is the fact that trypsin, in common with other pancreatic proteolytic enzymes, exhibits catalytic activity toward ester substrates,4. 5 and that, like certain other esterolytic enzymes, it is irreversibly inhibited by diisopropylfluorophosphate 6 7 and its analogues. * London University Postgraduate Travelling Student.CUNNINGHAM, J R . , TIETZL, GREEN A N D NEURATH 59 It is perhaps surprising that more than two decades after the first isolation in crystalline form, the characterization of trypsin as a protein should still be incom- plete. Modern methods of amino acid analysis have not been applied to trypsino- gen and only in fragments of trypsin; 8 no X-ray diffraction measurements have been reported to date; the moving boundary electrophoresis method has only recently been applied 8 , 9 and no detailed sedimentation analysis or free diffusion measurements have yet appeared.It was the purpose of the present investigation to provide some of the missing information by a study of the molecular-kinetic characteristics of trypsin and of its crystalline inactive precursor. For purposes of quantitative correlation of the chemical and enzymatic pro- perties of a protein, it is desirable that the corresponding parameters be determined under identical external conditions. With proteolytic enzymes, such an approach is impeded by their instability under conditions of maximum enzymatic activity. In part, this instability is related to the fact that these proteins are potentially capable of acting both as enzymes and their respective substrates and hence undergo autolysis.Since activation of the zymogens is believed to involve only mild chemical or structural changes,l these inactive precursors, when available in pure form, can be used to advantage to investigate the molecular-kinetic characteristics under conditions corresponding to maximum biological activity of the enzyme. This has been possible for a-chymotrypsin,lo9 11 since chymotrypsinogen is stable and can be purified with ease by multiple recrystallizations.1 However, since recrystallization procedures have failed for trypsinogen because of spontaneous activation, and since in higher protein concentrations the molecular-kinetic pro- perties of cc-chymotrypsin and its zymogen were not concordant,lly 12 other ap- proaches have been also explored in this work.One of these has been the use of enzyme inhibitors of sufficiently low molecular weight to introduce only second order changes in molecular parameters of the enzyme. Since no synthetic re- versible inhibitors are known for trypsin,S recourse was had to the irreversible inactivation of the enzyme by diisopropylfluorophosphate. Previous studies of the reaction of this reagent with acetylcholine esterase 13 and chymotrypsin 697 have facilitated the present investigation which has led to the crystallization of the inactive DFP-trypsin.* As will be shown below, this reaction has also aided in the preparation of recrystallized trypsinogen.Lastly, advantage was taken of the stabilizing effect of certain cations, such as calcium,8. 16,17 in pH ranges alkaline to pH 3. EXPERIMENTAL Twice crystallized trypsin of standard enzymatic activity toward benzoyl-L-arginine ethyl ester (BAEE) were commercial preparations (Worthington) prepared by the method of Northrop and Kunitz.1 They were preserved either as a powder containing 50 % MgS04 or salt-free, after exhaustive dialysis against N/1000 HCl followed by lyo- philization. Crystalline trypsinogen was prepared as described later in this paper. Diisopropyl- fluorophosphate (DFP) was obtained as a 1 M solution in isopropanol through the courtesy of Dr. E. F. Jansen and as a pure liquid through the courtesy of Dr. R. T. Major from Merck and Company. BAEE was prepared by previously described procedures.4 Sedimentation analyses were carried out with the Spinco Model E, electrically driven ultracentrifuge.In order to minimize temperature variations, most runs were limited to 1 h during which the temperature rise was about 0.5" C. Measurements of the peak displacement of the refractive index gradient curves were made in a Gaertner micro- comparator. Location of the peak was aided by the diffraction band symmetrically disposed about the maximum ordinate. Sedimentation constants, calculated in the con- ventional manner from a plot of loglox against time,ls where x is the distance of the boundary from the axis of rotation, were corrected for solvent viscosity relative to water and reduced from the average rotor temperature (about 25" C) to 20" C.* After completion of this work,14 the independent isolation of crystalline DFP-trypsin was published by Jansen and Balls.1560 TKYPSIN A N D RELATED PROTEINS Diffusion measurements were performed at 0.9 C i n an apparatus iiiade by Frank Pearson Associates, equipped with the Philpot-Svensson cylindrical lens system, two Schlieren lenses and a diagonal slit. A Tiselius electrophoresis cell was used in conjunction with a modified Kahn-Polson 19 boundary sharpening technique. Diffusion constants were calculated by the maximum ordinate-area method 20 from enlarged tracings of the midline of the refractive index gradient curves. Selected exposures were also analyzed by the method of moments.20 For each diffusion experiment, apparent diffusion con- stants were plotted against reciprocal time and extrapolated to the ordinate intercept.21 Diffusion constants were corrected for relative solvent viscosity and reduced to 20" C.Enzymatic activity was determined in a 0-005 M tris-(hydroxymethy1)-amino methane (THAM) buffer, pH 7-8, at 25" C with BAEE as substrate, using the potentiometric titration method previously described 4 to follow the progress of hydrolysis. Protein concentrations were determined spectrophotometrically at 280 mp by interpolation from a standard curve obtained for each protein with solutions of known Kjeldahl nitrogen content (N factor 6.7). RESULTS SEDIMENTATION BEHAVIOUR OF TRYPS1N.-The results of sedimentation velocity measure- ments of trypsin at four different pH values are given in fig.1 in which the sedimentation 3;2 - 3.0- 3 2.8- .$ 2.6- 2.4- 2 2- f A 0 0.5 I .o I .5 2 .o PERCENT P R O T E I N FIG. 1 .-Sedimentation constants of trypsin (open symbols and solid diamonds) and of DFP-trypsin (solid circles, squares and triangles) under the following conditions of measurements : 0 acetate buffer, pH 5.0 ; 5 maleate buffer, pH 6-28 ; A acetate buffer, pH 3.86 ; + HCI + NaCl, pH 3.0 constant s20, In these analyses, salt-free, lyophilized trypsin was directly dissolved in the desired buffer and immediately subjected to ultracentrifugation. Since a routine schedule was adhered to, the experimental con- ditions of these analyses are comparable and correspond to an elapsed time of 60 to 80 min between the preparation of the solutions and the midpoint of each sedimentation.The buffer solutions contained approximately 0.02 M of buffer salt and 0.18 M NaCl. The buffer salts were acetate at pH 3.86 and pH 5.0, maleate at pH 6.28 and tris-(hydroxy- methyl)-amino methane (THAM) at pH 7-81. Measurements at pH 3 were performed in HC1 containing 0.2 N NaCl. It is apparent from the curves plotted in fig. 1 for trypsin,* that above pH 3 the sedi- mentation behaviour of this protein is not of the type expected for a monodisperse solute, even though the sedimentation diagrams shown in fig. 2 reveal only a single peak. At any given pH, the shape of the curve relating sedimentation constant to protein concentra- tion is intuitively interpretable in terms of a concentration-dependent equilibrium between * Results similar to those shown in fig.1 were observed for the first time in preliminary sedimentation analyses of trypsin carried out by Dr. Seymour Kaufman and Dr. George W. Schwert in the laboratories of one of the present authors (H. N.) at Duke University. is plotted against protein concentration.FIG. 2.-Sedimentation patterns of 1-1 % DFP-trypsin (top row) and 0.9 % trypsin (rows 2-5) in pH 5.0 acetate buffer, containing, in case of trypsin, also 0.01 M Ca*+. In each row, from left to right, the patterns correspond to 40, 48, 52, 56 and 60 min of time elapsed after the fir st exposure. Average speed, 59,733 rev/min, average temperature about 25" C ; bar angle, from top to bottom, 50", 45", 40°, 45", and 40". The time elapsed between the preparation of the solutions and the midpoint of each run was 68, 210, 330 and 1375 min for rows 2, 3, 4 and 5, respectively.[To face page 60CUNNINGHAM, JR., TIETZE, GREEN AND NEURATH 61 monomeric and polymeric species, similar to that observed in analogous studies of chymo- trypsin.lO.12 However, in contrast to the latter system, the sedimentation curves repre- sented in fig. 1 do not converge in higher protein concentrations towards a single, pH- independent, relation but retain their individuality. In the low concentration range, the curves converge toward an extrapolated sedimentation constant of 2.5 S. In view of the known stabilizing action of calcium on trypsin,s, 16, 17,22 it was deemed of interest to repeat the sedimentation analyses just described in the presence of 0.01 M Ca2+, added to buffer solutions in the form of CaC12.The results of representative experiments performed at pH 5.0, 6-28 and 7.81, respectively, are plotted in fig. 3. The buffer solutions used in these experiments had the following composition : at pH 5, 0.02 M acetate, 0.18 M NaCl ; at pH 6.2, 0.1 M maleic acid, 0.15 M NaC1, adjusted to the pH with sodium hydroxide; at pH 7.8, 0.1 M THAM, 0.15 M NaCI, adjusted to the pH with hydrochloric acid. Because of the large scatter of points obtained at pH 5.0, no continuous representation of the concentration dependence of sedimentation constants was attempted. However, the position of these points is different from those obtained at the same pH in the absence of calcium. The experimental series represented by fig.3 was fully repeated with another preparation of the enzyme, and repeated in part with a third one. Though at each pH, the curves obtained for individual preparations were a * 8' 0 0 . . . . . . . . . . . . . . . . . . . . . . . . . 0 0.5 10 1.5 20 PERCENT PROTEIN FIG. 3.-Sedimentation constants of trypsin in the presence of 0.01 M calcium ions. 0 acetate buffer, pH 5 ; 0 rnaleate buffer, pH 6-28 ; 3 THAM buffer, pH 7.81. not strictly superimposable, the shapes of the curves were essentially the same, and con- verged to the same ordinate intercept. absence of calcium ions, the sedimentation pattern of trypsin showed a higher degree of boundary spreading than that of DFP-trypsin (see later). It was also evident that at later exposures of a single run, the refractive index gradient curves of trypsin appeared skewed and that both of these characteristics were more pronounced the higher the pH (fig.2). These observations, together with the noted effect of calcium ions, suggested that the inherent instability of the enzyme may influence the apparent sedimentation behaviour of trypsin, as it does the electrophoretic pattern,s even in pH regions in which the enzymatic activity is low. The results of survey experiments on the time-dependence of sedimentation rates (0.9 % protein) are represented in fig. 4. It is evident that at pH 3 (HCI containing 0-2 M NaCl), the sedimentation constant is essentially independent of time. However, at pH 3.86 (acetate buffer) the sedimentation constant decreases with time, and even more so at pH 5.0 (acetate buffer).At pH 7.8 (0.1 M THAM) in the absence of calcium ions, the sedimentation constant decreases to very low values but in the presence of calcium only a very small decrease is observed. It should be noted, however, that the initial (zero time) sedimentation velocity and the effects of calcium thereon, cannot be evaluated by this method. It is evident, therefore, that the curves represented in fig. 1 and 3 are significant only to the extent that the points by which they are defined have been obtained at comparable time intervals subsequent to the pre- parations of the solutions. Their absolute significance, however, is questionable and it TIME DEPENDENCE OF SEDIMENTATION BEHAVIOUR AND ENZYMATIC ACTIVJTY.-In the62 TRYPSIN AND RELATED PROTEINS may be surmised that at finite protein concentrations, and at pH 5.0 or higher, the true sedimentation constants of trypsin are higher than is indicated by the present results.A molecular instability of trypsin is also reveaIed by a decay of enzymatic activity. In the present measurements, a 5 % trypsin solution in 0.001 N HCI was diluted at zero time with the desired buffer to a final concentration of 1 % and incubated in a constant temperature bath at 25" C . After various time intervals, 0.2 ml aliquots of the enzyme solution were removed and immediately diluted with 0.001 N HCl to 10 ml. Esterase activities were then determined within less than 2 h after removal of the aliquot, in the presence and absence of 0-02 M calcium ions. The results of these measurements are shown in fig.5 in which ko is plotted against time, where ko is the zero order reaction constant per mg enzyme N. Aside from the activating effect of calcium, which will be described in detail elsewhere,23 the curves show that at pH 3.0 trypsin is essentially stable and retains its full enzymatic activity for several hours. At pH 5-0 and at pH 7.8, the enzymatic activity decreases almost instantaneously after preparation of the solutions, an initially large decay gradually levelling off. The effect is larger at pH 7-8 than at 38- 34- 3.0 - 2 6 - a 0' 2 2 - 3 1 8 - 14 10 MINUTES FIG. 4.-Dependence of sedimentation rates of trypsin on the time elapsed between preparation of the solution and midpoint of the sedimentation run. 0 pH 5, acetate buffer + 0.01 M calcium ions; 0 pH 5, acetate buffer; A pH 3-86, acetate buffet-; HCl + NaCI, pH 3 ; II pH 7.8 THAM buffer + 0.01 M calcium ions; I? pH 7.8 THAM buffer ; trypsin concentration, 0-9 %.pH 5-0, in accordance with previous measurements by Kunitz and Northrop 24 and the activating effect of calcium is retained throughout the course of these measurements. When, however, at pH 7.8, incubation is allowed to proceed in the presence of calcium ions, the decrease in enzymatic activity is markedly slowed down. These data provide support for the assumption that the time-dependence of sedimentation rates and of enzymatic activity of trypsin are essentially concordant manifestations of autolysis of the enzyme. of Jansen et al.,6 the reaction between trypsin and DFP at pH 5.0 follows bimolecular reaction kinetics, 50 % inactivation requiring about 5 h when DFP was present in a 100- fold molar excess.It was found in the present work that at pH 7-8 and 25" C, the re- action velocity was sufficiently high to warrant the expectation that in the concentrated protein solutions prevailing under conditions of crystallization of native trypsin, the reaction with DFP would be essentially complete before appreciable autolysis had occurred. This was found to be the case when the procedure recommended for the recrystallization of trypsin 1 was followed after trypsin and DFP were incubated for 1 h at room temperature. In a representative preparation, 7-5 g of trypsin (containing 50 % MgS04) were dis- solved in 15 ml of 0.02 N sulphuric acid.To this solution was added dropwise 10 ml of 0.4 M borate buffer, pH 9, containing 0-75 ml of 0.5 M DFP in isopropanol (DFP/ PREPARATION AND PROPERTES OF DFP-~~~~si~.-According to the original workCUNNINGHAM, J R . , TIETZE, GREEN AND NEURATH 63 trypsin mole ratio about 1-5). The pH was then raised to 7-8 with 0-25 N NaOH and an appreciable amount of a gelatinous precipitate was filtered off. After the clear filtrate was allowed to stand for I h, an equal volume of saturated MgS04 was added and the pI1 readjusted to 8.0. After standing in the cold for several hours, a copious crystal- line precipitate had appeared, microscopically indistinguishable as to shape from native trypsin. This was collected (4.6 g of filter cake), and redissolved in 5 ml of 0.02 N sulphuric acid.The pH was lowered to 3 with 5 N sulphuric acid, and after removal by filtration of a considerable quantity of insoluble material, the protein of the clear filtrate was recrystallized by the procedure just described except that DFP was omitted. The crystalline filter cake (3.8 g) resulting from the second crystallization was dissolved in sulphuric acid at pH 3.0, insoluble material removed by centrifugation, and the clear supernatant solution dialyzed against 0.001 N HC1 until salt-free and lyophilized (0.81 g protein, 22 % yield). Somewhat higher yields can be obtained by the method of Jansen and Balls,ls which was published while this work was in progress, and even higher ones (50 %) by modifica- 381 34 30 26 22 ;to 18 1 0 0 0 7.0 v 20" 240' 280 320 MINUTES FIG.5.-Dependence of the zero order hydrolysis rate constant ko of the tryptic hydrolysis of BAEE on the age of 1 % trypsin solutions. Trypsin solutions were allowed to stand for the times indicated on the abscissa, without calcium ions, but enzymatic activities were determined in the presence solid symbols) and absence (open symbols) of 001 M calcium. Conditions of ageing: G HCI + NaC1, pH 3.0; 0 acetate buffer, pH 5.0; A THAM buffer, pH 7.0 ; THAM buffer, pH 7-8iO.01 M Ca2+. THAM buffer, pH 7.8 ; tions of the present method subsequently developed in this laboratory. These will be described elsewhere. The single preparation which was used in molecular-kinetic studies described below contained 0.145 %P (as compared to 0.02 % for a control sample of native trypsin), whereas some preparations yielded 0.22 % P.These latter preparations were indistin- guishable in crystal form and sedimentation velocity from those of lower P content and all preparations were enzymatically inactive. On the basis of the lowest P content, the protein equivalent weight is 21,400, or 24,800 if allowance is made for the P content of the control.* Identical N content of 15-0 % was found for all preparations of DFP- trypsin and trypsin. DFP-trypsin does not react to any sigmfkant extent with the soybean or pancreatic inhibitor. This was shown by experiments in which the esterase activity of trypsin partially inhibited by soybean or pancreatic inhibitor, was determined in the presence of DFP-trypsin. DFP-trypsin was allowed to stand with the inhibitor before the addition of trypsin in order that any possible reaction between them should occur.However, as shown by the results given in table 1, no reaction occurred as the presence of DFP-trypsin did not affect the inhibition of trypsin by either soybean or pancreatic inhibitor. * All microanalytical determinations by Dr. A. Elek, Los Angeles, California.64 TRYPSIN AND RELATED PROTEINS TABLE 1 .-EFFECT OF DFP-TRWSIN ON COMBINATION OF TRYPSIN WITH PANCREATIC AND SOYBEAN INHIBITOR * pancreatic inhibitor soybean inhibitor concentration (pM) concentration (pM) trypsin 0.66 0.64 0-64 0.64 0-0 0.46 0.46 0.46 0.46 0.0 inhibitor - 0.21 0.21 0.21 - - 0.29 0.29 0.29 - 0.31 2.1 2-1 - - DFP-trypsin - - 1.0 2.0 2.0 k0 0.38 0.17 0.17 0-16 0.0 0.36 0.21 0.21 0.23 0.0 * 3 min incubation of inhibitor and DFP-trypsin, followed by another 3 min incuba- tion with trypsin prior to the addition of substrate (BAEE).Measurements in 0-005 M THAM buffer, pH 7.8, 0.01 M Ca2+, at 25" C ; substrate concentration 0.01 M ; ko is the zero order velocity constant per mg enzyme N for the hydrolysis of BAEE. SEDIMENTATION BEHAWOUR OF DFP-TRYPSIN.-The results of sedimentation rate measurements, performed under the same conditions as those previously described for trypsin, are plotted in fig. 1. In contradistinction to active trypsin, DFP-trypsin showed at all pH values a sedimentation behaviour essentially as expected for a monodisperse solute. The refractive index gradient curves revealed a single, symmetrical peak, as illustrated for a single experiment in fig. 2, and the individual sedimentation constants when plotted against protein concentration followed closely a linear regression of negative slope, independent of pH.Extrapolation to zero protein concentration yielded unambigu- ously a sedimentation constant of 2.50 S. ments were carried out in pH 3.86 acetate buffer, ionic strength 0.2. Preliminary nieasure- ments of 0-76 and 0.42 % protein solutions yielded D20, of approximately 9.310-3 x 10-7 cm2/sec. The refractive index gradient curves were practically indistinguishable from the normal curve of error. Further diRusion measurements are now in progress to define with greater precision the true diffusion constant of the protein. Assuming a partial specific volume of 0.73, the present sedimentation and diffusion rate values yield a molec- ular weight of 24,000 and a dissymmetry constant offif0 =- 1.2.PREPARATION OF CRYSTALLINE TRYPSINOGEN IN THE PRESENCE OF DFP.-Crystalliza- tion of trypsinogen occurs under conditions which are favourable for the autocatalytic transformation to trypsin.*,2 Owing to the presence of pancreatic trypsin inhibitor in the protein solution from which the initial crystallization takes place, the catalytic re- action is suppressed but it has not yet been possible to achieve recrystallization of tryp- sinogen in the absence of the pancreatic inhibitor. In the present work, the initial crystallization was carried out in the presence of DFP which resulted in such effective inactivation of the last traces of trypsin that subsequent crystallization of trypsinogen could be achieved without the further addition of inhibitor of any sort.Preliminary experiments were carried out to determine any deleterious effects of ex- posure to DFP on the extent to which trypsinogen can be subsequently activated. In a typical experiment, 200 mg of trypsinogen containing 7 % active trypsin (7 x 10-7 mole) was treated with 2 x 10-4 mole of DFP for 3 h at pH 8, 0" C. The solution was then adjusted to pH 3, dialyzed and lyophilized. The lyophilized protein contained 0.2 % active trypsin. The maximum extent of activation of this preparation at 0" C was determined in comparison to that of a control preparation of trypsinogen which was merely dialyzed and lyophilized. Using the esterase method of assay, the DFP-treated trypsinogen yielded 96 % of the maximum tryptic activity of the control preparation, the small decrease being possibly due to some conversion of trypsinogen to DFP-trypsin.The starting material for the preparation of recrystallized trypsinogen was the fraction Tg resulting after the crystallization of chymotrypsinogen from fresh beef pancreas glands." 1 The first crystallization of trypsinogen was accomplished essentially as described by Northrop and Kunitz 1 except that the acid, amorphous filter cake, after washing with MgS04, was dissolved in ice-cold, 0.4 M borate buffer, pH 9.0, which con- tained, in addition, 0.25 ml of pure DFP per 100 g of filter cake. After the addition of a saturated solution of MgS04, crystals of trypsinogen appeared after 48 h standing in the cold room.These were collected by centrifugation in a Spinco Model L preparative * We are indebted to Dr. C . E. Graham of the Wilson Laboratories for the prepara- tion of crude pancreatic protein precipitates. DIFFUSION CONSTANT AND MOLECULAR WEIGHT OF DFP-TRYPSIN.-Diffusion measure-CUNNINGHAM, J R . , TIETZE, GREEN AND NEURATH 65 ultracentrifuge at 40,000 g and an additional crop of crystals was obtained by the addi- tion of more MgS04 to the clear supernatant solution. The combined crystalline pre- cipitates were dialyzed against 0.001 N HCl until salt-free and lyophilized. The material was practically free of trypsin and could be activated to the extent of 66 %. Recrystal- Iization was carried out as described by Northrop and Kunitz 1 for the initial crystalliza- tion, in the absence of added DFP, but crystal formation was allowed to proceed at room temperature, yielding large triangular crystals of the form described by these authors.1 The final, dialyzed and lyophilized product contained less than 0.1 % active trypsin (haemoglobin method) and could be activated to the extent of 99 %.The details of this method, and modifications thereof, will be described elsewhere. SEDIMENTATION BEHAVIOUR OF TRYPSIN0GEN.-At pH 3-86 and pH 5.0 (acetate buffer, ionic strength 0.2) the sedimentation behaviour of trypsinogen is similar to that of DFP- trypsin. The boundary appears in the refractive index gradient curves as a single sym- metrical peak, and the individual sedimentation constants follow closely a straight line of negative slope which extrapolates to an ordinate intercept corresponding to s20, w = 2-50s (fig.6). Additional sedimentation analyses, at other pH values, are in progress 2.63 3 8 0' L-P 4 f 2.2 1 2ol------ 0 0 0 2 0.4 0.6 0.8 1.0 1.2 I 4 1.6 PERCENT TRYPSINOGE N FIG. B.-Sedimentation constants of trypsinogen. A pH 5.0, acetate buffer ; 0 pH 3.86, acetate buffer. and will be reported at a later time. Preliminary diffusion experiments likewise suggest that the molecular parameters of trypsinogen and DFP-trypsin are essentially identical. DISCUSSION Previous estimates of the molecular weight of trypsin vary over a wide range Osmotic pressure measurements by Kunitz and Northrop 25 have yielded a value of 36,500. No measurements were reported till 10 years later when Bergold26 published a value of 15,100.This was deduced from what appears to be a single sedimentation analysis in 0.1 N acetate buffer, pH 5.0, at a trypsin concentration of 0.29 % yielding s20, = 1.69 S, and a diffusion constant of 10.95 x 10-7, Sedimentation and diffusion measurements of the trypsin-soybean inhibitor com- pound by McLaren yielded a molecular weight of 41,000 which, on the basis of equal weight combination, corresponds to a molecular weight of 20,000 for each moiety.* Film molecular weights determined by spreading of trypsin on 35 % ammonium sulphate yielded a value of 41,000, considered to represent a dimer.28 Phosphorus analyses of DFP-trypsin, recently reported by Jansen and Balls,ls correspond to an equivalent weight of 20,700, whereas similar analyses by Kilby and Youatt29 correspond to the uptake of 2 moles of phosphorus by 34,900g of trypsin on combination with 0, S- diethyl 0-p-nitrophenyl thiophosphate.* We are indebted to Dr. A. D. McLaren for informing us of these results prior to publication. He advised us also that more detailed ultracentrifugal analyses of trypsin and soybean inhibitor have been recently performed by Dr. E. Sheppard of the Poly- technic Institute of Brooklyn (Thesis, 1951); these results were not available to us at the time of writing of this manuscript.66 TRYPSIN AND RELATED PROTEINS Measurements of the deuteron bombardment of crystalline trypsin have yielded a target molecular weight of 30,600, whereas similar measurements employing elec- trons yielded 34,000 for an amorphous enzyme preparation.30 An interpretation of the sedimentation behaviour of trypsin requires considera- tion of the molecular stability of the enzyme.When sedimentation constants are determined under conditions at which the protein is enzymatically inactive, i.e. trypsin at pH 3.0 or DFP-trypsin at any pH of the present measurements, the sedimentation behaviour is characteristic of that of a monodisperse solute, and, within the range of the present experiments, independent of the age of the solu- tion. However, when sedimentation is carried out under conditions favourable for enzymatic activity, the sedimentation rate becomes dependent on pH and on the age of the solution and reveals a complex variation with protein concentration.This complexity can be resolved in part if it is assumed that the enzymatic and molecular instability of trypsin are interdependent phenomena brought about by autolysis of the enzyme. This view, previously advanced by Northrop and Kunitz,l receives substantial support from the present work in which sedimentation rate and enzymatic stability were measured under comparable conditions of protein concentration and buffer composition. Comparison of fig. 3 and 5 indicates that sedimentation rates and enzymatic activity decrease with time faster at pH 5 than at pH 3-86. Accordingly, at each pH, individual correction factors have to be applied to the sedimentation curves depicted in fig. 1 in order to obtain a true sedimentation constant at zero time. However, in view of the uncertainty of the extrapolation of the time-dependence curves (fig.4) to zero time, one can only conclude that at finite protein concentra- tion, the sedimentation rates will be higher than is apparent from fig, 1, whereas their actual value remains indeterminate at present. Judging from the results of fig. 4, the correction factors will be higher at pH 5.0 than at pH 3.86, thus ac- centuating the differences which appear from fig. 1 for these two pH values. The relative position of a corrected curve at pH 6.28 or pH 7.8 (fig. 3) is uncertain. However, if the curves of the decay of enzymatic activity (fig. 5) are accepted as an indication of the time decay of sedimentation rates, it would appear that in the higher pH regions in the absence of calcium, the initial sedimentation constant of a 1 % trypsin solution would be even higher than at pH 5.0.For the reasons just given, it is at present impossible to interpret in quantitative terms the effect of calcium ions on sedimentation rates. However, at the pH of maximum activity (PH 7.8), in the presence of calcium ions, the sedimentation behaviour approximates that of DFP-trypsin (fig. 1) and the time effects of sedi- mentation constants and enzymatic activity largely vanish. It appears, therefore, that when trypsin is stable, active or inactive, it exhibits the sedimentation be- haviour of a monodisperse solute. However, when trypsin is active and unstable, it exists in a monomer-polymer equilibrium which is dependent on time and protein concentration.This is qualitatively similar to the behaviour of chymotrypsin but quanti- tative differences are apparent. Thus with trypsin, polymerization is minimum in the acid pH range, whereas with chymotrypsin, it is maximum in this range.lo912 Also, it has been reported that the sedimentation constant of chymotrypsin (B) tends to increase with time.31 In further contrast to chymotrypsin, the active centres of trypsin appear to be involved in the aggregation reaction since DFP-trypsin, in contradistinct ion to recent reports for DFP-chymotrypsin,* exists in the monomeric form only, regardless of pH and protein concentra- tion. It is inviting to speculate that the polymeric state of trypsin represents an enzyme-substrate complex in the path to autolysis and that, accordingly, this polymerization reaction is a case of biologically specific molecular interaction .The number of molecules involves remains to be elucidated. * Cited in ref. (12) ; see also ref. (32).CUNNINGHAM, JR., TIETZE, GREEN AND NEURATH 67 The sedimentation behaviour of trypshogen is essentially identical with that of DFP-trypsin and of trypsin at pH 3.0. If the best data at present available for the sedimentation and diffusion constants of these proteins are used, a molecular weight of approximately 24,000 is calculated for these enzymes, which is com- parable to that calculated for the monomeric forms of chymotrypsin and chymo- trypsinogen. This work has been supported by research grants from the Public Health Service, the Rockefeller Foundation and Eli Lilly and Company. 1 Northrop, Kunitz and Herriott, Crystalline Enzymes (Columbia University Press, 2 Kunitz, Enzymologiu, 1939, 7, 1. 4 Schwert, Neurath, Kaufman and Snoke, J. Bid. Chem., 1948, 172, 221. 5 Neurath and Schwert, Chem. Rev., 1950,46, 69. 6 Jansen, Fellows Nutting, Jang and Balls, J. Biol. Chem., 1949, 179, 189. 7 Jansen, Curl and Balls, J. Biol. Chem., 1951, 190, 557. 8 Bier and Nord, Archiv. Biochem. Biophys., 1951,33, 320. 9 Anderson and Alberty, J. Physic. Chem., 1948, 52, 1345. 10 Schwert, J. Biol. Chem., 1949, 179, 655. 11 Schwert, J. Biol. Chem., 1951, 190, 799. 12 Schwert and Kaufman, J . Biol. Chem., 1951, 190, 807. 13 Mazur and Bodansky, J . Biol. Chem., 1946, 163, 261. 14 Neurath, Cunningham, Tietze and Green, Fed. Pruc., 1952, 11, 265. 15 Jansen and Balls, J. Biol. Chem., 1952, 194, 721. 16 McDonald and Kunitz, J. Gen. Physiol., 1941, 25, 53. 17 Gorini, Biochim. Biophys. Acta, 1951, 7, 318. 18 Svedberg and Pedersen, The Ultracentrifuge (Oxford University Press, Oxford, 19 Kahn and Polson, J. Physic. Chem., 1947, 51, 816. ZoNeurath, Chem. Rev., 1942, 30, 357. 21 Longsworth, J. Amer. Chem. Suc., 1947, 69, 2510. 22 Bentley, Fed. Proc., 1951, 10, 161. 23 Green, Gladner, Cunningham and Neurath, J. Amer. Chem. SOC., 1952, 74, 2122. 24 Kunitz and Northrop, J . Gen. Physiol., 1934, 17, 591. 25 Kunitz and Northrop, J. Gen. Physiol., 1936, 19, 991. 26 Bergold, 2. Nuturfursch., 1946, 1, 100. 27 McLaren, Compt. rend. Lab. Curlsberg, Ser. chim., 1952, 28, 175. 28 Mishuck and Eirich, J . Polymer Sci., 1951, 7, 341. 29 Kilby and Youatt, Biuchim. Biophys. Acta, 1952, 8, 112. 30 Pollard, Buzzell, Jeffreys and Forro, Arch. Biochim. Biophys., 1951, 33, 9. 31 Smith, Brown and Laskowski, J. Biol. Chem., 1951, 191, 639. 32 Smith and Brown, J . Biol. Chem., 1952, 195, 525. New York, 1948), 2nd ed., chap. 5 and 6. 3 Kunitz, J . Gen. Physiol., 1948, 32, 241. 1940), part I.

ISSN:0366-9033    

DOI:10.1039/DF9531300058

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

CUNNINGHAM, JR., TIETZE, GREEN AND NEURATH 67 ELECTROPHORETIC STUDIES ON ENZYMATICALLY MODIFIED OVALBUMIN AND CASEIN BY GERTRUDE E. PERLMANN The Rockefeller Institute for Medical Research, New York, U.S.A. Received 13th May, 1952 Electrophoretic analysis which has been used for a characterization of enzymatically modified proteins, has proved to be a sensitive tool for following enzymatic reactions involving charged groups. It also presents a qualitative picture of the type of groups concerned. The modifications to be discussed include reactions in which carboxylic and phosphate groups are removed from the proteins. The present paper describes recent work in which electrophoresis has been used to follow the enzymatic modifications of the two proteins, ovalbumin and casein.68 OVALBUMIN A N D CASEIN Unlike most studies of this type, e.g.proteolysis with the aid of pepsin and papain,l-S only those enzymes have been used that attack specific linkages in the protein and thus leave the substrate relatively intact. Although the change in molecular weight accompanying the modification of ovalbumin, say, appears to be within the limits of error of the measurement,6 other properties of the protein, such as the electrophoretic mobility undergo appreciable alteration. This probably results from the fact that one, or more, of the relatively few groups that are split off in the presence of the enzyme contribute to the net charge of the intact protein. The reactions to be considered are (1) the ovalbumin-plakalbumin transforma- tion, (2) the dephosphorylation of ovalbumin and plakalbumin, and (3) the de- phosphorylation of casein.Since much of the work has been with ovalbumin as the substrate, its electro- phoretic behaviour will be illustrated with the aid of the patterns of fig. 1. Here a represents egg white, which is a mixture of several proteins.7 From this mixture Egg White a- -d Ovalbumin b. a- r---------;td FIG. 1 .-Electrophoretic patterns of egg white and ovalbumin in sodium phosphate buffer of pH 6.8 and 0.1 r/2. Electrophoresis was carried out at a 1 % protein con- centration for 14,400 sec at 6-4 V/cm. ovalbumin is crystallized as needles by ammonium sulphate precipitation,g but 6 shows that the crystalline material is electrophoretically complex, consisting of two components, A1 and A2.9 It will be noted that these two components are also present in the egg white in much the same proportions as in the crystalline protein.In some preparations a slower moving component A3 is present in the electrophoretic patterns. THE OVALBUMIN-PLAKALBUMIN REACTION.-AS shown by Linderstrpm-Lang and Ottesen 10911 ovalbumin can be transformed, by the action of a proteolytic enzyme from B. subtilis, into a new protein, plakalbumin, which crystallizes as plates. The reaction involves the liberation of alanine, valine, glycine, aspartic acid and glutamic acid.12 On electrophoretic analysis plakalbumin gives patterns similar in appearance to those of ovalbumin except that at an ionic strength of 0-1 in the pH range from 3 to 7 the mobilities of the two components PI and P2 are 0.6 x 10-5 cm2 sec-1 V-1 less negative than the corresponding constituents, A1 and A2, of the parent substance13 This is shown for theG E R T R U D E E .PEKLMANN 69 major components, A1 and PI, of each protein by the pH-mobility curves of fig. 2. Since the mobility change of 0-6 x 10-5 is the same as that observed when two equivalents of acid are added to a mole of ovalbumin at I72 = O.l,14 this change has been interpreted 13 as being in agreement with the chemical finding that an aspartic and a glutamic acid residue are lost in the A -+ P transformation.* DEPHOSPHORYLATION OF OVALBUMIN.-GrOUpS containing phosphorus are known to be present in both ovalbumin and casein. Since these groups may contribute to the net charge over a considerable range of pH, their selective removal might also modify the mobility of the protein without seriously affeciing other 6.0 4.0 2.0 - 2.0 - 4.0 - 6.0 Flakalbumin 0 Ovalbumin 4.0 60 8.0 FIG.2.-Mobilities of plakalbumin and ovalbumin as function of pH. properties. Moreover, if ovalbumin is assumed to have a molecular weight of 44,000, the phosphorus content of this protein does not correspond to an integral number of atoms of this element per mole. This fact, together with the electro- phoretic complexity of ovalbumin, led Linderstrdm-Lang and Ottesen to suggest that A1 contains two atoms of phosphorus per mole and A2 one.11 Thus an oval- bumin preparation such as that of fig. l b with 85 % A1 and 15 % A2, should con- tain 2 x 0.85 + 1 x 0.15 = 1-85 atoms of phosphorus per mole, a value in good * With the aid of a different enzyme preparation than that used in the work described here, Ottesen and Villee (Compt.rend. trav. lab. Carlsberg, 1951, 27, no. 18) observed a plakalbumin formation in which a di- and tetrapeptide are released ; one being alanyl- alanine, the other containing valine, alanine, aspartic acid and glycine in the ratio of 1 : 3 : 1 : 1. No extensive electrophoretic studies have been carried out yet on these pre- parations and therefore no comparison can be made. Further information about the ovalbumin-plakalbumin process may be found in an article by Ottesen and Wollenberger (Nature, in press) and a more detailed discussion is presented by Linderstrsm-Lang (Lane MedicuZ Lecture (Stanford University Press, 1952), in press).70 O V A L B U M I N AND C A S E I N agreement with the chemically dcternliiied one of 1-82.With all preparations in- vestigated over a period of several years such agreement was found.15. To test further the hypothesis of Linderstr$m-Lang and Ottesen, phosphatases were added to salt-free ovalbumin solutions of pH 5.4 and the reaction was followed Time Flectrophoretic Atoms phosphorus In hours composltiori per mole protein Compurea Obsewed 85% A] 14% A2 18, 1.8* t M = A3 47% A, 49% 143 135 4% A3 y 34 I FIG. 3.-Dephosphorylation of ovalbumin at pH 5.4 with prostate phosphatase as function of time (taken from J. Gen. Physiol., 1952, 35, 711). Each reaction mixture contained 4-6 % ovalbumin and 0.01 % enzyme. by electrophoretic and chemical analyses.Two representative series of experiments are illustrated with the aid of fig. 3 and 4. As can be seen from fig. 3, if prostate Time Electrophoretic Atoms phosphorus in hours composition per male protein Computed Observed 85% A1 14% A2 184 1.82 i2 2 O truce A3 Az 50% A2 40% A3 0.50 0.58 30% * A, FIG. 4.-Dephosphorylation of ovalbumin at pH 5-4 with intestinal phosphatase as function of time. Each reaction mixture contained 4.6 % ovalbumin and 0.006 % enzyme. * may represent a proteolytic degradation product. phosphatase is added to ovalbumin the proportion of the slower moving compon- ent A2 increases with time at the expense of Al, until A1 is completely transformed to a protein with the electrophoretic properties of A2 and with one phosphorus per mole.This monophosphorus ovalbumin, A2, crystallizes from ammonium sulphate as needles.GbKTRUDE E . PERLMANN 71 In the experiments shown in fig. 4, a phosphatase from calf intestine was added to ovalbumin. However, dephos- phorylation continues and a new component appears which moves more slowly than A2 and is designated as A3. That A3 does not contain phosphorus is revealed by the chemical analysis of the mixture and of A3 whose patterns are shown in lines 3 and 4 of fig. 4. After 24 h, ovalbumin has been converted to a phosphorus- free protein A3 which may still be crystallized as needles and whose mobility is that of the slowest component present as a trace in the original ovalbumin preparation. Although chemical differences other than the phosphorus content, e.g.small variations in the amino acid composition may exist between Al, A2 and A3, it is very probable that the mobility increments are due to the loss of charged phosphate groups and that the electrophoretic complexity of ovalbumin is adequately explained by the number of phosphorus atoms present in each component. The different mobilities of A1, A2 and A3 in a sodium phosphate buffer of pH 6.8 and 0.1 ionic strength, i.e. uAl = - 5.9 x 10-5, uA2 = - 5.2 x 10-5 and U A ~ = - 4.5 x 10-5cm2sec-1 V-1 are further illustrated with the patterns of fig. 5. These were obtained with a mixture of equal quantities of A1, A2 and A3 in a sodium phosphate buffer of pH 6.8 and 0.1 r/2. Fig. 5a is the pattern at a total protein concentration of 1.6 % and has the apparent composition of 45 % Al, Here A1 is again rapidly transformed into A2. rotol protein Klect~opho~etlc Atom4 phosphwu3 concentrution composition per mole protein per cent Computed Observed 45% Al 1.22 1.0 32% A ? 23% A3 Q ~ 1.6 FIG. 5.-Electrophoretic patterns of an artificial mixture of equal amounts of A1, .A2 and A3 in sodium phosphate buffer of pH 6-8 and 0.1 r/2.Electrophoresis was carried out for 12,600 sec at 6-1 V/cm. 32 % A2 and 23 % A3. If the phosphorus content of this mixture, column 3 and 4 of fig. 5, is now computed from this apparent composition, the value of 1-22 atoms per mole deviates considerably from the chemically determined value of 1.0. If, however, this same mixture is analyzed at a total protein concentration of 043 %, the pattern of fig. 5b is obtained and the apparent composition of 33 % Al, 34 % A2 and 33 % A3 approaches closely the true value.These results illustrate rather strikingly the large differences to be expected between the apparent and the true composition at high protein concentrations, especially if the mobility differences are small as in this instance.ls-l* Consequently, in all of the electrophoretic analyses reported here the total protein concentration has been kept as low as possible while still permitting the desired accuracy in the determination of the pattern areas. In these analyses phosphate buffers have been used because they afford a convenient means for stopping the action of phosphatases. In fig. 6, the electrophoretic mobilities of A1, the major component of oval- bumin, A2 and A3 are plotted as a function of pH.From the relative position of these curves it can be seen that the isoelectric pH values of the three ovalbumins are different and that the curves diverge until a constant mobility difference of Au = 0.6 x 10-5 is found in the pH range of 7 to 9. Comparison of this mobility decrement at pH 7.0 with the titration data again indicates that the net charge is altered by a value of - 2 at each step in the dephosphorylation. This result indicates that the two phosphate groups of A1 are esterified through the phosphate hydroxyl whose pK is > 12, i.e. each phosphate group is present as a monoester72 OVALBUMIN A N D CASEIN with two hydroxyls whose hydrogen ions are dissociated above pH 3. The con- vergence of the curves of fig. 6 at pH 5.0 suggests that the pK for the first of these hydroxyls is somewhat above its value in the free acid or simple organic esters, i.e.No //O \O- \O- protein-P-OH ~- H+ protein-P-0- PH 5 PH 7 However, electrophoretic analysis does not reveal the nature of the amino acid residue in the protein to which a phosphate is linked. Preliminary experiments have indicated that the two groups in A1 are attached to different amino acids.ls9 19 6.0 4.0 2.0 - 2.0 - 4.0 - 6.0 FIG. 6.-Mobilities of the ovalbumin component A1 and the dephosphorylated ovalbumins A2 and A3 as function of pH. This conclusion is foreshadowed by the results in fig. 3 and 4 since the prostate enzyme attacks only one phosphate group in Al, whereas the intestinal enzyme removes both. DEPHOSPHORYLATION OF PLAKALBum.-Since no phosphorus is lost in the ovalbumin --f plakalbumin transformation 1 1 9 20 it may be anticipated that plakal- bumin could be dephosphorylated in much the same manner as ovalbumin and with similar changes in mobility. Both the prostate and the irdestinal phosphatase dephosphorylate plakalbumin to monophosphorus P2 and phosphorus-free P3.As shown in fig. 7 by the super- imposed tracings of the patterns of these proteins, the relationship of PI, P2 and P3 is similar to that of the corresponding ovalbumins, AI, A2 and A3. The arrows This has proved to be the case.GERTRUDE E . PERLMAN 73 in fig. 7 are the positions taken from fig. 5 that boundaries due to Al, A2 and A3 have under identical experimental conditions. It is therefore not surprising that the mobility decrements of the plakalbumins over the entire pH range, as shown in table 1, are the same as those previously described for the ovalbumins,l5 but that the actual mobility values are less negative.This is further demonstrated by the isoelectric pH values given in table 2. For comparison those of the cor- responding ovalbumins are also listed. The shift of the isoelectric pH of the six proteins with the ionic strength is the same, but a constant difference of 0.08 pH d ascending 4 I 3 FIG. 7.-superimposed tracings of the patterns of plakalbumin and the dephosphorylated plakalbumins P2 and P3 in sodium phosphate buffer of pH 6.8 and 0.1 r/2. Electro- phoresis at 1.2 % protein concentration for 12,600 sec at 6.4 V/cm. The arrows indicate the boundary positions of the corresponding ovalbumins.units was found in the A ~ J A ~ and P1-+P2 transformation and of 0.1 in the A2-fA3 and p2-tP3 reaction. In the neighbourhood of the isoelectric pH the mobility change accompanying the removal of each phosphate group corresponds to a loss of about one negative charge, suggesting that under these conditions only one of PLAKALBUMINS P2 AND P3 IN BUFFER SOLUTIONS OF IONIC STRENGTH 0.1 TABLE 1 .-MOBILITIES OF THE PLAKALBUMIN COMPONENT AND THE DEPHOSPHORYLATED buffer 0.1 N HClS-0.5 N glycine 0-02 N NaAc+ 0-1 N HAc +0-08 N NaCl 0.1 N NaActO.1 N HAc 0.1 N NaAcS-0.09 N HAc 0-1 N NaAcf0.07 N HAc 0-1 N NaActO-06 N HAc 0.1 N NaAc t0.05 N HAc 0.1 N NaAc+ 0.01 N HAc 0.02 N NaCacf0-004 N HCac+O.OS N NaCl 0.02 N NaV+O.02 N HV +0*08 N NaCl 0.1 N NaVS-0.02 N HV 0.1 N NaVf0-005 N HV P H 3.0 3.9 4.64 4-72 4.8 4.84 4.95 5.64 6.78 7.8 8.6 9.2 U X 105 p1 f 6.54 -- 4-50 - 5-62 - 5-84 - 5.93 p2 6.53 - 3.93 p3 6.90 3.40 0.8 0.4 - 0.2 - 2.0 - 3-50 - 4.35 - 4-60 Ac = acetate ; Cac = cacodylate ; V = diethylbarbiturate.* taken from J. Gen. Physiol., 1952, 35, 711. 1 taken from J. Amer. Chem. Soc., 1949,71, 1146. C74 OVALBUMIN A N D CASEIN TABLE 2.-&MPARISON OF THE ISOELECTRIC pH VALUES OF OVALBUMINS ANI) PLAKALBUMINS APH ____ - -~ ionic strength PH A* A2 * A3* Ai-+Az A2jA3 0.1 4-58 4-65 4-74 0.07 0.09 0.05 4-63 4.70 4.80 0.07 0.1 0.02 4.68 4.75 4-85 0.07 0.1 0.01 4-7 1 4.80 4.90 0-09 0.1 P$ p2 p3 Pl-fPZ p2-'p3 0.1 4-72 4.80 4.88 0.08 0.08 0.05 4.77 4.86 4.95 0.09 0.09 0-02 4.82 4.9 1 5.0 0.09 0.09 0.0 1 4-86 4.96 5-06 0.I 0 . 1 0.1 0.05 0.02 0.01 Ai+b A2-fP2 A3+P3 0-14 0.15 0.14 0.14 0.16 0.15 0.14 0.16 0.15 0.15 0.16 0.16 * taken from .I. Gen. Physiol., 1952, 35, 711. $ taken from J . Arner. Chern. SOC., 1949, 71, 1146. the two hydroxyls of a phosphate has lost its proton, i.e. the phosphate group is present as -P-0-. The constant shift of 0.14 to 0.16 of the isoelectric pH \OH values in the Al-tP1, Az-tP2 and A3-tP3 processes, as pointed out previously, is due to the removal of the two carboxyl groups lost in the plakalbumin trans- formation. DEPHOSPHORYLATION OF CASEIN.-That casein is a mixture of several distinct proteins was first demonstrated by the solubility studies of Linders tr#m-Lang and Kodama.21.22 Mellander later showed that casein has three electrophoretic components,23 a-, B- and y-casein, which in 1944 were separated by Warner.24 The electrophoretic patterns of these purified fractions are reproduced in fig.8. Fig. 8a is that of unfractionated casein consisting of 75 % of the a-component and 25 % /%casein. The proteins of fig. 8b and c are those of the isolated fractions, the phosphorus content of the a- and /%casein being 1.0 and 0.6 % respectively. In the sodium phosphate buffer of pH 6.8 and 0.1 r/2, u, = - 7.6 x 10-5 and uB = - 3.4 x 10-5 cm2 sec-1 V-1. Although casein has thus far been considered to be resistant toward the action of purified phosphatases from mammalian tjssues,25-27 it has now been found that a-casein, the fraction with the higher phosphorus content and the more negative mobility, is readily dephosphorylated in the pH range from 5.6 to 6-6 by prostate phosphatase.28 This enzyme, however, has no effect on /%casein.During the dephosphorylation of a-casein the solubility of the protein decreases. Simultaneously, as shown in fig. 9, the electrophoretic behaviour of the protein changes and several new components with lower mobilities appear. In the experi- ments represented by fig. 9, 0-5 % a-casein in acetate buffer of pH 5.6 and T/2 0.1 was exposed to the action of the prostate enzyme and the enzymatic process followed by electrophoretic and chemical analyses. Fig. 9a is the tracing of the pattern of a-casein, 9b, c, d and e those after the protein has been in contact with the enzyme for 1, 3, 6 and 12 h, respectively.The electrophoretic composition and the loGERTRUDE E. PERLMANN 75 chemical analyses are listed in table 3. Here the components of the partially de- phosphorylated mixtures are identified on the basis of their mobilities, the values of which in a 0.1 ionic strength phosphate buffer are given in parentheses. As seen Unfractionated Casein a: (3 0 a a. ~ d a- -d a- casein a @-casein (3 C. a- -d FIG. 8.-EIectrophoretic patterns of unfractionated casein, a-casein and 8-casein in sodium phosphate buffer of pH 6.8 and 0.1 r/2. Electrophoresis was carried out at 0.5 % protein for 14,400 sec at a potential gradient of 4.95 Vlcm. a' -casein 12 ~OUPS e. FIG. 9.-Tracings of electrophoretic patterns of a-casein before and after treatment with prostate phosphatase.Electrophoresis was carried out in sodium phosphate buffer of pH 6.8 and 0.1 r J 2 for 10,800 sec at 4-75 V/cm. from fig. 9b and line 2 of table 3, after 1 h 6-7 % of the phosphorus is liberated. The main component has the same mobility as that of the a-casein, but two com- ponents, tentatively designated as u3- and a4- are present. After 3 h contact with the enzyme the boundary with the mobility of u-casein has disappeared. The major component of this mixture has a mobility of u = - 7.1 x 10-5 cm2sec-1 V-1.76 OVALBUMIN AND CASEIN TABLE 3.-DEPHOSPHORYLATION OF X-CASEIN WITH PROSTATE PHOSPHATASE AS FUNCTION OF TIME Each reaction mixture contains 0.5 o/, casein and 0.005 ;( enzyme in acetate buffer of pH 5.6 and 0.1 Ti2 time of apparent relative composition of components ( %) % % inorganic non-protein exposure to the enzyme a(-7.6) ai(-7.1) d2(-6.6) a3(-5.6) 4 - 3 .6 ) Lw5(-2.9) phosphorus nitrogen released formed (hours) - - - - 0 100.0 - - - 1 82.4 - - 3 - 74-2 - 6 Electrophoresis carried out in sodium phosphate buffer of pH 6-8 and 0.1 T/2. 8.5 9.1 - 6.7 2.2 17.2 8.6 - 14.9 3-6 44.0 6*8* 30.9* 13.7 4.6 25.3 6.7 12 - 41.1 4.5 38.0 6.1 10-3 28.2 12.6 - * not clearly resolved. As dephosphorylation continues, the apparent concentrations of the slower moving components increase. Due to the uncertainty of the molecular weight of casein, the number of phos- phate groups per mole is unknown, although the phosphorus content indicates that this number is much greater than in ovalbumin. However, if the removal of each phosphate group is accompanied by a definite mobility change, as with ovalbumin, the mobilities listed in table 3 may then possibly correspond to the loss of one phosphate group in the cc--ta~ transformation, one for the CCI+CC~, two for the ~~2-+tc3, four for the a3-fa4 and one for cc4-fcc5.The complexity of the partially dephosphorylated a-casein is hardly surprising in view of the high phos- phorus content of the starting material. Moreover, most of this phosphorus is linked to serine. In the last column of table 3 it is further shown that the dephosphorylation of the protein is accompanied by the formation of trichloroacetic acid soluble nitrogen. No explanation of this phenomenon can be given at the present time. Although the action of the prostate enzyme on ovalbumin was not accompanied by the production of non-protein nitrogen, a proteolysis of casein cannot be excluded, particularly since some trichloroacetic acid soluble nitrogen appears even after liberation of phosphorus has ceased.The mechanism of the dephosphorylation of a-casein is still obscure, but a few general conclusions can be drawn from this work. It is clear that cc-casein contains a certain number of phosphate groups with ionizable hydroxyls which contribute to the net charge and thus also to the electrophoretic mobility of the protein. Such phosphate groups, as shown by the investigation of the ovalbumin, are readily attacked by phosphatases. Finally, it should be stressed that electrophoresis can be of considerable im- portance in the study of enzymatic reactions which induce minute changes in a protein molecule.Moreover, the combination of this method with chemical analysis will facilitate studies directed toward gaining insight into protein structure. These conditions are not fulfilled with P-casein. 1 Svedberg and Eriksson-Quensel, J . Amer. Chem. Sac., 1934, 56,409. 2 Annetts, Biachern. J., 1936, 30, 1807. 3 Tiselius and Eriksson-Quensel, Biochem. J,, 1939, 33, 1752. 4 Petermann and Pappenheimer, J. Physic. Chem., 1941, 45, 1. 5 Petermann, J. Physic. Chem., 1942, 46, 183. 6 Giintelberg and Linderstrfjm-Lang, Cornpt. rend. trav. lob. Carlsberg, 1949, 27, no. 1. 7 Longsworth, Cannan and MacInnes, J. Arner. Chem. Sac., 1940, 62, 2580. 8 S~rensen and H+yrup, Compt. rend trav. lab. Carlsberg, 1915-17, 12. 9 Longsworth, J. Amer. Chem. Sac., 1939, 61, 529. 10 Linderstr$m-Lang and Ottesen, Nature, 1947, 159, 807. 11 Linderstr$m-Lang and Ottesen, Compt. rend. trav. lab. Carlsberg, 1949, 26, 403. 12 Eeg-Larsen, Linderstrgjm-Lang and Ottesen, Arch. Biochern., 1948, 19, 340.G E R T R U D E E. PERLMANN 77 13 Perlmann, J . Amer. Chem. SOC., 1949, 71, 1146. 14 Cannan, Kibrick and Palmer, Ann. N. Y. A c d . Sci., 1941, 41, 243. 15 Perlmann, J. Gen. Physiol., 1952, 35, 71 1. 16 Svensson, Arkiw. Kemi. Min. Geol. A, 1943, 17, no. 14 ; 1946, 22, no. 10. 17 Perlmann and Kaufman, J. Amer. Chern. SOC., 1945, 67, 638. 18 Longsworth, J. Physic. Chem., 1947, 51, 171. 19 Perlmann, Abstr. 119th Meeting, Amer. Chem. SOC., April, 1951. 20 Perlmann, Nature, 1949, 164, 961. 21 LinderstrQm-Lang and Kodama, Compt. rend. trav. lab. Carlsberg, 1925, 16, no. 1. 22 Linderstr#m-Lang, Compt. rend. trav. lab. Carlsberg, 1929, 17, no. 9. 23 Mellander, Biochem. Z., 1939, 300, 240. 24 Warner, J. Amer. Chem. Sac., 1944, 66, 1725. 25 Rimington and Kay, Biochern. J., 1926, 20, 777. 26 Schmidt and Thannhauser, J. Biol. Chem., 1943, 149, 369. 27 Anagnostopoulos, Pacht, Bourland and Grabar, Bull. SOC. Chim. Bioi., 1951, 28 Perlmann, J. Amer. Chem. Soc., 1952, 72. 33, 699.

ISSN:0366-9033    

DOI:10.1039/DF9531300067

出版商:RSC    

年代:1953

数据来源: RSC