摘要:

CHAIN CONFIGURATIONS IN CRYSTALS OF SIMPLE LINEAR POLYMERS BY C. W. BUNN AND D. R. HOLMES Imperial Chemical Industries Ltd., Plastics Division, Welwyn Received 1 1 th February, 1958 The factors responsible for the chain configurations found in crystalline regions of the simpler types of linear polymers are discussed. It is suggested that three factors are involved. (i) A bond orientation effect which favours staggered bond configurations in saturated molecules, for example, the plane zigzag chain in polythene and syndyotactic vinyl polymers, the helical staggered configuration in some of the isotactic vinyl polymers, the non-helical non-planar staggered configuration in rubber hydrochloride. (ii) Intra- molecular interaction between neighbouring substituents, or between substituents and chain atoms, which may lead to deviations from staggered configurations in overcrowded molecules, for example, straight chain fluorocarbons, polyisobutene, isotactic vinyl polymers with large substituent groups.(iii) Packing effects, which influence the choice between configurations of nearly equal energy, for example, rubber hydrochloride, polyesters, pol yet hers. The factors that determine the configuration taken up by chain molecules in the crystalline regions of a high polymer specimen may be divided in the first instance into forces within the molecule, which prescribe a certain minimum energy configuration for an isolated molecule, and the forces between neighbouring molecules, which may tend to pull the molecules into a different configuration. The purpose of this paper is to consider the parts played by these two factors in determining the configurations of polymer molecules of the simplest chemical structure (those with comparatively few atoms in the chemical repeating units), and further, to consider the parts played in intramolecular forces by the van der Waals interactions between non-bonded atoms and by other configurational influences.The way of approach to a consideration of the configurations of polymer molecules is paved by our knowledge of the configurations of those small monomer molecules that are capable of changes of configuration by rotation round bonds. In ethane derivatives the minimum energy configuration is always a staggered one, and energy barriers to rotation from one staggered configuration to the next are about 3000 cal/mole.There has been much discussion of the origin of these energy barriers; the van der Waals repulsions between atoms of the two trios attached at either end of the central rotating bond would of course favour the staggered configuration, but there has for a long time been a suspicion that this is not the only factor,l and recent calculations by Mason and Kreevoy 2 of the van der Waals contribution (using empirical, experimentally based, force laws) show that in general the van der Waals interactions account for only about half the magnitude of the observed barrier height in a number of molecules. The remainder is thought to be due to the quadrupole moments (or even higher multipole moments) of the bonding electrons ; 3 9 4 we may conveniently refer to this as a bond orientation effect quite distinct from the van der Waals repulsions between the atoms held by the bonds.The importance of this conclusion is that although in simple ethane derivatives with monatomic substituents the two effects favour the same con- figuration (or very nearly), in more complex moJecules (where van der Waals repulsions between substituents attached to next-but-one or next-but-two chain atoms are involved), the two factors may be opposed. 95 .- -96 CHAIN CONFIGURATIONS I N CRYSTALS In chain polymer molecules there are three alternative staggered positions for each successive chain bond, the trans (A) and the two gauche (B, + 120" from A, and C, - 120" from A), and the various possible ideally staggered configurations can be enumerated by effecting changes on these three.1 The configuration taken up by any particular molecule, considered in isolation, will be the one of lowest energy; there may be some departure from the ideal staggered configuration if the van der Waals interactions between non-bonded atoms are not symmetrical a bout the staggered position.In polythenes the CH2 chains in the crystalline regions are in the fully extended configuration, the carbon atoms forming a plane zigzag.5 There is little doubt that the plane zigzag is the minimum energy configuration for an isolated chain, because the all-trans plane zigzag chain is the only one that does not bring non- bonded atoms closer than the van der Waals contact distances. The energy relations are likely to be somewhat similar to those in n-butane, where the energy difference 6s 7 between trans and gauche configurations is around 800 cal/mole.(The energy barrier between successive staggered configurations is likely to be 3000-4000 cal/mole, again as in n-butane.) The molecular configuration in the polythene crystal must therefore be all-trans unless some other configuration packs so much better that it gives an energy advantage of at least 800cal/mole CH2 group. (Other differences in free energy, due to the different vibrations of the molecules of different configuration, are likely to be small compared with these two factors.) The energy differences associated with packing efficiency differences can be estimated roughly. In polymers of known crystal structure, the molecular packing fraction8 (mpf) at room temperature (the fraction of the total space occupied by the molecules if atoms are given their accepted van der Waals radii) is usually 0-70-0.73, though in the most poorly packed it can be as low as 0.66 and in the most efficiently packed9 about 0.76.For hydrocarbons, the energy content for a mpf of 0.76 would be about 300 cal/mole (CH2) lower than for 0-66 (This is based on an empirical expression.10); thus, even for the most extreme difference in packing efficiency, the energy difference is smaller than the differ- ence between all-trans and all-gauche configurations. However, in actual fact the all-trans polythene molecules are well packed, with mpf = 0.73, and the small margin between this and the likely upper limit of 0.76 would give an energy difference of less than 100 cal/mole.It is evident that any chain configuration other than the all-trans is unlikely to occur except in an unstable crystal form, or of course in amorphous regions where molecules are strained, (Actually a second crystal form of polythene is known, but it appears to be just an alternative packing of the all-trans chains,ll of lower density, with d = 0.965 and mpf 0.70). In isotactic vinyl polymers of general formula (- CH2--CHR--), with all the R groups in corresponding positions (either all in right-hand or all in left-hand positions), the molecules are found by Natta and his colleagues 12-17 to be in the helical staggered configuration (AB)3 (or the enantiomorphic (AC)3) with three chemical units in the geometrical repeating unit (length - 66&, when R is CH2, or This chain-type is illustrated in fig.la. The AB or AC suc- cession in the nomenclature of chain-types means that the chain bonds take alternate trans and gauche positions and that for the gauche positions the rotation is always in the same direction and leads to a left-hand helix (A& or a right-hand helix (AC)3. (Note that the same molecule can assume either a left- or right- handed form. 18) The reason for the adoption of this helical-configuration is clear : the all-trans plane zigzag chain would put the side-groups only 2.54 A apart, which is much too close, for a CH2 or CH3 group has an effective diameter of about 4.0A, and a benzene ring a thickness of about 36A.The helical form keeps the side-groups far apart, and is energetically preferable on this account; it is not free from strain, however, because distances between certain main-chain carbon atoms (c1-c4), and between the first carbon atom of the side-group -CH3, -CH2-CH3, -CH2--CH2-CH3, -CH2-CH2-CH(CH3)2, -CH =C . W. BUNN AND D . R. HOLMES 97 and a main chain atom (R1-43) would be 2-9 A if the bond angles were tetrahedral (1093"). The situation is relieved by opening the bond angles a little : in poly- propylene the chain repeat distance, 6.50 A, is greater than the " ideal " distance for a staggered configuration with tetrahedral bond angles (6-20 A), and implies bond angles of 114-5" if all are opened equally, while in polystyrene the repeat distance of 6.65 8, means bond angles of 1163".In this way the nearest distances between non-bonded carbon atoms become 3.2 8, in polypropylene and 3.3 8, in polystyrene. Note that there is another staggered configuration with the same FIG. 1 .-Configurations of isotactic vinyl polymers. (a) Side-groups -CH3-CH2. CH3, -CH2. CH2. CH3, -CH2. CH2. C(CH3)2, (b) Side-groups -CH2 . CH(CH3)2 or -CH2 . CH(CH3) . CH2 . CH3 ; helix, 7 units (c) Side-groups -CH(CH3)2 ; helix, 4 units in 1 turn. -CH=CH2, or --CsHs ; helix, 3 units in 1 turn ; chain-type (AB)3. in 2 turns. chain-form but with the side-groups attached to differently situated side-bonds ; but sterically this is even worse than the all-trans AA configuration, and can be ruled out. The energy differences between the helical configuration found in crystals of these substances and the other possible staggered configurations have not been estimated, but they must be large, because extremely short distances between non- bonded atoms (2.6 8, between carbon atoms) occur in the latter ; the energy differ- ences are likely to be much too large for the choice of configuration to be influenced by the energy differences associated with packing differences.We may note in passing, however, that these helical molecules pack less efficiently than the plane CH2 chain : the mpf is about 0-69 in polypropylene, polybutene and polystyrene. Although intermolecular forces are not likely to influence the choice between the different staggered configurations, there is one way in which they may play a significant role.In these isotactic vinyl polymers, it is unlikely that the minimum energy configuration in an isolated molecule would be ideally staggered in the sense that the gauche chain-bonds are rotated exactly 120" from the trans position ; D98 CHAIN CONFIGURATIONS IN CRYSTALS repulsions between non-bonded atoms in this type of molecule are not sym- metrical about the 120" position, and the rotation would actually be expected to be a little less than 120". This would lead to a helix with a non-integral number of chemical units (a little more than three) to each turn; it would either have a very long repeat distance or would be irrational. The X-ray diffraction patterns of these substances do not give any indication of such a structure; the repeat unit seems to be an exactly 3-unit one in each substance.It is true that any evidence for a long repeat unit, which would consist of additional (more closely spaced) layer lines, might be missed in the imperfect diffraction patterns given by these substances, for the additional layer lines would be weak; on the other hand, according to Natta and his colleagues, the diffraction patterns of polystyrene, poly-a-butene and poly-l:2-butadiene can be accounted for by rhombohedra1 unit cells with two chains to the cell, and this indicates true trigonal screw axes in the molecules. It appears that any intramolecular tendency for the chain to deviate from the ideal staggered form is offset by intermolecular forces which pull it into the trigonal form in the interests of economical packing-for long-period or irra- tional chains would be expected to pack less well than the ideal form with a trigonal screw axis.Polymers of this isotactic poly-cc-olefine series with branched side-groups in which the branching is close to the main chain have longer repeating units. Poly- 3-methyl-butene-1, in which the side-group is -CH(CH3)2, has a repeating unit 6-84 A in length which comprises four monomer units ; 15 the configuration (fig. lc) appears to be a helix formed on the same principles as that already dis- cussed, but opened out so that repetition occurs at the fourth monomer unit instead of the third. The reason for the difference seems clear : a bulky side-group leads to more acute overcrowding of the type discussed in the previous paragraph; the methyl groups of the -CH(CH& side-chain would be much too closc: to certain chain atoms if the configuration were the ideal staggered trigonal one, and the repulsion between R3 and chain-groups C7 and Cg leads to a rotation of the gauche (B) chain-bond from the staggered 120" position to an angle of about loo", and a rotation of the A chain-bond from 0" (the strict trans position) to about - 26". (If in addition the bond angles are opened further than in the trigonal chains, this affects slightly the A and B rotation angles necessary to attain the correct repeat distance.) In two polymers in which the branching occurs at the second atom of the side-chain (poly-4-methyl-pentene-1 with side-chain --CH2--CH(CH&, and poly-4-methyl-hexene-1 with side-chain -CH~-CH(CH~)-CHZ---CH~), the chain has a repeat length of 13-85-14.0 A, and appears 15 to be a helix of seven monomer units in two turns (fig.16). This is a type intermediate between the three-unit and four-unit ones already discussed, and can be accounted for by rotations of the same type but half the magnitude of those occurring in the four- unit helix-i.e. by angles of A = - 13" and B = 110". Sterically, this is exactly what we should expect-the overcrowding is less severe when the branching occurs further out along the side-chain. Finally, in this series, a branch still further away from the main chain in poly-5-methyl-hexene-1 (which has side- chain -CH2-CH2-CH(CH3)2) brings us back to the three-unit helix; the bulkiness at the branch is too far from the main-chain to affect its configuration. Polyvinyl isobutyl ether, which has a very similar side-chain but with oxygen in place of the first CH2 (-0-CH4H(CH3)2), also has the three-unit helix, as one might expect.The detailed configurations of the side-chains in the isotactic polymers are not yet known, except for poly-butene-1 , in which staggered configurations persist through both main and side chains.14 The syndiotactic form of one of these hydrocarbon polymers, with side-groups alternately in left and right positions, is known ; this is poly-1 : 2-butadiene, with side-groups -CH=CH2. Natta and Corradini17 found that the chainC . W. BUNN AND D. R. HOLMES 99 is very nearly a plane zigzag (all-trans type), with repeat distance 5.1 A, just twice the span of one zigzag.This is again precisely what one would expect, for with the chain in the plane zigzag configuration, the side-groups, being alternately in left and right positions, are well separated. A small deviation from the plane zigzag configuration, which can be described as a rotation of each chain-bond a few degrees from the strict trans position, is satisfactorily accounted for by re- pulsion between the first carbon atom of the side-chain and the next-but-one chain atom. The same type of chain19 occurs also in polyvinyl chloride (-CH2-CHCl-)p. In rubber hydrochloride (-CH2-CH2-CH2 -C(CH3)Cl-->,, there are two different sub- stituents-C1 and CH3-on the same chain car- bon atom ; the substituent groups are far enough apart along the chain to give no overcrowding difficulty between themselves ; the problem is the chain configuration at the substituted points.If we focus attention on the chain carbon atom that holds the substituents (e.g. C3 in fig. 2), the next- but-one chain CH2 group (Cl or C5) has the choice of three staggered positions; in one of these, it contacts C1 and CM3, in the second C1 and CH2, and in the third C H 2 and CH3. Since a chlorine atom is somewhat smaller than a CH3 or CH2 group and stands further out from the carbon atom to which it is attached, the first two would appear to be preferable to (i.e. less overcrowded than) the third, but it is not obvious which of the first two is the better. If CH2 is effectively smaller than CH3 (owing to the rotation of CH3), the second alternative (a CH2 contacting C1 and CH2) would be preferable; but if this were chosen consistently, the molecule would be so sharply folded that there would be impossibly short dis- tances between other non-bonded atoms.In actual fact, the CH2 in one direction along the chain (upwards in fig. 2, i.e. Cl) chooses the first (it contacts C1 and CH3) and the CH2 in the opposite direction (downwards in fig. 2, i.e. C5) chooses the second (it contacts C1 and C2), leading to a chain of type A3BA3C,20 with repeat distance 8 - 9 5 ? ~ This may well be the minimum energy configuration for an isolated molecule. But we FIG. 2.-Rubber hydrochloride ; chain-type A3BA3 C. may note also that it packs quite well, with a m.p.f. of 0.72 ; a plane zigzag chain con- figuration, which would be the chain form if the first alternative (CH2 contacting C1 and CH3) were chosen consistently, would pack less well because there would be gaps between successive side-groups which are not large enough to hold the side-groups of neighbouring molecules.Thus the occurrence of the A3BA3C chain form may probably be attributed partly to intramolecular and partly to intermolecular forces. In polyisobutene (-CH2-C(CH&-)p, which has pairs of methyl groups on alternate chain atoms, there is very severe overcrowding between the side-groups. If the chain were a plane zig-zag with normal bond angles, successive pairs of side-groups would be 2.54 8, apart, and this overcrowding cannot be relieved much by rotation round chain bonds to other configurations. Certainly there is no staggered configuration that gives distances greater than 2-6& and the intermediate bond-positions are not much better.It is not surprising that the100 CHAIN CONFIGURATIONS I N CRYSTALS configuration in the crystal is not a plane zigzag, nor any staggered configuration ; it is a helix with a repeat distance of 18-6& comprising 8 chemical units and making 5 turns in the geometrical repeating unit (fig. 3). This conclusion comes from a study of the crystal structure of the stretched material which, though still incomplete, has gone far enough to leave no doubt about the type of helix. It was based originally 21 on structure factor calculations (the unit cell is an ortho- rhombic two-chain one, with space-group P212121) ; more recently, Bessel function calculations of layer line intensities, both by ourselves 22 and by Liquori,23 have confirmed this conclusion, and we have now refined the crystal structure far enough FIG.3.-Polyisobutene ; helix, 8 units in 5 turns. to be able to say something about the deviations from the ideal type of helix and the bond angles.22 The ideal helix may be described as B16, with the angle B = 83" ; to account for the intensities of the X-ray reflections it is necessary to distort the bond angles, opening the CH2( chain bonds to 126", and closing the (CH&C< chain bonds to 107", and in addition to make the rotation angles B alternately 1023" and 51". It is not clear why the molecule assumes this particular type of helical con- figuration. A departure from the plane zigzag or any staggered configuration is necessary because in all the staggered configurations the overcrowding of the methyl side-groups is most severe ; but it would be possible to relieve the situation by a rotation of only 30" from the trans configuration, giving a helix with one turn in eight chemical units (the helix first suggested by Fuller 24) ; this puts one methyl group equidistant (2-6& from the two methyl groups of the next pair.C.W. BUNN AND D. R. HOLMES 101 Instead of this, we find rotations of 102+ and 51", which puts any one methyl group approximately equidistant from a methyl group of the next pair and a chain CH2 group. Possibly this means that the effective size of a CH2 group is less than that of a CH3 group because the latter rotates.Whatever the reason, this helical configuration is remarkably well defined by one of the best and most detailed of polymer X-ray diffraction patterns. Polyisobutene is a rubber-like substance at room temperature ; this means that the molecules are very flexible-that is, the energy differences between the range of configurations attainable by rotating round the chain bonds are small; for this reason, it may be that packing considerations play an important part in determining the configuration of the chains in the crystals : in other words, the reason why the B16 helix is found in the crystals rather than Fuller's helix may be that B16 packs better. The mpf in the crystals, if we use normal radii for the atoms, is quite high4-73-but this figure gives a false impression : owing to the intense overcrowding within the molecule, the external packing of neighbouring molecules is really less efficient than the figure of 0.73 would suggest. The ready flexibility of this very overcrowded molecule is of great interest- indeed, it is the flexibility rather than the configuration in itself that is of most practical importance: a study of molecular configuration is to be regarded as only the first step in approaching the energetics of change of configuration. In polyisobutene, models show that change of configuration by rotation round the chain-bonds does not alter very much the distances between methyl groups of neighbouring pairs, or between methyl groups and chain CH2 groups; all con- figurations are grossly overcrowded, and distances between non-bonded carbon atoms vary only between 2.7A and 2.8A.This means that energy differences between the various configurations are not very large. (Estimation of the mag- nitudes would be difficult owing to lack of information about the force laws at these distances, and about distortion of C-C-H bond angles.) But there is another factor, if we accept the implication of Mason and Kreevoy's work 2 that there is a bond-orientation effect favouring the staggered configuration : if a model is twisted about to the various configurations, it is seen that when all bonds are staggered, the methyl groups are most overcrowded, while the configurations giving the best clearance between methyl groups are far from staggered; in other words, there are two energy terms of opposite sign, the net result being low energy differences for a wide range of configurations, so that the molecule has great flexibility and the substance has rubber-like properties at room temperature.This suggestion was first made by one of us long ago ; 25 it seems worth reviving in view of recent work. PoIytetrafluoroethylene (-CF2-CF2-)p is another helical molecule of long period which is also of great practical interest owing to its unusual properties. The configuration in the fully ordered crystal below the 20°C transition point is a slowly twisting helix with a repeat distance of 16.8 A, comprising 13 CF2 groups; there is actually only half a turn in this repeat distance.26 Each chain- bond is twisted 20" from the precise trans (A) position.The reason for the de- parture from the plane zigzag configuration seems clear; the van der Waals radius of fluorine is 1.4A so that if the chain were a plane zigzag with repeat distance 2.54 .$, the fluorine atoms would be a little too close together ; the slight overcrowding is relieved by rotation at each chain-bond, which, together with a slight opening of the bond angles to 116", brings the shortest F-F distance up to 2-7A. It is interesting to find that in the partly disordered crystal just above the transition point (in point of fact, at 25"C), the molecules have a slightly different configuration, with a repeat distance of 19.5A and 15 CF2 units in the repeating unit: 27.28 the molecule is slightly untwisted. Whether this represents a new equilibrium configuration of the isolated molecule at the higher temperature, or whether the change is an effect of intermolecular forces (for the lateral packing is slightly more open above the transition, and the molecules are rotating about102 CHAIN CONFIGURATIONS I N CRYSTALS their chain axes), it is difficult to decide.At all events, the change is small, and it seems likely for two other reasons that intermolecular forces play only a slight part in determining the molecular configuration : one is that the packing is poor even in the fully ordered crystal-the mpf is 0.67, changing to 0-65 in the disordered form-and the other is that, in view of the very high melting point, the molecule appears to be very stiff,lo and is unlikely to be much influenced by the weak inter- molecular forces which are similar in magnitude to those between hydrocarbon molecules.There is much evidence indicating that the replacement of a CH2 group in a chain by an oxygen atom leads to an increased flexibility : the melting points of chain ethers and esters are consistently lower than those of hydrocarbons, in spite of increased intermolecular forces. Rotation round a CH2-0 bond appears to be much easier than rotation round a CH2-CH2 bond.10 We should expect therefore that the configurations of polyether and polyester molecules would be influenced strongly by intermolecular forces. The facts about the crystal struc- tures, as far as we know them, bear this out, for the configuration at the ether or ester parts of chains varies in different polymers even when there are no side- groups.In polytetramethylene oxide (-CH24H2 . CH2. CH2. O-->, and polytrimethylene oxide (-CH2 . CH2. CH2 . O-)p the repeat length and the general characteristics of the X-ray diffraction pattern indicate a plane zigzag configuration,22 while in polyethylene oxide (-CH2. CH2. O-)p the repeat distance is long and the chain probably helical,29* 32 though the structure has not been studied in detail. For polyesters we have much more information based on detailed crystal structure determinations. In Terylene the configuration in the aliphatic part of the molecule is not far from planar: there is a rotation round the O 4 H 2 bond of about 20" from the planar trans position.30 In polyethylene adipate and polyethylene suberate the corresponding angle31 is 66", so that the chain is pronouncedly non-planar. It is probable that these differences occur in response to the very different packing requirements of these two different types of molecule.(The packing efficiency is about average in all three crystals men- tioned, the mpf being 0.71-0.72.) In a number of other aliphatic polyesters the chain repeat distances 32 suggest a similar configuration, but in polyesters of the trimethylene glycol series the chain is shortened a good deal, and in some of them the repeat length can be increased by stretching fibres,33 suggesting that the chain is straightened by stretching. These changes are unlikely to occur in the CH2 sequences, and probably occur by rotation round the 0-CH2 bond. 1 Bunn, Proc. Roy. SOC. A, 1942, 180,67. 2 Mason and Kreevoy, J. Amer. Chem. SOC., 1955, 77, 5808. 3 Lassettre and Dean, J. Chem. Physics, 1948, 16, 151, 553 ; 1949, 17, 317. 4 Oosterhoff, Faraday SOC. Discussions, 1951, 10, 79, 87. 5 Bunn, Trans. Faraday Soc., 1939, 35, 482. 6 Szasz, Sheppard and Rank, J. Chem. Physics, 1948, 16,704. 7 Pitzer, J. Chem. Physics, 1940, 8, 71 1. 8 Kitaigorodski, Actu physicochim., 1947, 22, 309. 9 C. W. Bunn, not previously published. 10 Bunn, J. Polymer. Sci., 1955, 16, 323. 11 Teare and Holmes, J. Polymer Sci., 1957, 24, 496. 12 Natta et al., J. Amer. Chem. Soc., 1955, 77, 1708. 13 Natta, Corradini and Cesari, Rend. Accad, Naz. Lincei, 1956, 21, 24, 265. 14 Natta, Corradini and Bassi, Makromol. Chem., 1956, 21, 240. 15 Natta et al., Rend. Accad. Naz. Lincei., 1955, 19, 404. 16 Natta and Corradini, Rend. Accad. Naz. Lincei., 1955, 18, 19. 17 Natta and Corradini, Rend. Accad. Naz. Lincei., 1955, 19, 229. 18 Bunn and Howells, J. Polymer Sci., 1955, 18, 307. 19 Natta and Corradini, J. Polymer Sci., 1956, 20, 251. 20 Bunn and Gamer, J. Chem. Soc., 1942, 654.C . W. BUNN AND D. R. HOLMES 21 Bunn, J. Chem. SOC., 1947, 297. 22 D. R. Holmes, not previously published. 23 Liquori, 13th Int. Congr. Picre Appl. Chem. (Stockholm, 1953). 24 Fuller, Frosch and Pape, J. Amer. Chem. Suc., 1940, 62, 1905. 25 Bum, Proc. Roy. SOC. A, 1942, 180, 82. 26 Bunn and Howells, Nature, 1954, 174, 549. 27 E. R. Howells, not previously published. 28 Pierce, Clark, Whitney and Bryant, Abstr. A.C.S. Meeting (Sept. 1956). 29 Sauter, 2. physik. Chem. B, 1933, 21, 161. 30 Daubeny, Bunn and Brown, Pruc. Roy. SOC. A, 1954,226, 531. 31 A. Turner-Jones and C. W. Bunn, to be published soon. 31 Fuller, Chem. Rev., 1940, 26, 143. 33 Fuller, Frosch and Pape, J. Amer. Chem. Suc., 1942, 64, 154. 103

ISSN:0366-9033    

DOI:10.1039/DF9582500095

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

ORDER-DISORDER TRANSITIONS IN STRUCTURES CONTAINING HELICAL MOLECULES BY A. KLUG AND (the late) ROSALIND E. FRANKLIN Birkbeck College Crystallography Laboratory, London University Received 19th February, 1958 The most commonly considered types of disorder in a crystal containing long-chain molecules are those in which the individual chain molecules are either rotated about their long axes or else translated along the fibre axis. However, since many fibrous polymers are helical in structure, we should also expect the possibility of screw disorder; that is, a combination of the above two types. A disorder of this kind can be shown to exist in orientated gels of tobacco mosaic virus and in fibres of polytetrafluoroethylene (PTFE) at 2OOC. The method by which screw disorder may be deduced from X-ray diagrams is described, and the order-disorder transitions in the two substances discussed.The mech- anism of the transition in PTFE is of particular interest in connection with its remarkably low coefficient of friction. Attention is also drawn to a related type of disorder found in stretched fibres of deoxyribonucleic acid. Two types of disorder are commonly recognized in crystal structures consisting of long-chain molecules. The first arises when the individual molecules are randomly rotated about their long axes. Thus the transition that occurs in paraffin hydrocarbons 1 and related molecules 2 a few degrees below the melting point is believed to be due to the onset of rotation of the molecules about their long axes. The second kind of disorder is that in which the molecules are variably displaced along their long axes.When the number of monomer units in the " fibre " axis repeat period is large, it is not possible, by X-ray diffraction methods, to dis- tinguish between rotational and translational order. Besides straightforward rotational or translational disorder it is clearly possible to have a combination of the two types, that is, screw disorder. Indeed we should expect this to happen when the molecules have a helical structure and are packed rather closely together. Examples of a screw disorder of this kind can be de- duced from the X-ray diagrams of tobacco mosaic virus (TMV) and of the polymer polytetrafluoroethylene (PTFE). The effect with TMV has already been described elsewhere,3 and is recapitulated here in more general terms to serve as an intro- duction to the order-disorder transition4 in PTFE at room temperature.The latter is of interest in connection with the remarkable frictional properties of the substance . THE INTERLOCKING OF PARTICLES OF TMV It is well known that particles of TMV are rods of diameter 5 about 150 A and length 6 about 3000 A. More recent work 7 has shown that the virus protein is composed of sub-units set in helical array around the long axis of the particles. It has also been shown 3 that the previously accepted value 5 of 152 A for the diameter refers to the packing diameter between particles in the dry state, and that in fact the maximum radius 8*9 of the particle exceeds this packing radius. The TMV particle bears on its surface a helical array of protuberances, one for each sub-unit, projecting well beyond the mean radius, and so presents a system of helical ridges and grooves. Two neighbouring particles can thus interlock and approach each other to within a distance smaller than the maximum diameter.104FIG. 1.-A model to illustrate the inter- locking in a close-packed array formed by rods bearing helical grooves of small pitch angle. The central rod in the model can be screwed in and out by means of a screw-driver. [To face page 105A . KLUG A N D R . E. FRANKLIN 105 It is the nature of this interlocking and its effects on the X-ray diffraction diagram that we shall be concerned with here. X-ray diffraction diagrams of oriented, wet TMV gel indicate that the par- ticles are in random orientation about their long axes, since the pattern obtained is characteristic of the scattering by independent particles (continuous Fourier transform). Evidence that the particles are in fact probably oscillating or rotating about their long axes may be deduced from an observation of Bernal and Fankuchen.5 They showed that the distance between the particles in the so- called " equilibrium gels " could be varied by changing the pH or the salt con- centration and that the distance of closest approach in strong salt solution was found to be 173 A, a value which is greater than the packing diameter (1 52 A) of the particle.Even more striking is the fact that variation of interparticle distance with pH shows a distinct minimum of 185A at the isoelectric point.Since it is now known 9 that the maximum radius of the TMV particles is about 90 I$, it seems clear that the observed minimum distance represents the closest approach of two TMV particles rotating or oscillating about their long axes. When a gel of TMV is dried, the X-ray diagram shows that the particles move closer together to form a close-packed two-dimensional hexagonal array. In so far as the TMV particles may be regarded as uniform density rods, the structure will behave as a single crystal in two dimensions but the actual intramolecular X-ray pattern observed indicates that the particles are still in a state of rotational and/or translational disorder with respect to their long axes. As mentioned above, the interparticle distance in dry gel is a good deal less than the maximum diameter of the particles and this implies that a considerable degree of interlocking takes place.It has been shown by Franklin and Klug,3 from a study of the X-ray diagram of dry TMV, that the particles interlock because each bears a helical groove on its surface. It is perhaps not immediately obvious why the existence of grooves and ridges should produce any interlocking, for, when two parallel particles are brought into contact, the direction of the ridges on one particle crosses that of the grooves on the other. The interlocking in TMV is possible only because the helical groove involved makes an angle of only 25" with the horizontal and is thus very nearly flat. The flatness of the helix means that a particle may interlock with all the six neighbours surrounding it in a hexagonal array.This is illustrated by the model in fig. 1 which shows seven helically grooved rods fully interlocked. The central rod can (with the aid of a screw-driver) be screwed in and out among its six neighbours without altering the packing of the rods. The original paper should be consulted for details of the effect of the inter- locking on the X-ray diagram, and we shall here outline the type of reasoning used in terms applicable to general screw disorder in helical structures. THE DETECTION OF SCREW DISORDER IN X-RAY DIAGRAMS The model in fig. 1 forms a truly crystalline structure, and screwing one of the rods (considered infinitely long) in or out with respect to its neighbours would not destroy the crystallinity of the arrangement.Any real structure consists, of course, of discrete atoms, so that a screw movement of this kind would destroy the coherence between different particles. The X-ray diffraction pattern would thus for the greater part correspond to the continuous transform of the individual particles. There would be, however, a region of the X-ray diagram in which the crystalline character is still evident. It has been shown 10 that the diffraction pattern of a helical structure can be usefully thought of as a sum of contributions each of which corresponds to a particular " helical projection " of the structure. To define a helical projection we consider a space-filling set of helices all of the same pitch, and let each atom be projected along the helix passing through it on to either an axial or equatorial106 STRUCTURES CONTAINING HELICAL MOLECULES plane.Formally, the Bessel function contribution of order n on the Zth layer- line corresponds to the projection down a set of helices of direction denoted by (n, Z) and of pitch nc/Z. Consider now what happens when we have an assembly of molecules arranged at the lattice points of a crystal and a variable screw disorder is introduced. In general, the three-dimensional crystalline order is destroyed. But the projection of the molecules along the helix corresponding to the screw motion will remain unchanged. Hence that part of the X-ray diagram corresponding to this helical projection will still resemble diffraction by a crystal and show sharp reflections (i.e.the continuous transform of the molecule is sampled at the points of the reciprocal lattice of the crystal), while the rest of the X-ray diagram will show areas of diffuse scattering (continuous transform of the molecule). For simplicity, we have dealt first with the change from full crystalline order to a disordered state. It is, however, the converse process that takes place when orientated TMV gel is dried. The change here is from a state in which no orienta- tional order is present to a partially ordered state brought about by the interlocking be tween particles. A SCREW TRANSITION IN POLYTETRAFLUOROETHYLENE AT 20°C The molecular and crystal structure of PTFE has recently been worked out by Bunn and Howells.4 PTFE is crystalline below 20°C but at that temperature shows a first-order transition.By a comparison of the X-ray diagrams above and below this temperature, Bunn and Howells showed that the transition is one from fully crystalline three-dimensional order to a lower degree of order. They further concluded that the disorder consists either of a variable displacement of the molecules along their chain axes or a variable rotation about these axes. We wish, however, to point out that the experimental results clearly indicate that what happens at the transition temperature is the onset of a screw disorder. This may be inferred from the X-ray diagrams by reasoning along the lines given above. The molecular configuration determined by Bunn and Howells 4 is shown as a radial projection 10 in fig. 2.To obtain this diagram the atoms have been pro- jected radiaIly on to a cylindrical surface centred on the chain axis, and the surface then cut open parallel to the axis and opened out flat. The axial repeat period is 16-8 A, and in this distance are contained 13 > CF2 groups lying along six turns of the basic helix of pitch 2.8 A. The carbon atoms lie at a radius of 0.42 8, and the fluorine atoms at a radius of 1.6481. This is, of course, a purely geometrical description of the structure, but it is the most appropriate for our purposes. Bunn and Howells observed that when the temperature is raised to 25°C the equatorial reflections and those on the 6th and 7th layer-lines remain sharp, but that all those on intermediate layer-lines become diffuse. The areas of diffuse scattering are in the same place as the sharp reflections of the crystal structure, showing that the molecular configuration is unchanged, but that the overall crystallinity has been destroyed.The sharpness of the reflections on the 6th and 7th layer-lines indicates that, as far as these parts of the diagram are concerned, the structure has remained crystalline. That is, the corresponding helical projections are unchanged by the transition at 20°C. The direction of the helical projection corresponding to the 6th layer-line is shown by the full lines in fig. 2, and that corresponding to the 7th by the dotted lines. These are then the directions in which the screw motion responsible for the disorder takes place. In the notation of Klug, Crick and Wyckoff 10 the helical directions are (1, 6) and (- 1, 71, the Bessel functions on the 6th and 7th layer-lines both being of the first order.Although the results indicate that two screw displacements with respect to the original position in the low-temperature crystal lattice are possible, it should be noted that any one molecule can only undergo one of these screw motions at a time. It is not, however, possible to decide from the X-ray results whether thereA. KLUG AND R. E . FRANKLIN 107 are distinct domains in a crystal, each of which contains only one direction of screw disorder, or whether the two classes of disordered molecules are inter- spersed throughout one crystal. The question naturally arises as to why the screw disorder takes place. The answer is not entirely obvious, but must clearly be bound up with the helical nature of the molecular configuration. Several points seem worth noting.The fact that the transition occurs at a temperature (20°C) so far below the melting point (330°C) evidently means that the relatively rigid PTFE molecules are able to slip past, or roll round, one another very easily. Bunn and Howells attributed the ease with which this happens to the smooth profile of the approxim- ately cylindrical surface of the fluorocarbon chain. While there may indeed be i , i i i 4 i FIG. 2.-The radial projection (see text) of one axial repeat period of a molecule of polytetrafluoroethylene, drawn on a cylindrical surface of diameter equal to the inter- molecular distance. The sections of the van der Waals spheres of the fluorine atoms are shown as large circles.The full and dotted lines represent the helices (1,6) and (- 1,7) respectively. free rotation at higher temperatures, this is surely not the case at 20°C for, as we have seen, the molecules appear to be screwed in and out with respect to their neighbours. Since the molecules are held together only by van der Waals forces, this would happen only if there were in fact some degree of interlocking between neighbours. The packing of PTFE molecules may be conveniently illustrated by means of the radial projection in fig. 2. This was drawn on a cylindrical surface of diameter 5-6 8, equal to the intermolecular distance, so that the line of contact between two neighbours lies in the surface chosen. In the figure, we have also drawn the sec- tions of the van der Waals spheres of the fluorine atoms by this surface so that the diagram is a fairly realistic representation of the bumps or knobs on the outside of a PTFE molecule.It will be seen from the figure (or from Bunn and Howells’ drawing4) that the molecule bears grooves in the two helical directions (1,6) and (- 1,7) in which the screw disorder takes place. There are, of course, others as well, but the two helices mentioned are those of the smallest pitch angle. Neither of these two helical grooves is as flat as that present on the surface of the TMV108 STRUCTURES CONTAINING HELICAL MOLECULES particle and the ridge on a molecule of PTFE cannot interlock efficiently with the corresponding grooves on its neighbours.Interlocking is in this case facilitated by the presence of the two grooves since they are of opposite tilt and similar pitch. (The helix (- 1,7) has a pitch equal to 6/7ths of that of the helix (1,6) and is of opposite sense; see fig. 2.) This means that when two parallel molecules are in contact, the set of ridges belonging to the one helix can fit approximately into the grooves of the other. If the fit were exact, there would be no difficulty at all in surrounding a molecule by six interlocking neighbours. The approximate way in which two molecules can pack together may be illus- trated by making a copy of fig. 2 on transparent paper, and turning it over before I I I I I I I 1 I 1 I I f ! 1 I I FIG. 3.-The pattern formed by the fluorine atoms of two PTFE molecules in one of the many possible positions of contact.The line of contact may be anywhere between the dotted lines. The arrows denote the helical directions (1,6) and (- 1,7) along which the screw disordering displacements take place. superimposing the two diagrams (since it is the outsides of both cylindrical sur- faces that must come into contact). It is found that there are a large number of positions containing a narrow band in which the knobs of one molecule fall nicely between the knobs of the second. One of these positions is shown in fig. 3 which shows the two radial projections superimposed. The vertical line of contact can be taken to be anywhere between the pair of dotted lines, which subtend an angle of approximately 50" at the axis. However, by considering the contact between only two molecules at a time, it is not possible to understand precisely how the molecules pack in a hexagonal lattice, except on the general grounds given above.A molecule has 13-fold screw symmetry about its axis, so that the 6 contacts between a molecule and its neigh- bours cannot all be the same. Since 13 is not far from a multiple of 6, there mayA . KLUG AND R . E. FRANKLIN 109 be an approximately hexagonal system of contacts. It would not be profitable to pursue this matter further without a detailed study of the packing in the pseudo- hexagonal crystalline form of PTFE occurring at temperatures below the transition point. It ought to be mentioned, for completeness, that if the PTFE is racemic, helices of opposite sense will be present.However, it seems likely that in this case the racemate would be at least partially resolved on crystallization. Finally, on the question as to why it is along the helices of smallest pitch that the screw disordering motion takes place, it might be valid to introduce some kinetic considerations, besides the steric ones mentioned. It is easy to show that, for a given total displacement of a point on the surface of the molecule, the kinetic energy is the smaller, the smaller the pitch angle of the motion. Thus, if the disorder were not merely static, but some form of hindered oscillation (similar to that found in paraffin crystals 2), the particular screw motions adopted would be partly accounted for. This assumption might not at first sight seem very plausible at a temperature as low as 20°C, but the extremely streamlined nature of the fluorocarbon surface ought to be borne in mind.Bunn and Howells 4 also dis- covered a similar transition to that observed in PTFE in crystals of perfluoroacetane, c16F34. Here the transition temperature is - 170°C, indicating that, because of their lower molecular weight (800), the molecules are able to undergo the dis- ordering motions at a lower temperature than in PTFE. The low absolute value of the transition temperature does suggest that it is extremely easy to set fluoro- carbon molecules in a crystal into rotational or translational motion-or a com- bination of both-and that kinetic considerations might thus be relevant in dis- cussing the screw disorder in PTFE. PARTIAL DISORDER IN DEOXYRIBONUCLEIC ACID Other types of disorder are possible which lead to X-ray diagrams showing sharp reflections in some regions and continuous scattering in others.An example of this occurs when long chain molecules in a crystal are displaced along their long axes, by translations which are not random but related to the repeat distance along the molecular chain. An effect of this kind has been observed by Wyckoff 11 with stretched fibres of DNA in the B form. On the X-ray diffraction pattern even layer-lines are observed to have sharp spots on them, while odd layer-lines are diffuse. The simplest explanation of this again involves the interlocking of helical grooves, but in a slightly different way. The double-strand helix of DNA bears two approximately equally spaced grooves,l2 one deeper than the other.In spite of the fact that these grooves are rather steep, helical interlocking as described above can take place because both grooves are relatively very deep. The existence of the bulges and depressions on the surface of the molecule means that neighbouring molecules can pack together in such a way that the large ridge fits either into the deep groove or into the shallow one (and similarly for the small ridge). If it is assumed that these possibilities are equally likely in the stretched fibres examined by Wyckoff, then this is equivalent to giving each molecule a random displace- ment of either 0 or & 3c. This would lead to crystal reflections on even layer- lines and to continuous scattering on odd ones, as observed.The effect is analogous to that observed with some layer structures such as cobalt 13 or certain clays.14 Here the layers, which have hexagonal symmetry, have random lateral displacement of & 3 in the 110 direction, and only every third layer-line shows sharp reflections. With DNA, however, the experimental data are rather sparse, and more complicated models could doubtless be devised to explain the results. This work was supported in part by a Research Grant, No. E-1772 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.I10 STRUCTURES CONTAINING HELICAL MOLECULES 1 Miiller, Proc. Roy. Suc. A, 1932, 138, 514. for a review see Daniel, A&. in Physics, 1953, 2, 450. 3 Franklin and Klug, Biochim. Biophys. Acta, 1956,19,403. 4 Bunn and Howells, Nature, 1954, 174, 549. 5 Bernal and Fankuchen, J. Gen. Physiol., 1941, 25, 11 1. 6 Williams and Steere, J. Amer. Chem. Soc., 1951, 73, 2057. 7 Watson, Biochim. Biophys. Acta, 1954, 13, 10. Franklin and Holmes, Biochim. Biophys. Acta, 1956, 21, 405. 8 Caspar, Nature, 1956, 177,475. 9 Franklin, Klug and Holmes, in The Nature of Viruses (Ciba Foundation Symposium, 10 Klug, Crick and Wyckoff, Acta Cryst., 1958, 11, 199. 11 Wyckoff, Thesis (Massachusetts Institute of Technology, 1955). 12 for a review see Wilkins, Cold Spring Harbor Symposia, 1956, 21, 75. 13 Wilson, X-ray Optics (Methuen, London, 1949). 14 Brindley and Robinson, Trans. Brit. Ceram. SOC., 1947, 46,49 ; Miner. Mag., 194% J. and A. Churchill, London, 1956), p. 39. Franklin, to be published. 28, 393. Note added in proof More recent work on PTFE (for references, see Bunn and Holmes, this Discussion) has shown that the molecule is slightly untwisted just above the transition point at 20"C, so that the axial repeat period contains 15 CF2 groups distributed over 7 turns of the basic helix. The actual physical change in the internal configuration is, however, very small, and the argument for the intermolecular screw disorder is not affected.

ISSN:0366-9033    

DOI:10.1039/DF9582500104

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

ORIENTED GROWTH IN THE FIELD OF ORGANIC HIGH POLYMERS. BY J. WILLEMS Krefeld, 21 Tiergartenstrasse, Germany Received 30th January, 1958 In accord with the foundations for a structural proof of oriented overgrowth (epitaxy) laid down by Royer,l the existence of a two- or one-dimensional structure analogy of lattices seems valid for a11 types of epitaxy observed. An example of this interpretation in the usual manner of epitaxy for an organic substance, e.g. anthraquinone, on an inorganic substance, such as NaCl, on the basis of a two-dimensional structure analogy is shown in fig. 1 and 2.3 (It may be that the relation, in view of the survey of the anthraquinone structure (Structure Reports, 1947) are more complicated.) C C t 0-J FIG. 2.-Structural interpretation of the epitaxy of fig.1. (a) (001) of NaCl; (6) (010) of anthraquinone; (c) projection of the molecule upon (010). Binding forces between substrate and deposit in epitaxy can comprise all types of primary valences, i.e. heteropolar and homopolar bonds and metallic linkages, and secondary valences, i.e. orientation, induction and dispersioneffects, and hydrogen bridges. The secondary valence is of special importance for the epitaxy of organic molecules such as the high polymers. The type of binding forces exerted by the deposit units on each other need not to be identical or related to the type of binding forces exerted by the substrate units on each other; for example, epitaxy of paraffins, the lattices of which are substantially held together by dis- persion effect, on NaCl with a typical ion lattice, is easily obtained.4 The first oriented overgrowth with organic high polymers was observed by Richards,s who obtained epitaxy of paraffin wax crystals on the surface of strips of cold drawn polyethylene and polyethylene sebacate.But no epitaxy of paraffin wax was obtained on gutta-percha, rubber, polyethylene terephthalate, polyhexyamethylene adipamide (Nylon-6 : 6) and polyhexamethylene se bacamide (Nylon-6 : 10). 111112 GROWTH IN ORGANIC HIGH POLYMERS As to the different behaviour of the organic high polymers, Richards points out that the chain molecules of polyethylene and polyethylene sebacate are very similar in configuration and in arrangement of the chains in the unit cell to the molecules of paraffin, in contrast to the chain molecules of the other group of high polymers on which no epitaxy of paraffin was obtained.With the low molecular substances, epitaxy of compounds, the units of which are very different in configuration and in the arrangement in the unit cell, is easily obtained ; typical examples are the epitaxy of anthraquinone and paraffin on NaCl. Thus, from the findings of Richards arose the question whether high polymer substances differ principally in this point from low molecular substances. With this in mind, we tried to obtain epitaxy of crystals of aromatic compounds on aliphatic high polymers, i.e. with crystals of low molecular weight molecules greatly different in configuration compared with the molecules of the high polymer substrate.6 It was found that epitaxy of pentachlorophenol and hexaethylbenzene was easily obtained on the surface of a strip of cold drawn polyethylene in which sections of the polymer molecules in the crystalline regions were approximately parallel to the directions of cold drawing. The needles of pentachlorophenol were arranged in one position with the long axes of the needles approximately parallel to the direction of drawing, whilst the needles of hexaethylbenzene were arranged in two positions the long axes of the needles forming an angle of go", this angle being bisected by the direction of drawing of the strip (fig.3). Furthermore, epitaxy of pentachlorophenol (fig. 4), pentabromophenol, penta- chloroaniline and anthraquinone on the surface of cold drawn strips of poly- hexamethylene adipamide (Nylon-6 : 6) was obtained.In these overgrowths, pentachlorophenol and pentachloroaniline are oriented with the long axes of their needle-like crystals approximately parallel to the direction of drawing of the strip, and the needles of pentabromophenol and anthraquinone partly in the same manner and partly approximately perpendicular to this direction. Finally the epitaxy of pentachlorophenol on tendons of an arthropode (faralithodes camtschatica) may be mentioned. In this case the crystal needles of pentachloro- phenol were oriented with the long axes of the needles parallel to the fibre axes of chitin. As the drawn strips of high polymers used as substrates are birefringent, the investigation of the " law of intergrowth " by means of the polarizing microscope is rendered very difficult, and the complete law of intergrowth cannot as yet be ascertained.On the base of a one-dimensional structure analogy the following cases seem already to be explained in a satisfactory manner. The epitaxy of pentachloro- phenol (stable) with the long axes of the crystal needles b 11 c polyethylene (fibre axes) with b = 4-97A and 2c = 5-06A; 7 pentachlorophenol (stable) b 11 c chitin (fibre axes) with 2 x b pentachlorophenol = 9.94 8, and b chitin = 10-46 A.8 In the examples of epitaxy on Nylon-6 : 6, the explanation on the base of a one- dimensional structure analogy is less satisfactory ; for example, pentachlorophenol (instable) with the long needle axes b 11 c Nylon-6 : 6 (fibre axes) with 46 = 4 x 3-84 A = 15-36 8, and c = 17-20 A, corresponding to a mismatch (percentage excess of the lattice spacing of the deposit relative to the substrate) of 10.8 %.In the example described above, crystals of low molecular molecules were oriented on the surface of high polymers. These findings lead to the question whether it is possible to orient the macromolecules of a high polymer on a low molecular crystal. Indeed such an orientation was observed in the oriented overgrowth of polyethylene (Marlex, M = 10,500) on the (001) crystal face of NaC1.9 The polyethylene is oriented on (001) of NaCl in needles with the long needle axes parallel to [110] and [IT01 (fig. 5). Investigations by means of the electron microscope are being made at the present time in order to establish the law of intergrowth of this epitaxy, and the nature of the oriented " needles " of polyethylene observed.From the point of view of the recent investigations byFIG. 1.-Oriented overgrowth of anthraquinone upon a cleavage face (001) of NaCl x 300. [To face page 112FIG. 3.-Oriented overgrowth of hexaethylbenzene on cold drawn polyethylene. x 300. Direction of cold drawing FIG. 4.-Oriented overgrowth of penta- chlorophenol on cold drawn polyhexa- methylene adipamide Nylon-6 : 6. x 300. Direction of cold drawing + FIG. 5.-Oricnted overgrowth of poly- ethylene on (001) NaCI. x 500.J . WILLEMS 113 Fischer 10 and Keller 11 these needles may be either flat single crystals rolled up, or flat single crystals of uncommon thickness standing on edge, the thickness being determined by the nucleation 12 influence of the NaCl surface. 1 Royer, Bull. SOC. f r a q . Mill., 1928, 7 , 51. 2 Seifert, in Gomer and Smith, Structure and Properties of Solid Surfaces (Chicago, 3 Willems, Naturwiss., 1944, 32, 324. 4 Willems, Naturwiss., 1955, 42, 176. 5 Richards, J. Polymer Sci., 1951, 6, 397; cp. Willems and Willems, Nature, 1956, 6 Willems, Experientia, 1957, 13, 276. 7 Bunn, Trans. Faraday SOC., 1939, 35,482. 8 Meyer and Pankow, Helv. chim. Acta, 1935, 18, 589. 9 Willems, Experientia, 1957, 13, 465. 10 Fischer, 2. Naturforsch., 1957, 12a, 9. 11 Keller, Phil. Mag., 1957, 2, 1171. 12 van der Merwe, Faraday SOC. Discussions, 1949, 5, 201. 1953), p. 318. 178, 429.

ISSN:0366-9033    

DOI:10.1039/DF9582500111

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

STUDY OF SINGLE CRYSTALS AND THEW ASSOCIATIONS IN POLYMERS BY A. KELLER AND A. O’CONNOR H. H. Wills Physics Laboratory, University of Bristol, England Received 23rd January, 1958 Single crystals of polyethylene were prepared. Closely resembling paraffins, they have a flat tabular habit, thickening through the formation of spiral terraces. The molecules were found to be perpendicular to the layers about 1008, thick. The Same periodicity has been confirmed by low angle X-ray experiments and has been identified with the layer-thickness. We postulate that the molecules must be sharply folded in order to produce this orientation. The possible causes and consequences of this postulated folding are discussed. It is suggested, on the basis of preliminary results, that this folding might be a general feature in paraffins of sufficiently long chains.It is mentioned that similar chain-folding has also been found with polyamides where the chains are at a large angle to the length of fine fibrillar units. The existence of a new type of rotationally twisted crystallization in polyethylene has been discovered and it is demonstrated that this can lead to a rnoirk representation of the crystal lattice with the aid of the electron microscope. Other novel structural features in polyethylene crystals-variable in time and affected by the electron beam-are also mentioned. It is attempted to link np our observations on single crystals with the more usual fibrillar crystal habit leading to spherulite formation, accounting also for the frequently observed twisted structures within the spherulites.Until quite recently, crystalline polymers were regarded merely as consisting of a random assembly of crystallites and amorphous regions where one long chain molecule can pass through several amorphous and crystalline areas. The first indication of a texture on a different scale was the discovery of spherulites and the recognition that the spherulitic texture represents the general mode of crystal- lization in polymers (e.g. ref. (l), (2)). These spherulites are spherically symmetrical microscopic aggregates of radiating fibrous units. They develop through a par- ticular type of branching growth of fibrils through the intermediate stage of sheaves. The finest component units which could be identified within them were either microfibrils of about 100-200 A wide, or flat ribbons, or thin sheets.In its coarser features, the spherulitic morphology in polymers is very similar to that of other spherulite-forming substances. Frequently the existence of a helicoidal arrange- ment of the birefringent units along the spherulite radii can be deduced from the periodicalIy varying birefringence producing concentric extinction rings. This phenomenon is apparently widespread among other crystalline materials (e.g. ref. (3)) even if it has never been studied with modern techniques and its real nature and origin are not properly understood. Another puzzling feature in polymers is the existence of large spacings as revealed by low angle X-ray diffraction (e.g. ref, (1)). These spacings are of 80-200A, and reflections are usually observed in their first order only.They are not caused by any obvious chemical regularity along the chains and have been attributed to an alternation of amorphous and crystalline regions. In the present work we were following two lines. In the first place we attempted to obtain further information about the morphology by a combination of electron microscope and electron diffraction techniques. Primarily we were aiming at the identification of the basic crystalline units. Secondly, we were pursuing the problem of large spacings in the belief that this is connected with the problem 114FIG. 1 .-Electron micrograph of Marlex crystallized from trichlorethylene ; Au-Pd shadowed ; 8,800 x ; substrate carbon. FIG. 2.-Electron micrograph of Marlex crystallized from xylene ; Au-Pd shadowed ; 6 , 3 0 0 ~ ; substrate carbon.(The irregular striation in the background is due to extraneous matter.) [To face page 114FIG. 3.-Electron diffraction pattern given by twinned crystals of Marlex 50, prepared from xylene. FIG. 4.-Electron micrograph of Marlex crystallized from trichlorethylene taken with a very weak beam (jointly with A. W. Agar); 14,500~.A . KELLER AND A. O’CONNOR 115 of the morphology and that at some stage the two lines of approach will overlap. In fact, this overlap has been achieved. Work is still in progress. Here some of the principal results obtained up to the end of 1957 will be presented. EXPERIMENTAL ELECTRON MICROSCOPE-ELECTRON DIFFRACTION EXPERIMENTS We attempted to obtain electron diffraction patterns of areas selected under the electron microscope in order to identify the nature and orientation of the crystalline units visible under the electron microscope.For a long time our attempts were unsuccessful until we discovered that the electron beam destroys the diffracting power of the specimen without affecting the shape of the objects. Thus unless diffraction experiments are at- tempted this damage will remain unnoticed. The practical consequences of this damaging effect of the electrons are serious for our type of work. It forces us to use extremely low beam currents. The resulting low intensity makes working difficult, in fact it makes direct imaging at even moderate powers next to impossible under normal conditions.We think that almost certainly, most electron microscopy on polymers in the past has been done on damaged specimens without this having been noticed. The experiments to be described here were carried out on the linear polyethylene Marlex 50. Some brief reference will be made to experiments with polyhexamethylene sebacamide (nylon-6 : 10). Samples were prepared from solution. The polyethylene was dissolved hot and crystallized while in the solvent, by controlled cooling. The nylon was crystallized by adding a poor solvent (benzyl alcohol) to a solution of rn-cresol. AS seen in all these methods of crystallization, crystallization occurred within the solvent and not through evaporation. Polyethylene crystallized under the above conditions formed either lozenge-shaped crystals (and closely related forms), or sheaves, or both together 4,s.6 In high dilution it was possible to obtain the lozenge-shaped crystals as a representative form of the whole material. These crystals were visible under the optical microscope with phase-contrast illumination. Electron micrographs (fig. 1) reveal that they consist of laminar terraces, thickening through spiral pyramids just as with paraffins. The step heights were of the order of 1WA. The crystals were not always regular lozenges but, depending on the method of preparation, could be more or less dendritic 5 and sometimes also near-hexa- gonal. All observed shapes were simply related to the basic lozenge. In thick crystals, folds appeared which were mostly along the short diagonal of the lozenge (ref.(9, see also fig. 2). In later stages this fold took up the shape of a branching sheaf. Electron diffraction patterns, if taken under suitable precautions, were extremely sharply defined. They revealed that the c-axis, the direcion of the molecules, was per- pendicular to the plane of the crystals, and that the a- and b-axes were along the long and the short diagonal of the lozenge respectively.5 Accordingly the growing faces were the (1 10). The pattern in fig. 3 is given by an assembly of crystals where at least four are in (1 10) twin orientation. Twins of this kind were frequently observed, sometimes repeated in regular sequence. Fig. 3 also illustrates the effect of secondary diffraction. From our point of view the most significant secondary diffraction spots are those nearest to the centre.Fig. 3 was printed dark to show these. Their arrangement has the same sym- metry as the main spots, consequently they can be considered as corresponding to an identical lattice but with larger spacings. This is the origin of moirk patterns (see later). There are many features which are not yet understood, such as smaller substeps (visible also in fig. 1). Frequently reverse steps could aIso be seen bordering the main steps (fig. 2). The development of the growth spirals could be traced to its initial stage where only the initial ledge terminating at the screw dislocation is present (fig. 2). Quite lately, using a special technique, we succeeded in obtaining electron micrographs at appreciable magnification without destroying the crystallinity (Agar, Frank and Keller, to be published). The result was surprising.In places where the crystal was at least two layers thick a line, or cross, grating pattern appeared which was often curved (fig. 4). This pattern disappeared rapidly in the electron beam. There was also another structure apparent : a system of lines following approximately, but not exactly, the faces of the lozenges, extending throughout the interior of the crystals (fig. 5). They seem to be surface corrugations heightened by Bragg extinction. It appears as if the crystal were divided into four quadrants. This line-system disappeared not only in the electron beam but also within a few hours after the specimens were dried from solution.116 SINGLE CRYSTALS AND THEIR ASSOCIATIONS I N POLYMERS In one special preparation (an extract of somewhat lower molecular weight) the lozenge-shaped crystals possessed a leaf pattern (fig.6). It can be seen that this peculiar pattern is formed by the regular sequence of layers each rotated by the same amount in the same direction. The loci of the apices produce the leaf shape. It is noticeable that the shape of successive lozenges varies systematically. From a solution in o-chlorophenol, continuous films could be prepared, floating on the surface of the liquid, which showed a two-dimensional spherulitic structure under the polarizing microscope. The spherulites themselves possessed concentric extinction rings, usually attributed to a helicoidal structure. Electron microscopy was hardly possible, owing to the thickness of the films, nevertheless in favourable localities it was possible to establish that these spherulites consisted of regularly curling flat crystals (fig.7) giving rise to the periodic extinction effects. It will only be mentioned here, that using the method of preparation described above large sheaves of nylon-6 : 10 were obtained. These sheaves possessed a microfibrillar structure at the edges, the fibrils being about 1508, wide. It was possible to establish, by using selected area electron diffraction, that the molecules were ai a large angle (65"-75"), i.e. nearly perpendicular to the fibril axes. LOW-ANGLE X-RAY SCATTERING In these experiments a Kratky type collimator 7 was used, fitted with many special accessories required by our problems.All work to be described here was done on the polyethylene Marlex 50. The polymer was crystallized by cooling the hot solutions as already described and sufficient quantities were collected by filtering. In general, a porous tablet was obtained which revealed random orientation on conventional X-ray examination. The low angle X-ray pattern showed four well-defined rings corresponding to four orders of a spacing in the region of 1208,. The exact values vary between 115 and 1268, for different preparations. When the polymer was crystallized in dilute solution and filtered very slowly, a compact and coherent film formed. Wide-angle X-ray patterns revealed that the molecules were perpendicular to the macroscopic film surface. In fact, such a film can be considered- as far as molecular orientation is concerned-as a cross-section of a drawn fibie, except that no drawing took place, the film being an aggregate of spontaneously formed crystals.This orientation is obviously caused by the flat crystals sedinienting all horizontally. The same large spacings appeared again, giving four orders. However, this time they were in arcs, corresponding to planes parallel to the terraces within the crystals. It is thus beyond reasonable doubt that these spacings represent the same periodicity as re- vealed by the layering within the crystal. In some preparations another spacing of about 140-24OA also appeared. The relative intensity of the corresponding reflections was usually slight. Both the intensities and spacings corresponding to this second set of reflections varied from specimen to specimen.When the continuous film, mentioned above, was annealed near the melting point, the usual spacing of about 120 8, nearly disappeared, and at the same time there appeared another reflection corresponding to a spacing of 200-300 8,. Also the wide angle pattern was affected in so far as the c-axes (the molecules) were not preferentially perpendicular to the film surface any longer but were random in planes perpendicular to the film plane. This corresponds to the randomization around the b-axes which remained unaltered in the film plane. The degradation products thus obtained ranged from hard polymers to soft greases. We found that the correspond- ing large spacings as recorded by low-angle X-ray diffraction were progressively reduced from 120 8, to about 40 8, when going from the undamaged polymer to the softest wax.Marked reduction below 110 8, was found only as the waxy consistency was reached. We also thermally degraded some samples of Marlex 50. DISCUSSION Most probably the electrons cause a primary ionization with a surprisingly high efficiency (we estim- ate that one electron might affect one atom in the lattice), which affects the crystal- line order either directly or indirectly by inducing a chemical change. It is not the object of the present paper to investigate this matter more closely than is relevant to the electron optical examination of polymers. It is apparent that The first problem is the effect of the electron beam.FIG.5.-Electron micrograph of Marlex crystallized from trichlorethylene, taken with a very weak beam immediately after preparation (jointly with A. W. Agar) ; 7,500 x . [To face page 1 16FIG. 6.-Electron micrograph of a Marlex extract ; 7,200 x .FIG. 7.-Electron micrograph of a thick Marlex film showing banded spherulites in the polarizing microscope ; formed in a solution of o-chlorophenol ; 4,500 x .A. KELLER AND A . O'CONNOR 117 such examinations require special precautions owing to the damaging effect of the electron beam. Most experiments where this damage could be avoided were at the limit of present-day experimental possibilities. Clearly, image intensifica- tion would be needed. We think that the same difficulties might also very likely exist with many organic substances other than synthetic polymers, but unless electron microscopy was combined with electron diffraction within the microscope, this would not be noticed.The appearance of single crystals in their present form came somewhat as a surprise as such a close similarity between paraffins and polymers was contrary to expectation. The spiral terrace growth in paraffins is readily explained as the heights of the terraces represent one lattice translation and this in turn cor- responds to the length of one molecule. The screw dislocation initiating the spiral has a Burgers vector of unit length. In polymers the molecules are very long, measured generally in thousands of angstroms, consequently one would not expect a regularity of the order of lOOA along the chain length.It is true that electron micrographs only show small areas, and mostly only the thin parts of the crystals are clear enough for measurement. But the fact that the layers could be identified with a regular sequence of spacings in the low-angle X-ray patterns proves that this regularity is representative, and even more strictly observed than one would suspect from the micrographs. dn general, such a regular layer structure might be associated either with a chemical periodicity within the molecule, or with the actual molecular length. However, there is no possibility of a chemical periodicity within the polyethylene molecule and neither have we observed a variation with molecular length (above a certain minimum value). In view of the broad distribution of molecular weight,s it was possible to obtain fractions of different molecular lengths by solvent extraction at different temperatures.Although accurate molecular-weight determination haves not yet been made, the variation of solubility from fraction to fraction indicated a wide range of molecular lengths, yet no significant differ- ence in crystal habit was observed. We know from the work of Tung 8 that the average molecular length in Marlex 50 is around 6000A. Obviously these molecules cannot be straight and at the same time perpendicular to layers only about lOOA thick. As the parallel align- ment of the chains is practically perfect we are forced to conclude that the molec- ules must sharply fold back on themselves.5 This suggestion is not entirely new as it has been mentioned by Storks in 1938.9 Accordingly the real unit cell com- prises the full fold-period which contains a large number of chemical repeat units.The chemical repeat distance which has been previously identified with the unit cell would accordingly be only a subcell. Accordingly the step height and the corresponding large period given by the diffraction pattern would represent one lattice translation as for paraffins, except that here it would not correspond to the lerigth of the molecule, but only to a portion of it as defined by the fold. 1 t could be asked whether such a sharp fold, connecting adjacent chain portions ~ h i c h are closely packed in the lattice, is possible or not. For this we have to consider the lattice in c-projection, i.e.as viewed along the molecular chains (fig. 8). The molecules closest to each other are those lying in the (1lQ) planes. The planes of the carbon zig-zags of such adjacent molecules are at an angle fo 82". If a fold can bridge two such molecules in a (1 10) plane it could bridge any other pair. In fact we find from models that such a bridging is possible without affecting the interatomic distances or the valence angles. The fold would contain only very few, three or five (depending on where we start counting) carbon atoms. We have been discussing the fold as lying in the (110) plane. This in fact is the most likely possibility as these planes represent the growing faces of the lozenge. Each new molecular segment is expected to attach itself to this face and not to protrude further outwards, as a fold along any other direction would make it to do.This, however, implies that the crystal consists of four distinguishable118 SINGLE CRYSTALS AND THEIR ASSOCIATIONS IN POLYMERS wadrants with folds lying in the (110), (Ilo), (110) and (TlO) planes. (Naturally pairs of these would have identical orientations but would represent diametrically opposite quadrants.) In fact, our latest micrographs (fig. 5) reveal quadrants exactly of this type. Also there is evidence of such folds from past work. Muller 10 found that long cyclic paraffins pack as closely as linear ones, consequently the rings have collapsed into parallel chains which must be bridged by a very few atoms. Such a bridge would correspond to our postulated fold, preserving the close packing of the chains.The next problem is the cause of the regularity of the folding. One possible reason for a sharp distribution was given in ref. (5). Further, it is easy to see that there should be a lower limit. The fold will be stable if its additional free energy is balanced against the reduction of free energy due to adjacent chains becoming parallel. The particular length of the parallelization, where this condition is first achieved, will be the minimum fold-period and will represent the limit of solubility. There is no energetic reason why fold-periods larger than this should b T FIG. 8.-c-axis projection of polyethylene unit cell. not exist, except that large folds will not be stable if they protrude beyond the neighbouring ones which are all at one level.Thus large folds will tend to be reduced and it can be imagined that finally a lowest limit is reached. Even so, the strict regularity as demonstrated by the four orders of the X-ray pattern remains the most puzzling feature arising from this work. If the folds sre a result of the chains taking up a configuration of lower free energy, we would expect the fold-period to vary with different solvents, and with variations in crystallization temperature, etc. Preliminary observations, how- ever, have revealed a surprising invariance of the large spacings to such changes. In contrast to these observations, it has recently been noted that specimens change visibly (as judged by electron micrographs) within hours of preparation. For example, part of the structure in fig.5 represents protrusions which disappear com- pletely within a short time. This suggests that the periodicity recorded by X-ray diffraction may not correspond to the fold configuration within the liquid at the crystallization temperature, but rather to that within the dried material. Although no correlation between the variation of low-angle patterns and time, has so far been established, the low-angle patterns do occasionally indicate the existence of larger periods, which vary from specimen to specimen. This indicates that the regular spacing generally observed is not invariable. It is also in line with the above suggestion that the fold-period changes with time (i.e. recrystallization occurs), and it may be possible under certain conditions for the original fold to be preserved to some extent, resulting in the observation of two different low-angle reflections from the same specimen.The fringes in fig. 4 are moire' patterns produced by the super-position ofA . KELLER AND A . O’CONNOR 119 gratings. They arise through the successive layers not lying in exactly the same orientation. This is a direct consequence of double diffraction, an example of which is given in fig. 3. There it is seen that the beam diffracted for the second time corresponds to that given by an identical lattice of larger spacings. In fig. 4 these larger spacings come within the resolving power of the microscope. The full possibilities of this imaging of lattices has not yet been explored. It can be seen from the curving of the fringes that the lattice is deformed.As successive layers are in varying orientations it is reasonable to assume that the lattice itself is twisted. The accidental preparation of fig. 6 shows a regular sequence of rotation of suc- cessive terraces. This is probably the same phenomenon as the one in fig. 4 giving rise to the moirh, only there it is restricted to two or three layers. We are probably dealing with a lattice imperfection which has not been encountered before in the studies of crystal growth. The thickening along the b crystallographic axis leading to fibrillar and sheaf- like units might possibly represent the beginning of spherulite formation. If such fibrous units could form through a rolling-up of flat sheets along the b-axis, the b radial orientation 2 within the spherulites is accounted for.Formation of needles from flat crystals through curling up is well established with paraffins 11 and might represent the link between the flat crystal habit of long chain paraffins and the usually fibrillar habit of polymers. This fibril-formation in paraffins apparently occurs when the molecule is branched 12 or if the molecular weight is not uniform.11 The former observation might explain why flat crystals could not be obtained with the highly branched high-pressure polythene. This rolling-up could well produce regular helices which would account for extinction rings in banded spherulites. This is borne out by the spherulitic film from o-chlorophenol and the corresponding electron micrograph (fig.7) (these photographs could only be taken in the disrupted parts of the thick films where much of the regularity must have been disturbed). The change in orientation in annealed films, obtained through the sedimentation of single crystals, is consistent with the postulated rolling-up along the b-axis. Accordingly this corresponds to a transformation of flat crystals into a spherulitic morphology. In paraffins the chains are straight and perpendicular, or slightly inclined, to the surface of the flat crystals. It appears now that as the length of the chains is increased they may not stay straight indefinitely but at a certain length a sharp folding back would occur. As paraffins beyond C100H202 have not been syn- thesized we could not test this assumption directly.However, our degradation experiments showed this process in reverse. The polymer with very long chains gave a large period of 120& but after a certain stage of degradation the period gradually decreased. We suggest that at this stage the molecules became shorter than the fold-period, and from this stage onwards not the folding but the length of the molecule is responsible for the step height and for the large spacing in the diffraction pattern. Our experiments on nylon, only mentioned here, indicate that the folding is not restricted to polyethylenes. The nylon molecules were found to be nearly perpendicular to the microfibril and by the same argument, as used in connection with the thin layers in polythene crystals, this can be explained by a regularly folded molecular configuration.Although this would not account for the uniform width of the microfibrils, it would bring this fact into correspondence with the uniform thickness of the polyethylene crystal layers. Details of this work will be published elsewhere. ADDENDUM-LATEST RESULTS Molecular-weight measurements carried out by Dr. Howard (British Nylon Spinners Limited) confirmed that our material remained essentially unchanged in the course of our specimen preparation leading to single crystals. Conse- quently the formation of thin layers, much thinner than the length of the molecules,120 SINGLE CRYSTALS AND THEIR ASSOCIATIONS IN POLYMERS could not possibly be due to some systematic breaking-down of the molecules, the only alternative to chain-folding.In the course of our latest work it became apparent that the 120 8, fold period was not invariant as it appeared from the first observations. The large spacing, as determined most accurately by the low-angle X-ray technique, increased with increasing crystallization temperature, the photographs having been taken at room temperature after the crystalline precipitate was separated from the solvent. The dependence of the spacing on the temperature of crystallization is shown by fig. 9. In each case, 300 ml of 0.01 % concentration of Marlex in xylene at 100°C were poured into glass tubes suspended in a large oil-bath maintained at a constant temperature. It is this bath temperature that is plotted in fig. 10 as crystallization temperature. Obviously some crystallization must occur in the cooling process, T 140,.O U Q .5 130- Ef 2 120- 0 2- X U 0- c - O 110. 4 100,- 90- 7 / / / "/ X I / X+ / / / x 4 / I I / / / x - - L 1 to a degree which is greatest at the lowest temperatures of crystallization, when the solution has to pass through a temperature range in which the rate of crystal- lization is high. While this introduces some uncertainty in the abscissae at lower temperatures, it does not affect the general trend shown by the dotted curve. The lowest spacing of 92A was obtained when the hot solution was cooled rapidly to 10°C, and at the highest temperature where crystallization still occurred (91°C) two spacings- appeared simultaneously, one at 140 A and the other at 100 A,* both in several orders. The curve in fig.9 suggests that the factor determining the fold period might be the size of the critical nucleus, i.e. the smallest nucleus which is still stable at the particular temperature of crystallization. The size of the critical nucleus should be inversely proportional to the supercooling which would lead to a curve of the type as in fig. 9. Lately we have extended our work to some isotactic polymers. Details will be published later. Here it will only be stated that isotactic poly-4-methyl- pentene-1 could be obtained in the form of flat square-shaped crystals consisting of thin layers. The molecules were found to lie perpendicular to the thin layers, the symmetry of the packing being tetragonal. By the same argument as used in connection with polyethylene it follows that the molecules must be folded regularly at about 100 A intervals.Poly-4-methyl-pentene-1 could also be obtained in * only the final one is included in fig. 9.A. KELLER AND A . O'CONNOR 121 fibrillar form under suitable conditions of crystallization, in which case the molecules were found to be perpendicular to the length of the fibrils. This finding is in line with those on fibrous aggregates in polyamides, and again requires a folding of the chains. Poly-4-methyl-pentene- 1 has a relatively large side group on every second carbon atom of the main chain. Consequently, chain folding is not restricted to the simplest paraffin-type chain. In the course of a further experiment we deposited spherulites of isotactic polypropylene from solution. These aggregates were too thick for electron microscopy, but on melting and subsequent cooling thin fan-shaped laminae grew out of the molten blobs. Electron diffraction revealed that the molecules were again perpendicular to the thin layers which again requires folding of the chains. Thus we have obtained evidence that the molecules can take up a folded codiguration also when crystallizing from the melt at least under certain special conditions of preparation. Crystallization from the melt in general would require further investigations. The authors wish to thank Prof. F. C . Frank for his constant stimulating interest in this work. We also wish to acknowledge the co-operation of Dr. A. W. Agar in the part relating to moire' patterns. 1 Die Physik der Hochpolymeren, vol. 3, ed. Stuart (Springer, Verlag, Berlin, Got- tingen, 1955). 2 Keller, J. Polymer Sci., 1955, 17, 291, 351. Keller and Waring, J. Polynter Sci., 1955, 17, 447. 3 Michel-Levy and Munier-Chalmas, Bull. soc. franc. miner,, 1892,15,159. Wallerant, Brill. SOC. franc. miner., 1907, 30,43. Gaubert, Bull. soc..franC. miner., 1909,32, 421. 4 Till, J. Polymer Sci., 1957, 24, 301. 5 Keller, Phil. Mag., 1957, 2, 1171. 6 Fisher, Z. Naturforsch., 1957, 12a, 753. 7 Kratky and Sekora, Monatsch., 1954, 85, 660. 8 Tung, J. Polymer Sci., 1957, 24, 333. 9Storks, J. Amer. Chem. Soc., 1938, 60, 1753. 10 Muller, Helv. chim. Acta, 1933, 16, 155. 11 Rhodes, Mason and Sutton, Znd. Eng. Chem., 1927, 19, 935. Edwards, Ind. Eng. Chem., 1957, 49, 750. 12 Robert, Alexanian and Buzon, Proc. Third Petroleum Congr. (La Haye, 1951) p. 259.

ISSN:0366-9033    

DOI:10.1039/DF9582500114

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

MOLECULAR AND GROUP ASSOCIATION EQUILIBRIA IN POLYMERS CONTAINING WIDELY SPACED INTERACTING GROUPS BY H. MORAWETZ Polymer Research Institute, Polytechnic Institute of Brooklyn, Brooklyn, N.Y. Received 16th December, 1957 Mixtures of methyl methacrylate copolymers containing about 5 mole % of methacrylic acid or dimethylaminoethyl methacrylate, respectively, form molecular association complexes in butanone solution which remain stable up to the highest dilutions accessible to osmotic or light-scattering measurements. In benzene solution, molecular association of the acidic polymer molecules with each other is also observed and the dependence of the size of the molecular aggregates on the concentration of cosolvents can be used to estm ate group association equilibrium constants. With styrene + methacrylic acid copolymers infra-red spectroscopy can be used to determine the extent of carboxyl dimerizationi and this is found to be independent of the solution concentration of the copolymer, being governed by the local carboxyl concentration in the isolated polymer coil.For a fixed degree of carboxyl dimerization, the extent of molecular association is higher in thermo- dynamically better solvents. In bulk samples of the copolymers the carboxyl dimerization equilibrium becomes frozen in the neighbourhood of the second-order transition tem- perature. While fairly extensive studies have been carried out on molecular association equilibria of low molecular weight species, little is known about the principles governing molecular association of high polymers.Yet it is clear that for each type of association known to exist with small molecules, analogous complex formation should be observed with polymers carrying the appropriate interacting groups. In non-polar media, typical examples comprise the hydrogen-bonded association complexes of hydroxylic 1 9 2 and carboxylic 3 compounds or the ion- pair formation of amines with carboxylic acids.4~ 5 When the solvent molecules form strong hydrogen bonds with one another as in the case of water, solutes may aggregate for three different reasons. (i) Particles carrying large charges of opposite sign will tend to associate particularly at low salt concentration. (ii) Molecules with bulky non-polar residues will be drawn to each other so as to minimize the number of broken hydrogen bonds between the solvent molecules (" the hydrophobic bond ").6 (iii) Hydrogen-bonded solute complexes map persist even in aqueous solutions provided the hydrogen bonds between solute molecules are sufficiently strong.7 Consider a chain molecule soluble in a given medium.If we now modify the polymer by introducing strongly interacting groups at a wide spacing along the macromolecular backbone, groups attached to the same polymer chain as well as groups attached to different chains will participate in complex formation. The intermolecular association persists frequently to the highest dilutions accessible to osmotic and light-scattering studies and leads to a dependence of the apparent molecular weight on the properties of the solvent medium.899 At higher con- centrations at which the polymer molecules may form a continuous network throughout the system, intermolecular group association may result in gelation. Such gels are thermally reversible, liquefying at temperatures at which the group association is reversed.10 Although molecular association of polymers and thermally reversible gel formation have been investigated by a number of workers in the past, no attempt 122H.MORAWETZ 123 seems to have been made to study systematically the dependence of these phenomena on the variables of polymer structure. The present report summarizes some initial observations made on polymers prepared specifically to illuminate the effect of the type and spacing of interacting groups and the length of the molecular chain on the properties of polymers both in solution and in bulk.EXPERIMENTAL The preparation of the methyl methacrylate copolymers and the styrene copolymers as well as their characterization has been reported previously.ll.12 Zimm-Myerson osmo- meters with wet regenerated cellulose membranes type 300 (Sylvania Division, American Viscose Co.) were employed for determinations of the apparent number-average molecular weights. The true number-average molecular weight of polymers carrying carboxyl groups was determined, employing as a solvent either pure pyridine, or any other solvent to which had been added 1.6 volume % of N,N-dimethylbenzylamine with a trace of t- butyl catechol stabilizer. Jnfra-red absorption spectra were determined with a Perkin-Elmer double- beam spectrophotometer, and the ratio of the optical densities at the absorption maxima of the 5.70-5-75 p and 5.85-5.88 p bands (corresponding to the carbonyl stretching vibration for monomeric and hydrogen-bonded carboxyl)13 was used as an index of carboxyl dimerization.Concentrations were calculated from the optical densities using molar extinction coefficients (1. equiv. -1-mm -1) obtained from pivalic acid solutions as 37 and 51 for monomeric carboxyl, 41 and 70 for dimerized carboxyl in 1 : 1 : 2 : 2-tetrachloroethane and carbon tetrachloride, respectively. Spectra of carboxyl-containing polymers in bulk were obtained by the pressed potassium bromide disc technique using an electrically heated cell 14 for holding the sample in the spectrophotometer.The molar extinction coefficients of monomeric and dimeric carboxyl in the solid polymers was estimated as equal to pivalic acid values in benzene, 65 and 63 1. equiv. -1-mm -1. RESULTS AND DISCUSSION MOLECULAR ASSOCIATION OF METHYL METHACRYLATE COPOLYMERS CARRYING CARBOXYL AND AMINO GROUPS In this study 11 a methyl methacrylate copolymer with 4.9 mole % methacrylic acid (no. av. mol. wt. MA = 32,300) and a copolymer containing 5.8 mole % dimethylaminoethyl methacrylate (no. av. mol. wt. MB = 135,000) were employed. Reduced osmotic pressure plots of mixtures of the two copolymers in any given ratio were strictly linear, but the apparent average molecular weights 2 obtained from the intercept were much higher than calculated from the molecular weights of the components of the mixture, indicating extensive molecular association.A degree of association D defined as the average number of molecules associated to an osmotically active particle is given by D = [aA/MA + (1 - .A) /MB] (1) where O ~ A is the weight fraction of the acidic polymer in the polymer mixture. Fig. 1 shows the dependence of D on aA for butanone solutions at 30.2"C and 50.1 "C and for benzene solutions at 49-7°C. It may be seen that in butanone D rises to a sharp peak for mixtures of equal weights of the two polymers, while in benzene solution the association of acidic and basic polymers is complicated by the associa- tion of acidic polymer molecules with each other. The nature of the association complexes involving a tertiary aliphatic amine and a carboxyl group has been investigated by Barrow and Yerger in their spectroscopic study of solutions containing triethylamine and acetic acid in carbon tetrachloride or chloroform.4 The evidence showed that the amine associates both with the monomeric acetic acid and with acetic acid dimer, and led to a hydrogen-bonded ion-pair structure for these species.The 1 : 1 complex was found to be more stable in the more polar chloroform and this was explained by solvation of the ion pair. It should be noted that the present data obtained at around 50°C show stronger association of the basic and acidic copolymers in benzene than in the more polar124 MOLECULAR AND GROUP ASSOCIATION EQUILIBRIA IN POLYMERS butanone. This is all the more significant since butanone was found to be the thermodynamically better solvent for the non-associating basic copolymer (the slopes of the reduced osmotic pressure plots were 1.6 X 10 7 and 1.0 x 10 7 ergs cm 3/82 for butanone and benzene respectively) so that more strongly associating groups would be required for the formation of molecular aggregates in this medium.At any rate, the dependence of the stability of ion-pairs on the nature of the medium is relatively slight compared with the very large effect on the formation of association complexes which depend entirely on hydrogen bonding. It is thus clear that the molecular aggregates of the carboxyl-bearing copolymers will rapidly dissociate as the hydrogen-bonding capacity of the interacting groups is being saturated by the solvent medium. b s - W c n 0 0 1 4 .b 8 1.0 "A FIG.1 .-The molecular association of mixtures of an acidic acid and a basic copolymer of methyl methacrylate. 0 - butanone at 30-2"C, 0 - butanone at 50.1"C, 0 - benzene at 49.7"C. MOLECULAR ASSOCIATION OF METHYL METHACRYLATE + METHACRYLIC ACID COPOLYMER IN BENZENE CONTAINING BASIC OR HYDROGEN-BONDING COSOLVENTS In agreement with previous investigators, we have found no indication of incipient dissociation of the molecular aggregates formed by polymer molecules on diluting solutions in the range accessible to osmotic measurements (from 1 g/100 ml to 0.1 gll00 ml). In fact, light-scattering measurements showed that the association complexes remained stable 15 down to a concentration of O.Olg/ 100 ml. These observations, suggesting that the degree of association of these molecules carrying large numbers of interacting groups remains constant over a wide range of solution concentration, are not easily interpreted in terms of a physical model.Let us consider for simplicity a solution of a polymer containing a single type of interacting group. We may then explain the constant size of the molecular aggregates by assuming that (i) most of the interacting groups are associated intramolecularly or else hidden within the polymer coil so that the "effective functionality "f'of the polymer (i.e. the number of sites available for intermolecularH . MORAWETZ 125 association) is less than two. (ii) The intermolecular association of the available sites proceeds practically to completion. This requirement may necessitate the cooperation of two or more favourably spaced interacting groups in each " site ".This model relates the degree of association D to the effective functionality f of the average polymer molecule by f = 2 ( 0 - l)/D, in analogy with the treatment of the condensation of poly-functional monomers 16 and it satisfies the condition that D be independent of solution concentration. If a cosolvent B is now added which may form complexes with the interacting group A of the polymer according to the equilibrium [ABl/[Al [BI = K, 4 I 1 4 I I I I I (3) 9 FIG. 2.-Molecular association of methyl methacrylate + methacrylic acid copolymer at 294°C in benzene containing: 0 dimethylaminoethyl acetate, methyl acetate, 0 acetic acid and 8 butanone. the effective functionality will be reduced to f' which determines the new degree of association D' by a relation analogous to (2).Assuming that f' is proportional to the number of free A groups, i.e. flf' = ([A] + [AB])/[A], we have then fly-'= (0 - l)D'/(D' - l)D = 1 + K(B). (4) Experimental data have been obtained17 on the effect of cosolvents on the molecular association in benzene at 29.8"C of a methyl methacrylate copolymer containing 4-9 mole % methacrylic acid. The copolymer had an osmotic molecular weight of 34,500 in pyridine and its degree of association in pure benzene at 294°C was 6.86. The cosolvents methyl acetate, acetic acid, dimethylaminoethyl acetate and butanone were chosen for study so as to obtain information on the relative stability of carboxyl complexes with the different types of groups which may have determined the extent of molecular association in the solutions of mixed acidic and basic polymers described in the previous section.The results plotted in fig. 2 show that acetic acid is much less efficient than methyl acetate in reducing the molecular association of the copolymer. This is undoubtedly due to the fact that very little of the added acetic acid is in the monomeric form which would associate with the carboxyl groups of the polymer, while all of the carbonyl groups of the ester are available for such association. It follows that this acidester copolymer must be assumed to associate largely due to intermolecular interactions of a carboxyl126 MOLECULAR AND GROUP ASSOCIATION EQUILIBRIA I N POLYMERS with an ester group rather than the formation of carboxyl dimers.The plots of f/f' against [B] are linear as required by (3) for the ester and the amine and the slopes correspond to association constants of 6.2 and 86 l./mole, respectively. (The latter figure is appreciably lower than the reported value of 800 l./mole for the association of triethylamine with acetic acid in carbon tetrachloride,4 but part of the discrepancy may be accounted for by the lower acidity of the carboxyl groups in the copolymer). The acetic acid data cannot be interpreted in this simple manner, because of the dimerization equilibrium of the cosolvent and the interaction of its monomeric form with both ester and carboxyl groups of the polymer. It should also be noted that the results point to much stronger hydrogen bonding to methyl acetate than to butanone ; this is contrary to the conclusion of Gordy and Stanford 18 who found that the infra-red OD absorption band of deuterated methanol was shifted more strongly in ketone than in ester solutions. However, this apparent discrepancy with our results may be due to the fact that in Gordy and Stanford's experiments the hydrogen-bonded acceptor varied at the same time as the solvent medium, while in our studies the group interactions were all studied in dilute benzene solution.0 INTRAMOLECULAR GROUP ASSOCIATION The study of the solution behaviour of styrene copolymers with methacrylic acid lends itself to a more detailed interpretation since only one group association A _ - ;7 I I I I 1 I I 5 10 I 5 2 0 I S 3 0 3 5 I .0 I \ 0 Pivolicocid refer tocopolymer deriqnotion equilibrium-the dimerization of carboxyl groups-must be taken into account and since in addition to the osmometric determination of molecular association, infra- red spectroscopy can be used to determine the fraction of carboxyl dimerized. An extensive study of this system has been carried out 12 and it was found that the extent of carboxyl dimerization has a value determined by the copolymer composi- tion and the solvent medium but is, within wide limits, independent of solution concentration. This is illustrated in fig. 3 in which the extent of the carboxylH. MORAWETZ 127 association is characterized by dl/d2, the ratio of optical densities at the absorption maxima of the carbonyl stretching vibration at 5.70-5.75 p and 5-85-5.88 p cor- responding to monomeric and dimerized carboxyl, respectively.The result is understandable for polymer solutions which are sufficiently dilute so that the individual molecular coils are far from each other and intermolecular group association is improbable compared with complex formation involving groups attached to the same molecular chain. Under these circumstances, the degree of group association should be goverened by the " effective local concentration " of the interacting groups within the space occupied by an individual polymer chain.19 The data obtained to date also seem to indicate that dl/d2 depends only on the density of the associating groups along the chain and is independent of chain 3.5- 3-0 D 2.5- FIG.4.-Molecular aggrega- tion and carboxyl dimeriza- tion in styrene + methacrylic acid copolymers. 2.0 0 C,H,Ci, Solutions 0 C CI, Solutions - - length.19 Moreover, the constancy of the extent of carboxyl dimerization seems to hold even up to concentrations at which there is appreciable interpenetration of the molecular coils. These observations suggest that most of the complexes form between groups spaced relatively close to one another along the polymer chain. A comparison of the fraction of dimerized carboxyl with the osmotically determined degree of molecular association D is given in fig. 4. In all cases D was remarkably low considering the large number of dimerized carboxyl carried by the copolymer. A typical case was a copolymer with zn = 102,000 carrying 90 carboxyls.Although 70 % of these groups were dimerized in tetrachloroethane solution, only 1.5 chains were associated, on the average, to an osmotically active unit. It can also be seen from the data in fig. 4 that for any given degree of group association, the molecular association of the polymer is higher in tetrachloroethane than in the poorer solvent carbon tetrachloride. It is understandable that the better solvent medium, in which the polymer chain is more highly extended, should favour intermolecular as against intramolecular group association. CARBOXYL ASSOCIATION OF STYRENE + METHACRYLIC ACID COPOLYMERS IN BULK The extent of carboxyl association in bulk samples of styrene + methacrylic acid copolymers has also been measured by infra-red spectroscopy.20 The results128 MOLECULAR AND GROUP ASSOCIATION EQUILIBRIA I N POLYMERS given in fig.5 show the variation of a, the degree of dissociation of the carboxyl dimer, with the temperature of the sample. During the first heating cycle (dashed line), o! remained unchanged up to a temperature of around 100°C; on further heating o! decreased, but on cooling down to room temperature it assumed a lower value than that observed originally. In subsequent heating cycles the samples behaved quite reversibly. The temperature dependence of a above 100°C corresponds to a heat of dimerization of 8-10 kcal in good agreement with values reported for low-molecular- weight carboxylic acids.3 At lower temperatures the dimerization equilibrium is effectively frozen due to the very high viscosity of the system.The freezing of the equilibrium occurs close to the second-order transition temperature of 82°C for polystyrene 21 and it would be interesting to ascertain whether the coincidence of a I -4.6 Mole$ Mathacrylic acid Mole% Methacrylic acid ------------- FIG. 5.-Dissociation of car- boxyl dimers in bulk samples of styrene 3- methacrylic acid copolymers. 0.4 - 0.t I I I I I I I 4 0 6 0 8 0 100 11L 140 Tcmp.[OC) discontinuity in the temperature coefficient of thermodynamic polymer properties and the freezing of chemical equilibria of groups attached to the polymer backbone is a general phenomenon. Such a correlation has been suggested previously by Zhurkov and Levin 22, 23 who studied hydrogen bonding in polymers carrying hydroxyl groups.The fact that cc was higher in the polymer samples as prepared by precipitation from dilute solution than after the first heating cycle shows that the polymer was originally even further from equilibrium than after the annealing operation. This may well be a general effect to be taken into account whenever one deals with polymer samples prepared in this manner. Financial support of this investigation by the Of€ice of Naval Research is gratefully acknowledged. 1 Wolf, Dunken and Merkel, 2. physik. Chem. B, 1940, 46,287. 2 Huckel and Schneider, 2. physik. Chem. B, 1940,47, 227. 3 Allen and Caldin, Quart. Rev., 1953, 7, 255. 4 Barrow and Yerger, J. Amer. Chem. SOC., 1954, 76, 5211. 5 Barrow and Yerger, J. Amer. Chem. SOC., 1955,77,4474, 6206.H . MORAWETZ 129 6 Kauzmann in The Mechanism of Enzyme Action, ed. McElroy and Glass (The Johns 7 Arshid, Giles, Jain and Hessan, J. Chem. SOC., 1956, 72. 8 Trementozzi, Steiner and Doty, J. Amer. Chem. Soc., 1952, 74, 2070. 9 Nord, Bier and Timasheff, J. Amer. Chem. SOC., 1951, 73,289. 10 Ferry, Adv. Protein Chem., 1948, 4, 1. 11 Morawetz and Gobran, J. Polymer Sci., 1948, 12, 133. 12 Chang and Morawetz, J. Physic. Chem., 1956, 60, 782. 13 Hadzi and Sheppard, Proc. Roy. SOC. A, 1953,216,247. 14 Longworth and Morawetz, Chem. and Ind., 1955, 1470. 15 Gobran, Ph. D. Thesis, (Polytechnic Institute of Brooklyn, 1954). 16 Mark and Toboisky, Physical Chemistry of High Polymeric Systems, (Interscience, 17 Morawetz and Gobran, J. Polymer Sci., 1955, 18, 455. 18 Gordy and Stanford, J. Chem. Physics, 1940, 8, 170. 19 Morawetz, J. Polymer Sci., 1957, 23, 247. 20 Longworth and Morawetz, J. Polymer Sci., 1958, 29, 307. 21 Williams and Cleereman, Styrene, its Polymers, Copolymers and Derivatizv, ed ., 22 Zhurkov and Levin, Doklady Akad. Nauk. S.S.S. R., 1950, 72, 269. 23 Zhurkov and Levin, Vestnik Leningrad. Univ., 1950, no. 3,45. Hopkins Press, Baltimore, 1954), p. 71. New York, London, 1950), p. 368,387. Boundy and Boyer, (Rheinhold Publ. Co., New York, 1952). p. 478. E

ISSN:0366-9033    

DOI:10.1039/DF9582500122

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

SOME COMMENTS ON THE THEORY OF DENATURATION BY STUART A. RICE, Dept. of Chemistry and Institute for the Study of Metals, University of Chicago, Chicago 37, Illinois AKIYOSHI WADA, Dept. of Chemistry, Ochanomizu University, Bunkyo-ku, Tokyo, Japan AND E. PETER GEIDUSCHEK Dept. of Chemistry, University of Michigan, Ann Arbor, Michigan Received 23rd January, 1958 Some aspects of the theory of denaturation are discussed with the aim of combining structural features with elementary statistical-mechanical considerations. The relevant contributions to the free-energy changes on denaturation are discussed and two specific examples given. In the first, partially interrupted configurations of an a-helix are treated, and these results along with other considerations used to calculate the volume change on the reversible denaturation of BSA at low pH.The agreement with experiment is good. As an example of an irreversible reaction, the thermal denaturation of DNA is treated. It is suggested that the reaction proceeds via a nucleation mechanism in which the critical nucleus is a gap of some 29 adjacent pairs of broken secondary bonds. A calculation of the entropy of activation for this model is also in agreement with experiment. Great progress has been made in recent years in the experimental elucidation of the structures of proteins. The combined use of degradative end-group analysis, detailed X-ray studies, and the judicious construction of models based on the known structures of small molecules have all contributed to the picture of a protein as at least a partially micro-crystalline species.1 The work of Pauling and others strongly suggests that the crystalline portions are helical, the precise nature of the helix being somewhat more in doubt.Support for these con- clusions can be drawn from a wide variety of sources. Of particular interest, however, are the extensive studies of Doty2,3*4*5 and co-workers on the syn- thetic polypeptides. These compounds have been demonstrated to have the cc-helix configuration under certain well-defined conditions and the relative roles of helical and non-helical polymer configurations and their relation to solvent structure and monomer structure has been thorougkily explored. The extension of these studies to proteins has recently been reported.6 Protein denaturation studies have a long and extensive history.1 The very term " denaturation " is not too well defined ; we use it to denote the response of the native protein to heat, acid, alkali, and a variety of other chemical and physical agents which cause marked changes in the protein structure.The inference of details of the structure from this type of study has not been notably successful. hTevertheless, there are numerous empirical observations which have been crudely correlated with not too precisely defined structural parameters. Aside from the widespread discussions of the " loosening of the structure in the activated state " most semi-quantitative correlations have been limited to correlations of reaction order and titration curves.1 At least part of the difficulty with studies of denatura- tion has been the lack of any attempt to correlate the energy and entropy of activation with possible structural changes in a detailed manner.It is the purpose of this brief note to discuss several aspects of the denaturation process along with some crude statistical considerations with the intention of providing some examples of how correlations between structure and rate can be made. Our comments are not intended to be complete, but rather to suggest how 130S. A . RICE, A. WADA AND E. P. GEIDUSCHEK 131 the problem might be tackled. We first consider equilibrium properties, and then turn to a discussion of rate problems. SOME GENERAL CONSIDERATIONS At the outset it is necessary to define the term denaturation. By denaturation we shall mean that class of reactions which leads to changes in the structure of the macromolecule with no change in molecular weight.Dissociation reactions could be included, but introduce factors we do not wish to emphasize herein. The outstanding feature of most denaturation reactions is the transition from a region of extremely slow reaction to a region of immeasurably fast reaction with a small change of external conditions. An obvious possible cause is that a nuclea- tion process is involved. We consider herein only this nucleation mechanism for denaturation. Let us consider the possible free-energy changes which may occur in the de- naturation. Lack of space and the existence of the excellent discussions by Schellman and Kauzmann make an extensive treatment unnecessary and this section will be brief.If it is agreed that the denaturation results in the destruction of the secondary bond structure of the protein, then the following factors must be discussed. Changes in the secondary bond structure will undoubtedly involve changes in vibrational frequencies of the groups directly bonded. If the frequencies con- cerned happen to be torsional oscillations, then the considerations of Laskowski and Scheragag indicate an entropy change of 2-5 cal/mole deg. for each group which was hindered in its oscillation before denaturation and is essentially free after- wards. Of similar origin, in a statistical sense, are the changes of chain skeletal degrees of freedom and the extra freedom resulting from the unbending of a folded helix.A major contribution to the free energy (energetic and entropic) will arise from the changes in hydrogen bond or other secondary bond structure. The breaking of each hydrogen bond will require about 5 kcal/mole enthalpy change, most of which will be recovered from the solvation of the group which follows its immersion in water, This effect of solvation is only one example of the dominant role played by the solvent in determining the position of equilibria. This is evident in the calculations of Schellman7 and in the experimental work of Doty and Yang.5 The participation of solvent in the reaction involves both energetic and entropic changes as, for instance, in the " freezing-out " of hydrated water molec- ules. The two other sources of free-energy changes are changes in charge-charge interactions and changes in interaction with urea, small ions, or any other adid- tives.These latter two contributions are closely interrelated. The electrical free energy is very difficult to evaluate quantitatively. That it can pIay a dominant role in denaturation reactions is demonstrated by, for example, the reversible swelling of bovine serum albumin. The role of urea binding has been known for many years. Finally, a major source of free-energy change is the existence in structures like DNA of many equivalent sites along the chain at which denaturation may and probably does occur. In general, in a statistical treatment we do not count indistinguishable zrrangernents of broken bonds more than once. This then leads to a consideration of the multiplicity of possible ways that a sequence of bonds can be broken and therefore to an entropic contribution to the free energy.This case is exactly analogous to the computation of the number of vacancies in a lattice, or the entropy of mixing of vacancies and lattice sites. The foregoing are the factors which must be considered in any detailed cal- culation. In addition, a specific structure must be assumed or known for the macromolecule and a mechanism assumed for the reaction. There are always, of course, extra factors which are peculiar to individual structures and which have not been considered above. Examples of such special properties will appear in the ensuing discussion. To illustrate how the principles of statistical mechanics132 COMMENTS ON THE THEORY OF DENATURATION may be applied to denaturation reactions we consider first the reversible folding and unfolding of a single polypeptide a-helix and secondly the thermal denaturation of DNA.PARTIALLY INTERRUPTED CONFIGURATIONS OF AN a-HELIX AND THE FREE ENERGY OF PROTEINS As is well known, the polypeptide chain has been shown to exist in at least two forms, the a-helix first proposed by Pauling and Corey,l and a random-coil configuration. The conversion from one form to the other can be accomplished by changes in solvent, temperature or a combination of the two. In the a-helix form, each residue in the chain forms an intramolecular hydrogen bond with its third neighbour, the free energy of formation being rather small, and the entropy and entlialpy of formation also being small.Solution in solvents which lower the enthalpy of the random-coil configuration by solvation of the monomer groups will favour the random-coil configuration as will high temperatures.2-5 It is interesting to inquire whether there also exist intermediate configurations, or partially interrupted helices. In other words, do coil sections exist not only at the ends of chains but also in the central portions? To treat the helix-coil transition, let 2 be the degree of polymerization, Nh the number of residues in helical configurations, Mh the number of helical sections, Nc the number of residues in the random coil configuration and Mc the number of random-coil sections. The partition function for an interrupted helix may be written in the form : Nh=O M h - 0 with a the minimum number of residues required for helix formation, where g (Z, Nh, M,, k f h ) is the number of ways of constructing the polymer from Nh helical residues, k f h helical sections, etc., Qoh and Q,, are the internal partition functions of the helical and coiling residues and Qeh and Qec are the partition functions for the corrections due to end-effects which occur at each helix-coil junction.We now define so that eqn. (1) may be rewritten in the form z Nhfa N h - 0 Mh=O Q = 2 g ( z , Nh, Mh, Mc) exp (50 + “h 4 BhMh + BcMc). (3) If the minimum number of residues required for a helical section is taken for sim- plicity as equal to the minimum number of residues required for a randomly coiled section, a,* then the number of configurations is readily seen to be * It is clear that there will be no restrictions on the minimum length of random ends.In the middle of the chain, the fact that random regions arise from helical regions implies that ah= a,.S . A. RICE, A. WADA AND E. P. GEIDUSCHEK 133 since this is simply the number of ways of placing Nh - aMh balls and 2 - Nh - aM, balls in Mh and M, boxes respectively. By using the standard techniquc of picking the maximum term, again for simplicity putting Mh = M, = M, (they can only differ by & 1 in any event) and noting that the minimum number of residues required to form a helical fragment, a = 4, it is possible to show that 4 h{(z - Nh 4M)(Nh - 4M)) - 3 In {(Z - Nh - 3M - I)(Nh - 3M - 1)) -2In(M- 1}+B=O, (5) where we have used Stirlings approximation and have defined B = Bh + B,.From eqn. (5) it is possible to compute the mean number of helical sections for given Nh. Some typical figures are given in table 1. TABLE MEAN NUMBER OF HELICAL SECTIONS Mh FOR GIVEN Nh Nh = 100 900 B - 0 - B = 1 23 B = 2 19 B = 3 17 B = 4 15 B = 5 12 z= lo00 200 800 42 39 35 30 25 20 300 700 58 53 47 40 32 24 400 600 70 62 54 45 35 26 500 500 76 67 57 47 36 27 It should be noted that the actual values of the end-corrections are very difficult to evaluate. Schellman estimates that where the 4 arises from noting that an cc-helix will release one residue after four hydrogen bonds are broken, and where AH0 and AS, are the differences in enthalpy and entropy per residue between the helical and randomly coiled configurations. The total free energy of the given configuration is obtained from eqn.(1) with the maximized set of parameters Nh and M. Note that the average size of the ordered and disordered chain sections is only slightly larger than the minimum size. Since there exist minimum sizes of both ordered and disordered regions, the maximization of the entropy of mixing requires substantial numbers of each species. This then implies that the average species size will remain close to the minimum required size. It is interesting that a consideration of this sort has also arisen in the interpretation of the denaturation of DNA. Consider now the denaturation of a globular protein consisting of several folded polypeptide helices held together with disulphide bonds. Take as the reference state a single unfolded polypeptide strand of the same molecular weight in the helical configuration.The free energy of formation of the protein from the standard state may be divided up as where each term refers to a step in the hypothetical process . (a) Take the standard polypeptide and disorder some regions of the chain, the free-energy change being AApatid helix as calculated in the preceding section (less the entropy of mixing since the requirement that S-S bonds are formed preselects the regions which must be disordered).134 COMMENTS ON THE THEORY OF DENATURATION (6) Fold the partially disordered polypeptide into the desired configuration. If there are no changes in internal structure in this process, AAf,ld is composed of the changes due to changing moment of inertia, and the decrease in the skeletal freedom of the disordered regions. (c) Form the disulphide bonds which permanently link the folded helix, A&s. ( d ) Compute the change in charge-charge interactions due to the new arrange- ment of charges, possible changes in binding and penetration of external electrolyte, etc., AAelec.Having formed the protein from the standard state, we are in a position to investigate its denaturation. We consider as an example the reversible expansion of serum albumin at low pH's. The free-energy change for this process may be written as (7) since we assume that no S-S bonds are broken. The first term, AAd-ption, the free-energy change on destroying the remaining helix, is just the limiting case of eqn.(1) when there are no helical portions of the polypeptide left and is calculable from the preceding. The second and third terms are calculable in principle but very difficult to evaluate in practice. As estimates we quote the results : 10 AAdenat = AAdisruption f AAexpansion + AAelec + AAbinding, K' q2,'Z e- K'(ro1 AAelec == - $a' - + + kT[a' In a' + (1 - a') in (1 - a')] D' 2D"(ro) + (1 - a') In (native) +g u*o 1 nUln(*-- - A eiec ' ( [ q2e-K'<rO>]) D"<ro)kT ' p2 = 1 - 4a'(l - a') 1 - exp - a' = no/Z, (9) (10) and (denatured) (native) AAbinding = 1 nukT [In K, - In c,] - 1 n,kT [ln Ku - In c ~ ] , (1 1) where v is the number of crosslinking S-S bonds, VO, V,, V are the original, maximum, and present volumes of the protein, X1 is a free-energy parameter describing the interaction between solvent and residue, q is the magnitude of a charge, NO the number of unbound charges, K' the Debye screening length inside the protein, D" the dielectric constant inside and D' that outside the protein, ( Y O ) the average charge separation, nu the number of charges with ion pairs of kind 0, K, the corresponding ion-pair dissociation constant and co the concentration of ion 0.Detailed derivation of these results may be found in a paper by Rice and Harris 10 on the theory of polyelectrolyte gels, of which the denatured protein is an example. A,], (native) can be estimated by well-known techniques from the theories of Rice and Harris,llp 12 Katchalsky and co-workers,l3 or Kirkwood and Tanford.14 The latter is especially developed for the spherical protein case which corresponds closely to many native proteins.0 = 0 o = oS . A. RICE, A. WADA AND E. P. GEIDUSCHEK 135 To illustrate the utility of the estimates quoted we shall compute the expansion of serum albumin on denaturation as follows. For simplicity we consider only the electrostatic, expansion and binding terms. That is, we disorder the molecule at constant volume, and compute the expansion of this disordered state. Using the values 101 charges and 28 S-S crosslings in 606 residues, binding constants for small ions of order of magnitude 10-1, gives a predicted expansion of 2.5 which is in qualitative accord with experiment. ( ( V / V O ) ~ ~ ~ = 2.3; r/2 = 0.15). The expression given for the free-energy change in denaturation is very complex but one qualitative feature stands out.The method we have used to separate the contributions to the free-energy change suggests that the major competition is between AAelec and AAdisruptio,, since &Iexp is small and the change in binding probably contributes little change to the free energy. Until AAel, is sufficiently large to disrupt the helix, very little happens, and there is no expansion. Once some disruption occurs, the volume can change markedly and the transition will appear to have a fairly sharp onset. These qualitative considerations are not unexpected, our formulation being just a convenient way of expressing them. Similar applications can be made to other types of protein reactions which, for instance, may not involvc any changes in electrostatic energy.We have thus far considered only the thermodynamic properties of the transition native-denatured protein. We now turn to a discussion of the kinetics of de- naturation with emphasis on the role of structural features. To study the rate of the transition requires detailed knowledge of the structure of the macromolecule as well as a mechanism for the reaction. Because a good working model of the structure of DNA exists and since the thermal denaturation of DNA has been recently studied 16 we shall illustrate our methods with this compound. THE THERMAL DENATURATION OF DNA l5 Watson and Crick 17 have proposed that the native DNA molecule is com- posed of two polynucleotide strands wound in a helix about a common axis. The stability of this helix is attributed to the presence of highly specific pairs of hydrogen bonds between the purine and pyrimidine bases attached to the phospho- sugar chain skeleton.If the source of the stability arises from other secondary bonding, our basic considerations remain unaltered. The configuration of the native molecule is rigid, not only the possible position of the molecular backbone being restricted but also the possible torsional oscillations of the mutually bonded purine and pyrimidine bases being greatly reduced. It is well known that high temperatures, acid or alkali cause an irreversible change in the properties of the DNA molecule. Recent investigations have shown that in each case the product of the reaction is a highly collapsed molecule of the same molecular weight as native DNA.161 181 19 Moreover, in each case the transition from native to denatured state occurs in a very narrow range of pH or temperature. This behaviour is suggestive of a co-operative transition, a transi- tion which ordinarily requires a nucleation step.In almost all such cases, the rate-determining step is just the nucleation step. The co-operative nature of the transition in this case is due to the necessity of having a minimum sequence of broken bonds contiguous with one another before sufficient flexibility is intrcduced into the structure to permit contraction. From a priuri considerations one would believe that secondary bonds which are broken singly'in a very long linear array, are isolated from one another at small extents of reaction. This is a consequence of the entropy of mixing which maximizes the probability of a dispersed con- figuration.On the other hand, it is extremely difficult to imagine what physical process could occur which would cause a transition when there are only a few isolated breaks. Our naive considerations would seem to require some large- scale local disturbance as exemplified by a large gap of contiguous broken bonds. We are thus led to the conclusion, on physical grounds, that the rate-determining step in the denaturation of DNA is the opening of a large gap of adjacent broken136 COMMENTS ON THE THEORY OF DENATURATION secondary bonds, i.e. a large-scale local disordering. Whether the reaction occurs by the simultaneous rupture of these bonds, or by the creation of the gap from previously isolated breaks by the mechanism of thermal fluctuations remains undetermined both experimentally and from the model calculations.This factor is unimportant for the evaluation of the energy of activation since our reference state is always taken as native DNA. The concentration of isolated breaks plays no role and the quasi-equilibrium is completely specified by the concentration of critical gaps. For our present purposes the important feature is the necessity of the existence of a critical nucleus for denaturation. We represent the rate of thermal denaturation of DNA by rate = constant exp (- W*/kT), (12) where W* is the reversible work of forming the critical nucleus. The reversible work of formation of a nucleus may be divided into several contributions : P i - 1 where there are P pairs of broken secondary bonds, HO is the enthalpy change on breaking one pair of secondary bonds, AS2 is the entropy change due to the in- creased torsional freedom in the denatured molecule, AS3 is the entropy increase due to the greater freedom of the chain skeleton after a pair of bonds has been broken, and R In 2 is the entropy contribution from the mixing of the broken gap and the unbroken pairs of bonds as mentioned previously.AS, may be estimated from the entropy of fusion of ice, AS2 from the considerations of Laskowski and Scheraga,g and AS3 from an application of the theory of rubber elasticity from which where Pi involves the number of links in the ring and the position of the restricting bond, dV the volume in which two links are restricted to move when bonded to one another, 1 the length of a link and XO, YO, 20 the mean separations of the chain elements in question when in the denatured state.The experimental value of PHo is 146 kcal/mole. In order to calculate P, a value of HO must be assigned. This will clearly depend upon the nature of the secondary stabilization. If the Crick-Watson model is assumed, then a calculation based on hydrogen-bond strengths appears warranted. By this method we estimate P to be P = 29 f 12, the uncertainty arising from the uncertainty in HO = 5000 & 2000 cal/mole.20 In a separate paper we point out that a DNA model involving only hydrogen bonds is extremely unlikely. In that case there appears to be no a priori method of estimating Ho, but our formal considerations remain unaltered.Using the value of P estimated from the H-bond model the computed entropy change from the contributions considered is 342 -+ 318 cal/mole deg. and the observed value is 326 cal/mole deg. The uncertainty of the computed value is so largc that the agreement is probably fortuitous. The important feature is that our qualitative picture of the reaction mechanism is at least not in conflict with the experimental results. DISCUSSION The last section illustrates to some extent the greatest difficulty in the applica- tion of the methods proposed. Although the statistical theory is crude and in- sensitive to many details, there are insufficient data to test even this rudimentary version. Secondary stabilization energies are imprecisely hown, the role of the solvent may only be guessed, the statistics of short chains and branched chainsS.A. RICE, A. WADA AND E. P. GEIDUSCHEK 137 is largely unstudied, and so forth. We do nut believe that it is therefore not worth the effort to make naive analyses of the kind presented. Most of the models considered will not be definitive or even mutually exclusive. The great ad- vantage of even so crude a theoretical approach as the one presented is its ability to correlate structural data with mechanism, and to predict phenomena which may further test the model. For example, further examination of the statistical model mentioned for DNA reveals that there should be a range of reversible contraction before the onset of the apparent irreversible collapse and denaturation. The experimental evidence on this point is ambiguous.ls* 21 If it could be demon- strated unequivocally that the DNA molecule either did or did not reversibly collapse as predicted, there would be another datum with which to fit the model and modify it.It is for this reason that we believe the approach indicated in this paper to be worth while despite the present shortcomings of the available experi- mental techniques and data. The material of this paper has developed over many years and as a result of interaction with many people. Chief amongst these have been Prof. P. M. Doty of Harvard University, Prof. F. E. Harris of the University of California at Berkeley, and Prof. A. M. Holtzer of Washington University. We also wish to express our thanks to all the other investigators who have patiently heard us and con- structively criticized our thoughts. 1 see, for example, The Proteins, ed. Neurath and Bailey (Academic Press, New York, 2 Doty, Bradbury and Holtzer, J. Amer. Chem. SOC., 1956, 78, 947. 3 Doty, Holtzer, Bradbury and Blout, J. Amer. Chem. SOC., 1954, 76, 4493. 4 Doty and Lundberg, J. Amer. Chem. SOC., 1956, 78,4810. 5 Doty and Yang, J. Amer. Chem. SOC., 1956, 78,498. 6 Yang and Doty, J. Amer. Chem. SOC., 1957,79,761. 7 Schellman, Compt. rend. trav. lab. Carlsberg, Ser, chim., 1955, 29, 223, 230. 8 Kauzmann, Ann. Rev. Physic. Chem., 1957, 8,413. 9 Laskowski and Scheraga, J. Amer. Chem. SOC., 1954, 76, 6305. 10 Rice and Harris, 2. physik. Chem., 1956, 8, 207. 1 1 Rice and Harris, J. Chem. Physics, 1956, 24, 326. 12 Harris and Rice, J. Physic. Chem., 1954, 58, 725. 13 Katchalsky and Lifson, J. Polymer Sci., 1953, 11, 409. 14 Kirkwood and Tanford, J. Amer. Chem. SOC., 1957,79, 5333. 15 A preliminary and cruder version of the following analysis was presented in the dissertation of S . A. Rice, Harvard University, June 1955. 16 Rice and Doty, J . Amer. Chem. SOC., 1957, 79, 3937. 17 Watson and Crick, Nature, 1953, 171, 737. 18 Ehrlich and Doty, to be published. 19 Geiduschek, in press. 20 Davies, Ann. Report Prog. Chem., 1946, 43, 1. 21 Sturtevant, Geiduschek and Rice, this Discussion. 1954).

ISSN:0366-9033    

DOI:10.1039/DF9582500130

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

THE STABILITY OF THE HELICAL DNA MOLECULE IN SOLUTION BY JULIAN M. STURTEVANT, STUART A. RICE AND E. PETER GEIDUSCHEK Dept. of Chemistry, Yale University Institute for the Study of Metals and Dept. of Chemistry, University of Chicago Dept. of Chemistry, University of Michigan Received 23rd January, 1958 The stability of DNA is examined in terms of the response to changes of pH, ionic strength, temperature and various auxiliary chemical agents. Some new calorimetric studies are reported and analyzed. A detailed model of the acid denaturation is con- structed in agreement with both calorimetric and configurational data. The major feature of this model is the necessity for denaturation to occur via the opening of many “ gaps ” rather than the entire molecule “ unzipping ”. It is shown that the conventional Watson- Crick model in which hydrogen bonds are the sole source of stability is probably not completely correct.The possible effects of differential solvation and some other sources of secondary bond structure are discussed in the light of the available experimental evidence. As the structure and properties of the desoxyribose nucleic acids have in the past few years become one of the most widely attended problems of molecular biology, so the number and variety of experimental studies of their denaturation * has been extensive, dealing with a variety of samples isolated from different tissues by a number of different preparative methods. The destruction of the secondary structure has been studied by a variety of techniques such as titration,1-6 ultra-violet extinction,7-10 biological activity,ll and macromolecular analysis (light-scattering, viscosity, double refraction of flow, flow dichroism).l2-19 By means of these experiments the conditions for the stability of “native” DNA have now been fairly adequately characterized: in aqueous salt solution, the stability is determined by the pH, ionic strength and temperature, and may be further modified by the addition of urea, guanidinium chloride, sodium salicylate 13 and uridine phosphate.4 In addition, destabilization of the helical structure is observed in a large variety of non-aqueous solvents.17~ 20 In all of these systems, the transition observed is relatively sharp so that it is useful to think of the de- naturation in terms of a co-operative phenomenon occurring in a narrow tem- perature, pH or solvent composition range. To a rough approximation, it is therefore possible to summarize the available data on the stability of DNA in terms of a phase diagram (fig, 1).The data of fig. 1 are taken from investigations on a variety of DNA preparations, employing many different preparative procedures and observational methods which are therefore not absolutely comparable. In addition, the pH and temperature ranges over which denaturation has been found in these investigations are replaced, in fig. 1, by their midpoints. Nevertheless the phase diagram does summarize, semiquantitatively, the conditions of pH, temperature and ionic strength (HCI, NaCl, NaOH only, data on polyvalent * The word “ denaturation ” is used in the sense of destruction of secondary structure.It is now fairly well established that it is possible to destroy the helical secondary structure of DNA without depolymerization. In this article we shall not consider those denatur- ations which involve, in addition, modifications of the primary valence bond structure, such as enzymatic degradation, oxidation or alkylation. 138J. M. STURTEVANT, S. A. RICE AND E. P . GEIDUSCHEK 139 salts not included) under which denaturation OCCUTS. The addition of urea, guanidiniurn chloride and sodium salicylate, etc., operates exactly in the same manner as a rise in temperature in restricting the conditions under which the helical form of DNA is stable. Most of our own investigations 13~21 fall in the range of conditions which have also been studied by other investigators and so add little to the fund of available descriptive material concerning the denaturation of DNA.However, they do permit us, subject to certain assumptions, a somewhat speculative analysis of the thermodynamics of the denaturation process. In addition, the information which we have obtained in these studies persuades us to review critically the interpreta- tion of solution data on DNA in terms of the hydrogen-bonding model of DNA stability. These two entirely separate aspects of our work are developed below. I /' / =' ( C -- \ \ \ \ \ \ 1 I I 1 I l l 5 7 9 II A- BH FIG. 1.-" Phase diagram " for DNA as a function of pH, ionic strength (NaCl, HCI, NaOH and DNA only ; data on solutions containing divalent ions not included) and temperature.The dotted portions of the transition isotherms are drawn in regions of the diagram in which no experimental data are available. THE DENATURATION PROCESS EXPERIMENTAL DATA Results of two separate investigations will be discussed. In one of these studies, two samples of calf thymus DNA (SBl1 and V-1) were denatured at pH 6-5, high ionic strength (0.15) and high temperature in the presence and absence of 8 M urea, 3-2 M guanidiniurn chloride and 0.8 M sodium salicylate, and the denaturation was followed in terms of the reduced specific viscosity of 0.01 % DNA solutions (extrapolated to zero shear). Experimental details have been published previously. In the other study, a sample of salmon testis DNA (GC) was denatured at low pH and high ionic strength at 5", 25" and 40°C and the denaturation was followed calorimetrically, viscosity, light- scattering and titration curves being measured concurrently.The experimental details are published elsewhere. Both samples SB11 and GC were prepared by the method B of Simmons,22 sample V-1 was prepared by Dr. R. Varin according to the method of Schwander and Signer.23 Table 1 summarizes the macromolecular properties of the preparations used. The data on which our calculations are based are summarized in fig. 2, 3 for the " thermal denaturation " 13 and fig. 4, 5 for the " acid " denaturation.21 In the ensuing calculations we shall assume that in partially denatured states of DNA, an equilibrium exists between helical and disordered molecules and sections of molecules.The justification of this assumption is, at best, incomplete. However, the following items may be cited.140 (i) Partly denatured states of DNA do exist. These couId be due to the known heterogeneity of DNA. However, no discontinuities in the denaturation kinetics have ever been observed. (ii) A partial reversibility of denaturation has frequently been observed 4, 13,16.19.24 (but is seldom explicitly remarked upon). Our own observations on this point are also, unfortunately, fragmentary but they have been made both with the calf and salmon DNA. STABILITY OF THE HELICAL DNA MOLECULE I N SOLUTION TABLE 1 .-MACROMOLECULAR PROPERTIES OF NATIVE AND DENATURED DNA sample SB11 (calf) 0.2M NaCl denatureda sample GC (salmon) native denaturedb (M), x 106 7.7 7.7 6.3 x 106 6.4 X 106 (P2)l.s. 3000 980 2800 1400 native 0.1 M NaCl [rll dl/g 72 4 63 (7) 14207 w 22.5 22.9 22 - (a) denatured by heating 15 min at 100°C in 0.15 M NaC1, 0.015 M citrate pH 6.5 ; (b) denatured by acidifying to pH 2 6 at 25°C in 0.1 M NaCI; measured at room <M>, weight average molecular weight.(~2)1.~. light-scattering average of the radius of gyration. (c) private communication J. A. V. Butler to P. M. Doty quoted from ref. (13). measured at room temperature. temperature after reneutralization. 0 FIG. 2.-" Heat " denaturation of DNA (calf, sample V-1). Viscosity measured at 25°C after 1 h heating at the temperature noted on the abscissa in 0-15 M NaCl, 0.015 M citrate buffer, pH 6.5 (intrinsic viscosities extrapolated to zero shear).J .M. STURTEVANT, S . A . RICE A N D E. P. GEIDUSCHEK 14 1 9 W m c c a 9 0 )-r a N c U FIG. 3.-" Heat " denaturation of DNA (sample SB11) in aqueous solutions of urea, guanidinium chloride and sodium salicylate. Intrinsic viscosities (as a fraction of the initial intrinsic viscosities extrapolated to zero shear) measured at 25°C after 1 h exposure 6- 5- a 5 4- 0 U 4 I 3 - 2- I - to the elevated temperature noted on the abscissa. 50c, v 2 = o I, DNA -c 475 FIG. 4.-Enthalpy and viscosity changes accompanying the acidification of salmon (sample GC) DNA at 5°C and 0.1 ionic strength. Curve H: enthalpy of mixing HCl and DNA solutions in 0.1 M NaCl. Curve [s] : reduced viscosity measured at 0.007 to 0.015 % DNA and extrapolated to zero shear (units : dl/g).142 STABILITY OF THE HELICAL DNA MOLECULE IN SOLUTION Fig.6 presents the data on the extent of reversibility (as judged viscometrically) of the acid denaturation of sample GC at 5°C and 0-1 ionic strength. It will be noted that the DNA recovers completely from minor changes of configuration, as at pH 2.6, and that after reneutralization from pH 2-4, the reduced specific viscosity increases by a factor of 9. In the very dilute solutions of these experiments, no aggregation takes place, so that the viscosity increase must be due to a re-extension of the acid-collapsed molecule when the pH is readjusted to neutrality. 3 3 PH 4 FIG. 5.-Enthalpy of acidification of DNA solutions in 0.1 M NaCl at 5", 25" and 40°C. i d I ----%k- -- 3.5 PH FIG. 6.-The partial reversibility of macromolecular configuration changes in the " acid " denaturation of DNA (GC) at low pH and low temperature.Reduced viscosities (as a fraction of the reduced viscosity at neutral pH, extrapolated to zero shear) as a function of pH. 0, viscosity measured at the lowest pH of the denaturation process ; 0, viscosity measured after 20 min exposure to the DH noted and reneutralization with NaHCO3. INTERPRETATION The data which have been presented in fig. 2 to 5 are capable of interpretation in terms of the fraction of the DNA denatured under the various conditions of the thermal and " acid" denaturation experiments. In the acid denatura- tion experiments, the reactions are complete in 0.05 to 50sec, while in thermalJ. M. STURTEVANT, S. A . RICE AND E.P. GEIDUSCHEK 143 denaturation the duration of the reactions is approximately 5 to 45 min. In all cases the values recorded in fig. 2 to 5 are therefore the equilibrium values. In the simple equilibrium calculations that follow, we shall assume that (i) the entire sample may be regarded as a single species, (ii) the different partially denatured states of the system represent the various equilibria of the native and denatured forms of the DNA molecule. DENATURATION AT NEUTRAL pH AND HIGH TEMPERATURE In order to determine the fraction of material denatured from the reduced specific viscosity, it is necessary to make certain assumptions-namely, that the intrinsic viscosity of totally denatured DNA is negligible relative to native DNA (see table 1) and that the DNA is denatured one molecule at a time.* With these assumptions, the fraction of native and denatured DNA, and therefore the equilib- rium constant at any temperature may be calculated for the equilibrium From the temperature dependence of these equilibrium constants the heats and entropies of denaturation of samples V-1 and SBll in 0.15 M NaC1, as well as SBll in 8 M urea, 3.2 M guanidinium chloride and 0.8 M sodium salicylate are given in table 2.In the introduction we remarked on the considerable steepness of the denaturation transitions ; in our equilibrium interpretation this sharpness dictates the very high values of the heat of entropy of reaction shown in table 2. N +D. (1) TABLE 2.-HEATS AND ENTROPLES OF THERMAL DENATURATION AT PH 6.5 sample medium AH kcal/mole AS cal/mole deg.v-1 0 1 5 M NaCl O-015 M citrate + 42 + 86 SBll 0.15 M NaCl 0.015 M citrate 110 3 00 SBll 0.15 M NaCl + 8 M urea 110 319 SBll SBI 1 0-15 M NaCl + 3-2 M 0.15 M NaCl + 0.8 M guanidinium chloride 110 3 14 sodium salicylate 110 308 DENATURATION AT LOW pH, AND 5, 25 AND 40°C The interpretation of the calorimetric measurements of acid denaturation in terms of equilibria is considerably more direct. We note (fig. 4, 5) that at each temperature, the entire enthalpy change takes place within a very narrow pH range in which the macromolecular configuration change also occurs. In these experi- ments the DNA is changing its charge state, and the observed enthalpy changes represent both heats of ionization and heats of denaturation. However, con- siderable evidence, mostly from titration work,ls 2 ~ 4 is available to support the contention that the heats of ionization of adenine, guanine and cytosine in de- natured DNA are very close to zero.The absence of any enthalpy changes above the denaturation pH at all the temperatures studied strongly suggests that the heats of ionization in native DNA are zero also. In that case the total heat ab- sorption for the acidification of DNA may be assigned as the heat of denaturation. Over the temperature range studied, the maximum heat absorption for denaturation is given as (2) if, further, we make the assumption that enthalpy changes are proportional to the extent of denaturation then the data of fig. 5 yield the fraction of DNA de- natured as a function of pH.The same pH dependence of the denaturation is * The second of these assumptions is probably incorrect. However, in its effect on the calculated temperature dependence of the denaturation equilibrium constants, it compensates the effects of the assumption that the DNA is homogeneous. AH(T) = 2230 + 34-5 (T - 293) cal/mole P.144 STABILITY OF THE HELICAL D N A MOLECULE I N SOLUTION found at all three temperatures, and the data are adequately represented by the equation, N f 7.3 H+ + D, (3) for which the equilibrium constants are 1017.7, 1022.2 and lG-025 at 5, 25 and 40°C. From the temperature dependence of K we may calculate the heat of denaturation per " mole ". Since, in addition, the calorimetric enthalpies per mole of nucleo- tide are available, the number of nucleotides (m) in a mole of the denaturing unit may be calculated.Integration of the Van't Hoff equation (allowing both AH/ nucleotide and in to be linear functions of the temperature) yields the data given in table 3. TABLE 3 m, the number of AHfdenat. mole AS/d.m. molet'of nucleotide (kcal/mole) (cal/mole deg.) in a denaturation mole '* T"C K 5 1017.7 + 72 25 1022.2 + 85 40 1025.0 -t 78 + 343 47 + 390 36 + 366 27 A possible check on these numbers is available from the titration curves at 5, 25 and 40"C, if we may assume that heats of ionization in both the native and denatured DNA are zero. For eqn. (3) and table 3 yield the information that for every 4 nucleotides denaturing at 25 and 40°C, 0.68 and 0.99 moles H+ respec- tively, are bound. Since the extent of denaturation at any pH and temperature is known, the titration curves at 5, 25 and 40°C can be used to give the same information.The values obtained are 0.70 f 0.11 mole H+/4 moles nucleotide for denaturation at 25"C, and 96 f 0.2 at 40°C. The agreement is surprisingly good. The calorimetrically determined enthalpy of denaturation per mole of nucleo- tide may also be applied to the thermal denaturation in 0.15 M NaCl, but in this case we need the enthalpy at 90°C. Estimating m 4500 cal/mole P at this tem- perature, we find that there are 24 nucleotides in the denaturation mole of sample SBll for the thermal process. The apparent disagreement between this value and the value estimated on the basis of a hydrogen-bonded structure is probably due to the enormous error of the hydrogen-bond estimate and the large (but some- what smaller) error of the long linear extrapolation used herein. The simplest significance that we can give these numbers is to say that the denaturation mole is that size denatured region which is most frequently fcund in partly denatured DNA.That there should be a lower limit to the size of the thermodynamically stable denaturation unit is to be expected. It might be thought surprising that the system behaves as though it denatured through the formation of many fresh, independent, minimum-sized disordered regions rather than propagating from a small number of progressively growing denatured regions. * If the extent of denaturation is not too large, it should be possible to write the denaturation in terms of the following equilibria : * The simple considerations of the preceding paper indicate that if explicit cognizance is taken of the role of the entropy of mixing of denatured and native regions, the position of minimum free energy corresponds to a mixture in which there are substantial numbers of helical and disordered regions.J.M. STURTEVANT, S. A. RICE AND E. P. GEIDUSCHEK 145 where is the number of nucleotides in the smallest allowed denaturation nucleus. All denatured regions smaller than this are of such infre- quent occurrence that they make no contribution to the denaturation equilibria. is the number of protons that binds to the cc sized nucleus upon denaturation. is the number of nucleotides that will denature when a single proton binds adjacent to an already existing denatured region.is the concentration of native DNA in monomer moles/l. is the concentration of denatured DNA in monomer moles/l. - nr' is the concentration, in moles/l., of cc-sized regions of native DNA. u c 2 p . . . . CC is the concentration of y-sized denatured regions and y = a, a + p, is the equilibrium constant for nucleus formation. is the equilibrium constant for nucleus continuation. In terms of these equilibria, the ratio of denatured to native material at equilibrium is given by and the pH dependence of log (DlIN1) is given by Eqn. (5) and (6) provide, implicitly, the pH dependence of the denaturation equilibria as a function of D1/N1 and of the parameters Ki, K,, E, p and h. To simplify this, let cc = hp, i.e.assume that the segment of DNA chain denatured is proportional to the number of protons bound. We may then note that at small extent of denaturation and that further increase of this derivative with D1/N1 depends on the ratios of the two equilibrium constants Ki and Kp. We are looking for those conditions that will produce the experimentally observed situation-a substantially constant value of log (D1iN1) over the range in which log (Dl/Nl) can be observed at all accurately. If the standard free energy change for the denaturation of one nucleotide is the same for reactions (4a) and (46) then Ki = (Kp)h, and the re- quired behaviour is not found. However, for Ki = (2Kp)h and Ki = (3Kp)h the variation over the range of denaturation [log (Dl/Nl)] which is covered experi- mentally, is satisfactorily reproduced.For Ki = (24Jh the value of h in best agreement with the experimental data is 6.5, and for Ki = (3Kp)h the best value is 7. In both of these cases the formation of critical size nuclei is favoured over the growth of already existing nuclei. However, the differences are extremely small, amounting to only 420 and 670 cal per p unit respectively. At 25"C, each p unit contains approximately 3 nucleotides, so that the difference in the intrinsic stabilities which we have to assign in order to fit the observed behaviour is extremely small. d PH146 STABILITY OF THE HELICAL DNA MOLECULE IN SOLUTION An alternative denaturation process which might appear at least plausible, is that of a nucleation in which the initial denaturation (eqn.(44) kernel, once formed, grows very readily, i.e. Ki < (Kp)h. For this case the pH dependence of log (Dl/Nl) becomes essentially infinite even at very small per cent denaturation. The experi- mental data on the acid denaturation (treated in terms of the equilibria of a single species) are therefore incompatible with this alternative. * While the above calculations do give us the assurance that we are seeking- namely that the observed pH dependence is compatible with a model in which there are no absolute restrictions on the size of the denatured region-they are not to be mistaken for an exact representation of the denaturation process. We have allowed neither for the possibility of fusion of different denatured regions, nor for the instability of the very small ordered regions (< a nucleotides) that will tend to separate denatured segments at high percentage denaturation.At high fractions denatured, the model will therefore fail. In addition, it is im- portant to recall the assumptions under which we made these calculations : we assumed that the entire DNA sample was characterized by a single equilibrium constant (or a single pair of Kj and Kp). In fact, however, it is well known that DNA is chemically heterogeneous in the sense that macromolecular fractions of different chemical composition may be separated. In that case, there are two possibilities. The first is that the calculated values of the order with respect to pH, and the denaturation mole size, are averages for the entire sample. The second possibility, which is much more serious, is that each fraction of the sample has a much steeper pH dependence of the denaturation and that the observed value of h - 7 is the result of a distribution of samples denaturing very sharply at slightly different pKs.In that case the estimated size of the denaturation mole will be too small. Indeed, the possibility exists that our assumption of a single species fails sufficiently badly for us to have made a mistake of a qualitative as well as a quantitative nature; we may have rejected the nucleation model on false grounds. At least qualitatively the matter is capable of an experimental test. If the nucleation model (Ki < (Kp)h is correct and the sample denatures one molecule at a time, then the average macromolecular properties such as in- trinsic viscosity and light-scattering average radius of gyration will decrease linearly with the monomer mole fraction of denaturation.Jf the model proposed in eqn. (4a)-(4c) is valid, then a sample of DNA having, say, a 5-10 % mole de- naturation will have suffered some damage in almost every single molecule; the early stages of denaturation will, in other words, be accompanied by very marked changes in the macromolecular properties of the sample. In so far as the data are available, they appear to favour the latter alternative. Both the intrinsic viscosity (fig. 4) and the average radius of gyration decrease markedly during the very early stages of the acid denaturation of salmon DNA. Finally, we are obliged to rationalize the very large difference in the heats of thermal denaturation of the two samples of calf thymus DNA (V-1 and SBll).It will be noted (fig. 2) that sample V-1 is somewhat more easily denatured than SBIl. Presumably it has been made more heterogeneous (with respect to * Another possible nucleation process is one in which only the formation of the initial denatured kernel involves proton binding, i.e., followed by No! 3. hH+ S Da with Kj 4 (Kp)h. Such a process will give the required pH dependence of denaturation. However, the agreement with calculations based on the titration curves is completely destroyed-the experimentally determined titration curves are incompatible with this alternative.J . M. STURTEVANT, s. A. RICE AND e. p. GEIDUSCHEK 147 denaturability) by some artifact of the preparative procedure, and this heterogeneity leads to a less steep temperature dependence of the whole sample and to a low estimate for the size of the denaturation mole.* DENATURATION STUDIES AND THE STABILIZATION OF DNA SECONDARY STRUCTURE The opinion is often expressed that denaturation studies of DNA provide additional evidence for the hydrogen bond stabilization of its secondary struc- ture?, 6 Our own studies on DNA denaturation and the recent developments in the interpretation of polymer and protein electrostatic interactions 2% 26 have gradually persuaded us that this is indeed far from being the case.In fact, it appears clear that the data which are customarily cited as providing evidence for hydrogen-bond stabilization in solution are capable of alternative interpretations, and in some instances are indeed not entirely compatible with a pure hydrogen- bond model.The denaturation of DNA by acid and base has been most frequently inter- preted in terms of hydrogen-bond breaking by protons. That macromolecular collapse does not follow immediately upon binding the first few protons to the DNA chain has been known from the very beginning; the interpretation usually put upon this fact has been that only a fraction of intact H-bonds is necessary to maintain the extended helical configuration of DNA.4 In fact, the calorimztric data presented in fig. 4 and 5, show that no enthalpy changes occur until up to 65 % of all possible protons (at 5°C and 0.1 ionic strength) are bound to the purines arid pyrimidines.If the heats of proton binding of the purines and pyrimidines are zero, then it can only be concluded that no hydrogen bonds have been broken by the first 65 % of the pr0tons.t But in that case the denaturation that ultimately occurs as a consequence of the proton binding cannot be uniquely interpreted in terms of a hydrogen-bond model. Similarly the observed change of proton afiinity that occurs upon denaturation could be the result not only of irreversible hydrogen-bond cleavage but also of any of the other changes of the environment of the amino groups-improved hydration, greater accessibility of small ions, opportunity for ion-pair formation with near-neighbour phosphate groups-which occur as a consequence of the molecular collapse. In fact, it appears likely that a very important factor in the destruction of the double helix is the mutual repulsion and poor hydration of positively charged adehyl, guanyl and cytidyl groups in the relatively non-polar core of the native molecule.: *Another, and far less likely, alternative is that the entire secondary structure of sample V-1 is less stable so that while the size of the denaturation mole may be ap- proximately the same the heat of denaturation per nucleotide is less than in sample SBI 1.t The alternative that 35 % of the H-bonds have stabilization energies of 9.0 kcal/mole while 65 % of the H-bonds have essentially zero enthalpies of stabilization, is clearly im- plausible. $ It is of interest to attempt to calculate the electrostatic contribution to the free energy, even though the results of such a calculation will not be quantitatively reliable due to lack of knowledge of the internal dielectric properties of the molecule.To compute the electro- static energy, the attractive interactions between the protons on the bases (the " core ") and the phosphate groups on the outside of the cylinder formed by the DNA helix, as well as the proton repulsions, must be considered. Upon transition from helix to coil we note that the average nearest neighbour distance between protons on the purines and pyrimid- ines is at least doubled. Denaturation will therefore be accompanied by a decrease in the proton-proton repulsions where A p P = E T ~ P - E'~P. coil helix' -1 - E r e p = [ exp (-- KQ) + 3 exp (- 2~t-0) + . . Dr0148 STABILITY OF THE HELICAL DNA MOLECULE IN SOLUTION Another group of data which are often cited as compelling evidence for the existence of a H-bonded structure in DNA is the denaturation in aqueous solutions of " hydrogen-bond breaking agents " such as urea and guanidinium chloride.4~ 139 19 However, the data of table 2 show that in one case at least, the heat of denaturation of DNA in 8 M urea, 3.2 M guanidinium chloride and aqueous salt solution are the same. If the action of these substances were mainly to disrupt intramolecular DNA hydrogen bonds, then one would expect appreciable lowering of the en- thalpy of denaturation relative to aqueous salt solution.It is known that formamide, which is a H-bond breaking solvent for poly- peptides, causes the denaturation of DNA.On the other hand, so do methanol, ethanol, dimethyl formamide and acetonitrile whose ability to disrupt hydrogen- bonded aggregates is not remarkable. Evidently factors involved in the de- naturation by non-aqueous solvents are also too complicated to be described in terms of H-bond breakage. Finally, reference might be made to the admittedly incomplete data on the infra-red spectral changes that accompany DNA denaturation. We refer to the shift of absorptions in the 1600cm-1 region to lower frequencies when DNA is denatured.27 In a simple H-bond interpretation of such a shift, one would have to admit that the purines and pyrimidines were more strongly H-bonded in the denatured state and that heats of denaturation of such a structure would have to be negative! These are some of the points which we find it difficult to reconcile with a purely H-bonded model of DNA.It is certainly recognized nowadays that the solvent medium may radically affect the stability of hydrogen bonds and that in a molecule of the structural complexity of DNA, other stabilizing and destabilizing factors must certainly be present. The problem for the interpretation of informa- tion on the stability of DNA is the following. Is the known stability of DNA due to substantially complete purine-pyrimidine base pairing, strengthened by solvent effects? Or are the differential solvation effects of water so strong that hydrogen bonding makes only a relatively small contribution to the maximum attainable stability? In the latter case, the observed stability of DNA might arise from a very imperfectly purine-pyrimidine paired structure.At the present time, the choice between these alternatives cannot be made. and D is the effective dielectric constant, K the Debye screening constant, e the proton charge, and ro the average charge separation, which depends on the fraction of protons bound and on the molecular configuration (native or denatured). The attractive inter- actions between purines, pyramidines and phosphates also change upon denaturation. We approximate this by considering the molecule as a coaxial condenser for which the electrostatic potential is where p is the linear charge density of the core (= l/ro), and r is the diameter of the cylinder. As an example we take the case of denaturation at 0.1 ionic strength, 25"C, in which the native molecule with about 1.4 protons bound per 4P atoms denatures to give a product having approximately 2-1 protons bound per 4 P.The total energy change in this case is AE = - E;!: +E;fp - E r f = -5 kcal/mole P, when the effective dielectric constant is arbitrarily assigned a value of 10. This is far more than sufficient to compensate for the loss of secondary structure stabilization in denatur- ation, but the estimate is undoubtedly only qualitatively reliable. However, it is important to note that one may choose D in such a way as to give agreement with the titration curve of (native) DNA at 5"C, while at the same time providing the electrostatic free-energy changes on denaturation that are required to drive the denaturation to completion. 1 Cox and Peacocke, J. Chem. Soc., 1956, 2499, 2446. 2 Cox and Peacocke, J. Polymer Sci., 1957, 23, 765. 3 Duggan, Stevens and Grunbaum, J. Amer. Chem. SOC., 1957,79,4859.J . M . STURTEVANT, S . A. RICE AND E. P. GEIDUSCHEK 149 4 Cavalieri and Rosenberg, J. Amer. Chem. SOC., 1957, 79, 5352. 5 Gulland, Jordan and Taylor, J. Chem. Soc., 1947, 1131. 6 Mathieson and Matty, J. Polymer Sci., 1957, 23, 737. 7 Shack, Jenkins and Thompsett, J. Biol. Chem., 1953, 203, 373. 8 Thomas, Biochim. Biophj7s. Acta, 1954, 14, 23 1. 9 Lawley, Biochim. Biophys. Acta, 1956, 21, 488. 10 Cavalieri and Stone, J. Amer. Chem. SOC., 1955, 77, 6499. 11 Zamenhof, A h . Biophys. Biophys. Chem., 1956, 6, 85. 12 Doty, J. CeIf. Coinp. Physiol., 1957, 49, (suppl. l), p. 27. 13 Rice and Doty, J. Amer. Chem. Soc., 1957, 79, 3937. 14 Peacocke and Schachman, Biochim. Biophys. Acta, 1954, 15, 198. 15 Shooter and Butler, Trans. Faraday SOC., 1953, 52, 734. 16 Reichman, Bunce and Doty, J. Polymer Sci., 1953, 10, 109. 17 Geiduschek and Gray, J. Amer. Chem. Soc., 1956, 78,408. 18 Cavalieri, Rosoff and Rosenberg, J. Amer. Chem. SOC., 1956, 78, 5239. 19 Alexander and Stacey, Biochem. J., 1955, 60, 194. 20 Geiduschek, to be published. 21 Geiduschek and Sturtevant, submitted for publication to the J. Amer. Chem. SOC. 22 Simmons, Atomic Energy Commission Report UCLA 184, 1952. 23 Schwander and Signer, Helv. chim. Acta, 1950, 33, 152 1, 24 Geiduschek, J . Polymer Sci., in press. 25 Tanford and Kirkwood, J. Amer. Chem. SOC., 1957,79, 5333. 26 Tanford, J. Amer. Chem. SOC., 1957, 79, 5340. 27 Blout and Lenormant, Biochini. Biophys. Acta, 1955, 17, 325.

ISSN:0366-9033    

DOI:10.1039/DF9582500138

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

MOLECULAR ASSOCIATION INDUCED BY FLOW IN SOLUTIONS OF SOME MACROMOLECULAR POLYELECTROLYTES BY M. JOLY Service de Biophysique, Institut Pasteur, Paris Received 23rd Janiiary, 1958 The molecular solutions of several types of macromolecular polyelectrolytes are stable only at rest. As soon as a laminar flow takes place in the solutions, aggregation of the macromolecules occurs giving rise to particles of colloidal dimensions. Such an aggregate formation is reversible or not as a function of the velocity gradient depending on the nature of the solutions studied, but even where reversible, the phenomenon shows hysteresis. These behaviours are investigated by streaming birefringence measurements, mainly with several kinds of protein systems. A tentative interpretation of the flow-induced associ- ation is given on the basis of the collision and orientation theory.The sizes, mean-lives and formation kinetics of the aggregates are derived in terms of intermolecular forces and velocity gradient. A few consequences of this aggregdtion, such as dynamic turbidity are briefly indicated. It has been well known for a long time that laminar flow increases the velocity of aggregation of colloidal particles in unstable sols. An explanation of this phenomenon was first proposed by Smoluckowski 1 on the basis of the collision theory. A survey of the behaviour of suspended particles under laminar shear has been recently given by Mason and Bartok.2 For macromolecular poly- electrolytes, it is sometimes observed with suspensions which are perfectly stable at rest that the aggregation occurs as soon as they are submitted to a laminar flow of sufficiently high velocity gradient.Consequently the size of the particles in these solutions increases with the rate of shear. On the other hand, the shearing forces, if large enough, can break the particles,3 and a decrease of the pzrticle size is observed at high velocity gradient. This disruption has been shown with tobacco mosaic virus particles.4 By superposition of these two processes the size of particles in the flowing solution can successively increase and decrease with increasing velocity gradient. Such a behaviour has been described in mixtures of tobacco mosaic virus and sodium dodecylsulphate.5 The purpose of the present paper is the experimental study of the variations of the particle size (as a function of the velocity gradient), due to the aggregation or disaggregation induced by flow in dilute solutions of various polyelectrolytes. The size of the particles is determined by streaming birefringence. EXPERIMENTAL MATERIALS The acrylic acid + acrylonitrile copolymers (Acan) have been prepared and analyzed by Clavier.6 Actomyosin and myosin, prepared by Schapira and Dreyfus,7 were re- spectively obtained by the methods of Greenstein and Edsall8 and of Mommaerts.9 Metam yosin, a new muscle protein, was isolated and purified by Marcaud-Roeber.10 L-meromyosin was prepared by Broun and Kruh11 by partial trypsin hydrolysis of a myosin extracted according to Szent-Gyorgyi 12 and purified according to Mommaerts.9 Horse serum proteins, obtained by salting-out with (NII4)2S04 were purified by Barbu.13 Salmine sulphate was a commercial Roussel product, and histone sulphate was prepared by Rybak 14 from calf thymus by HCl extraction, picric acid precipitation and ethanol purification. METHODS The solutions under investigation are forced to flow between two coaxial cylinders, the inner of which rotates at various but well-defined speeds.For each value of the 150M. JOLY 151 velocity gradient g , or as a function of the time t for a given value of g , the extinction angle x is determined with an accuracy of & 0.1" by the differential method of Frey- Wyssling and Weber.15 The design of the apparatus has been given elsewhere.16 All the systems investigated here are polydisperse.Therefore the streaming birefring- ence measurements allow us to determine only the order of magnitude of the particle size. Indeed, as we have shown 17.18,19 for very elongated or of quasi-globular particles rigid enough to be undeformed by the flow in the experimental conditions, the length or the diameter of the ellipsoids hydrodynamically equivalent to the most frequent particles in a given suspension and the corresponding polydispersity ratio can be derived from the values of the apparent particle length or diameter for two suitably chosen values of the velocity gradient. But this derivation is only valid with the assumption that the particle- size distribution does not depend on the rate of shear. Now this is not the case with the present systems since they are characterized by their ability to form aggregates by flow.Consequently we can only consider the values of the apparent length I, or diameter d, of the particles and their variations as a function of g, which gives only the order of mag- nitude of the true value of the particle size since we cannot determine the polydispersity ratio in the present case. Noting that the apparent length I,(g) of the particles of any system for a given velocity gradient is defined as the length of the particles in an infinitely dilute monodisperse system of rigid ellipsoids which for the same g would give the same value of x as obtained with the system studied. Such a definition does not imply that the true particle shape is an ellipsoid. The values of I, or d, are deduced from those of the rotational diffusion constant D by means of the Gans re1ation;zo the relationship between x, g and D has been determined with high accuracy.21 For monodisperse systems, I, evidently does not vary with g ; for polydisperse systems all particles of which consist of the same substance, I, or d, decreases with increasing g and this variation is reversible.Consequently, if the apparent size increases with g over a given range of rate of shear, it can be concluded that the true size of the particles increases with g. This could be due to the deformability of the particles, but in such a case the apparent size variation would be reversible with respect to g , except perhaps for a small hysteresis. But when such a variation is largely irreversible, one can affirm that there is aggregation induced by the flow.Likewise, when I, or d, irreversibly decreases with increasing g for sufficiently high values of it, this means that, even if this flowing system is a poly- disperse one, the particles are more or less broken by the shearing forces. RESULTS Already in 1912 it was shown 22,23 that the agitation of a Cu(OH)2 sol increases the size of the suspended particles, and much more recently very large fibres of the 1 : 1 copper + cystin complex were obtained 24 by stirring dilute solutions. Concerning the shear-breaking of particles in flowing solutions, we shall not consider the rupture of covalent linkage at very high rate of shear 25 but only that of low energy bonds like hydrogen bridges or van der Waals forces as observed with tobacco mosaic virus,4 denatured serum albumin 26.27 or deoxyribonucleic acid.28 An interesting case is when the shear-broken particles are small liquid droplets suspended in a liquid phase, as observed by Winsor 29 with mixtures of cyclohexane, sodium tetradecane-5 sulphonate, water and nonylamine or cyclohexanol, by Silberberg and Kuhn 30,31 with mixtures of 1.1 % polystyrene and 1.7 % ethylcellulose in benzene a few degrees above the critical temperature of dissolution, and by Barbu and Joly 13, 18 with compressed serum albumin solutions.Our purpose is to determine the quantitative correlation between the size variation, the flow characteristics and the other parameters of the solutions. ACRYLIC ACID + ACRYLONITRILE COPOLYMERS With a 0.1 % aqueous solution of a sample of Acan, the molecular weight of which is 11.5 x 104 and an acrylic acid content of 20 %, do of the aggregates grows from 375 8, to 405 8, when g increases from 104 to 2 x 104 sec-1.With higher concentrations, the aggregation occurs very rapidly at low value of g. For instance, with 1 % solution, d, = 3300 8, for g = 400 sec-1. At higher velocity gradients, the aggregates are disrupted by the shearing forces and the particle size decreases: g = 1200 sec-1, d, = 2300 A ; g = 5500 sec-1, d, = 1300 A. One obtains similar results with such copolymers of various compositions and lower molecular weights, but all these systems do not show a good reproducibility, even in presence of KC1 or dimethylformamide, and the results are not accurate enough to characterize the effects of the different parameters.152 FLOW ASSOCIATION OF MACROMOLECULES HORSE SERUM ALBUMIN IN PRESENCE OF TRIVALENT IONS Similar behaviour has been observed with horse serum albumin (SA) in presence of trivalent cations in the range of velocity gradient for which the flow remains laminar (fig.1, curves 7 and 8). As said before, the flow aggregation is demonstrated by the irre- versibility of the d,(g) variations. Thus for g increasing from 450 to 9900 and then de- creasing from 9900 to 450 sec-1, d, measured at g = 450 sec-1 before and after g variation increases from 3450 to 3800 A with 0.6 % SAY pH 5.6 in 0.0002 M A1C13 : from 2700 to 3500 8, with 2.4 % SA, pH 6.0 in 0.001 M AICl3 and from 2900 to 3200 A with 4 % SA, PH 3.8 in 0.002 M AlC13.i f 3000 d 0 2COG I000 0 FIG. 1 .-Particle-size variations induced by flow. 1. Compressed horse serum albumin. A, 1 h ; 8000 kg/cm2 ; 37°C. (1) 4 ”/, ; pH 6-9 ; c = 1 %. (2) id. pH 7-4 ; B, 16 h ; 10,OOO kg/cm2; 37°C. (3) 6.9 % ; pH 6.3 ; c = 1-2 %. (4) 8.3 % ; pH 4.35 ; c = 1-66 %. (5) id. pH 4.4. (6) id. pH 4.45. (c = con- centration of flowing solution.) 11. Horse serum albumin with trivalent cations: (7) 0.6 %; 0.0004 M AlCl3; pH 6.9. (8) 2.4 %; 0.0002 M FeC13; pH 6.2. COMPRESSED HORSE SERUM ALBUMIN A similar study has been done with SA solutions submitted to very high hydrostatic pressures. This treatment causes the separation in the solutions of submicroscopic droplets more concentrated in protein than the surrounding medium.13.18 As shown in fig. 1, the flow behaviour of these very viscous droplets is quite analogous to that of SA in presence of trivalent ions.The comparison of the curves 4, 5 and 6 shows the con- siderable effect of the pH value near the isoelectric point on the ability of the droplets to coalesce by flow collisions. Curves 1 and 2 indicate that the pH effect is still important quite far from the isoelectric point. MYOSIN AND RELATED MUSCLE PROTEINS The amplitude of the size variations with g is often larger with elongated particles than with globular particles, as seen, for instance, in fig. 2. Curves 1 and 2, for concentrated and highly aggregated rabbit myosin solutions, show the powerful effect of the pH on the resistance of the particles to flow break-up. Curve 3 shows the rapid aggregation of the sheep foetus metamyosin molecules, the apparent length of the aggregates being twice as large for a four-fold increase in velocity gradient.The high sensitivity to the flow of this muscle protein is clearly seen in the variation with flow duration of I, measured at constant g. For instance, with a 0.01 % solution of adult rabbit metamyosin, the values of la measured after 2, 4, 6 and 8 min of laminar flow at g = 5500 sec-1 are respectively 1050, 1950, 2950 and 4300 A ; afterwards the solution becomes turbid. Rabbit L-mero- myosin (curve 4) is much less dependent on the velocity gradient. PROTEIN COMPLEXES The competition between the building-up and the breaking-up of the aggregates is clearly evidenced by the shape of the Za(g) curves obtained with mixtures of acid and basicM.JOLY 153 proteins that give rise to complex f0rmation.32~33 These curves exhibit in the range of laminar flow a wide and almost symmetrical maximum (fig. 3). The comparison of the curves 3 and 4 shows the remarkable influence of salt on the aggregate stability of such protein complexes. On the other hand, it is to be noticed that by long stirring at low rate of shear these solutions can give rise to macroscopic fibres. l a 4000 2000 0 I 3 6000 8ooooo 1000 2000 - 3000 4000 5000 60 9 sec-' FIG. 2.-Particle-size variations in flowing solutions of muscle proteins. I. 0.11 % rabbit myosin, 1-0 M KCI. (1) pH 6.8. (2) pH 6-7. 11, 0.05 % sheep foetus metamyosin. (3) pH 6.8; 0.4M KC1 + 0.1 M phosphate buffer. 111, 0.01 % rabbit L-meromyosin.(4) pH 6.65 ; 0.6 M KCl + 0.1 M phosphate buffer. 5000 L- I 0 1000 2000 -3000 9 sec-' FIG. 3.-Particle-size variations due to flow. (1) 0.16 % SA + 0.04 % SH; (2) id. + 0.01 % colchicin; (3) 0.06 % SA + 0.09 % SS ; (4) id. + 0.04 M NaCl; (5) 0-18 % EG + 0.012 % SS; 0.25 M NaCI; (6) 0-25 % EG + 0.018 % SS; 0.18 M NaCl; 0.07 % ATP. (SA: horse serum albumin; SH: histone sulphate ; SS : salmine sulphate ; EG : horse serum euglobulin). RABBIT ACTOMYOSIN It is of interest to compare the flow aggregation of the preceding artificial complexes with the behaviour of a natural protein complex like actomyosin. In fig. 4 the curves have been roughly classified according to the existence of a flat or cf a maximum in the Z, variation at rather high velocity gradient, in relation to resistance of the aggregates to the shear1 54 FLOW ASSOCIATION OF MACROMOLECULES break-up. All these curves exhibit a flat part at low g and then an abrupt variation of slope in a narrow range of g, the sign of the curvature changing at a rate of shear of about 2OOOsec-1.This suggests the need of a critical extent of particle orientation to enable the collisions to be efficient for aggregation. As with myosin solutions there is an important effect of pH on aggregation and dissociation as shown in the curves 10-14. The effect of ionic strength on aggregate stability clearly appears in curves 6 and 13, 12 and 16, and 18 and 20 ; it depends on the protein concentration and pH. d l a 7000 9300 i; 8000 fa 6000 - - 4 003 I I 3000// 5 I8 10 0 FIG.4.-Particlesize variations in flowing solutions of rabbit actomyosin. I. 0.01 % ; 1.0 M KCI; (1) pH 8-65. 11. 0.025 % ; 1.0 M KCl ; (2) pH 8.3 ; (3) pH 8.4. 111.0.05 %. A, H20 ; (4) pH 3.65 ; (5) pH 4. By 0.25 M KCl ; (6) pH 8-4 ; (7) pH 8-5. C, 0.5 M KCI ; (8) pH 6.7 ; (9) pH 8-2. D, 1.0 M KC1 ; (10) pH 6.8; (11) pH 7-5; (12) pH 8.3 ; (13) pH 8.4; (14) pH 8-6. E, 1.5 M KCl; (15) pH 6-9; (16) pH 8.3. F, 2.0 M KCl; (17) pH 6.95. IV. 0.12 %. G, 0.25 M KC1; (18) pH 7.2. H, 0.5 M KCl ; (19) pH 6.5 ; (20) pH 7-2. 5000; ' - - I 1000 2000sec-,3000 4000 9 HEATED HORSE SERUM ALBUMIN Solutions of horse serum albumin denatured by moderate heating have been exten- sively studied from the view-point of flow aggregation. The main experimental results are summarized in fig.5, 6 and 7. The curves of the fig. 5 relate to particles the Iength of which never decreases with increasing g in the range of laminar flow ; the curves plotted in the upper part of the graph concern solutions heated in presence of salt. With all these solutions the apparent length of the aggregates fist increases more or less rapidly with g and then remains practically constant at a value of g that is smaller the higher the protein concentration, ionic strength and heating temperature, the nearer the pH is to the isoelectric point and the longer the duration of heating. The length of the flat part of the curves increases with increasing protein and salt concentration and with tem- perature; the corresponding aggregates are all larger as the pH value approaches the isoelectric point.Fig. 6 and 7 show the behaviour of SA aggregates the size of which more or less rapidly reaches a maximum before it decreases; fig. 6 refers to solutions heated without salt and fig. 7 in presence of salt. Generally the solutions for which the particles pass through a maximum size for a defined value of g are characterized by a higher ionic strength or a pH nearer the isoelectric point than those of the solutions the ldg) curve of which does not exhibit a maximum. For almost all these solutions &(g) very sharply increases with g at low rate of shear, denoting an easy flow aggregation, whereas after the maximum, la rather slowly decreasesM. JOLY 155 FIG. 5.-Particle-size variations in- 500 duced by flow in heated horse serum albumin solutions.I, 10 rnin at 80°C. A, 0.75 % ; (1) 0-002 M NaN03 ; pH 7. B, 2 % ; (2) pH 7.3. C, 2.4 %; (3) pH 7 ; (4) pH 5oo 7.4 ; (5) id., 0.0004 M NaC1; (6) pH 6-9; 0.0002 M BaC12; (7) pH 3.9; 2ooo 0.02 M acetate buffer. D, 2-9 % ; (1 1) pH 3.9; (13) pH 4. E, 3 %; (20) 0.01 M Na2S04; pH 8.6. F, I 500 3.9 % ; (21) pH 3.7 ; (22) pH 7.9. 11, 5 min at 100°C; 2.4 %; (8) 0.02 M acetate buffer ; pH 3-9. III,20 min I 090 at 55°C; 2.9 %; (14) pH 4.4. IV, 10 rnin at 60°C; 2.9 %; (15) pH 530 4.4. V, 10 rnin at 70°C; 2.9 %; I 5 0 0 rnin at 100°C. G, 2.5 ; (9) pH 4.4. l a I000 i f a (10) pH 3.9; (16) pH 4.4. VI, 10 H, 2.9 %; (12) pH 3.9; (17) pH 7.1 ; IOOC -Qa 500 (19) 7.5. 2 ooc i &I I50C I ooc 5 OC ii 2000 1, I500 1000 50C FIG. 6.-Particle-size variations induced by flow in heated horse serum albumin solutions.I, 10 min at 80°C. A, 1.6 %; (1) pH4.4. B, 2 % ; (2) pH 4-3 ; (3) pH 6.7. C, 2-4 %; (4) pH 4.3; (5) pH 4.4; (6) pH 6.6; D, 2.5 % ; (7) pH 7.8. E, 2.9 %. (8)pH4-1; (9)pH7-1. F,3*1 %; (10) pH4; (11) pH 7. 11, 8.1 % SA heated 10 rnin at 60°C and diluted to 3.3 % after storage G, pH 4-3 ; (12) 22 h ; (13) 28h ; (14) 19 days. H, pH 4.5; (15) 19 days. 111, 2.5 % SA heated 10 rnin at 80°C and pH 7.8; measured at (16) pH 7.8 ; (17) pH 7.2.156 FLOW ASSOCIATION OF MACROMOLECULES with increasing g as a consequence of a strong resistance of the aggregates to the flow break-up. As a first approximation the maximum is higher and reached at a lower value of g as the protein concentration and ionic strength are greater and the pH value is nearer the isoelectric point.The preceding examples indicate that flow association is a frequent phenomenon. Thus, it is often observed with liquids extracted from biological medium. For instance, the sap obtained by grinding of cooled tobacco leaves infected by mosaic virus contains particles which aggregate very easily by laminar flow of this sap. Therefore it might be asked if in some cases the structure of living matter is not due in part to its state of motion in the cells.34 it l a I500 2ooc I000 A l a 1500 2 000 1000 2 500 8 1, 2000 I 5 0 0 1000 I 5 I IL 2500 5000 ,7500 I00 9 sec- FIG. 7.-Particle-size variations induced by flow in heated horse serum albumin solutions. I, 2.4 %; 10 min at 80°C; (1) pH 4 ; O.OOO4 M KCN ; (2) pH 4.15 ; 0.02 M ace- tate buffer; (3) pH 6.6; 0.002 M NaCl; (4) pH 6.9 ; 0.0005 M BaC12.A, pH 7 ; ( 5 ) 0.01 M NaF; (6) 0.01 M NaCI. B, 7.4; (7) 0.001 M NaCl; (8) 0.002 M NaC1; (9) 0.004 M NaCl ; (10) 0.01 M NaCI. 11, Various concentrations ; 10 min at different temperatures. C, 0.19 % ; pH 7 ; 0-005 M Na~S04; (11) 80°C; (12) 100°C; (13) 0.38 %; pH 7 ; 0.005 M Na2S04; 80°C; (14) 0.6 % ; pH 4-15 <A0-02 M acetate buffer ; 100°C; (15) 1-5 %; pH 7 ; 0.002 M NaN03 ; 80°C; (16) 1.6 %; pH 4.4; 0-01 M CaC12; 60°C. 111, 3.1 %; 10 min at 80°C. D, pI-1 4 ; (17) 0-0017 M NaCl; (18) 0.002 M LiCI. E, pH 7 ; (19) 0.0008 M NaCl ; (20) 04017 M NaCl ; (21) 0.0033 M NaCl. DISCUSSION Besides the irreversibility of the l,(g) or d,(g) variations, a supplementary proof that the increase of particle size with g is due to aggregation and not only to particle deformation, even when the extent of this variation is small, is indicated by the following fact.The increase of 1, or d, is very often accompanied by an extremely sharp increase of the turbidity, and frequently, if the flow duration is long enough and the rate of shear sufficiently low not to break up the aggregates, precipitation occurs. For interacting globular particles a tentative interpretation has been recently given 35 for the flow aggregation, assuming that the rate of shear is low enough to avoid breaking up the particles. The calculations were based on an extension 13 of the treatment given by Tobolsky 36 in polymerization kinetics by introducing a flow collision term derived from the relationship given by Mason.37~ 38 As a first approximation the variation of the mean volume of a globular ag- gregate built up by the flow is given byM.JOLY 157 for low values of the rate of shear and concentration. 00 is the volume of each initial solute particle before solution flow and aggregation ; no is the total number of initial solute particles or molecules per ml; A = (2kT/3n~) F(U), 7 being the solvent viscosity and F(U) the probability of a particle surmounting the potential barrier of height U which separates two initial particles before collision; B~(kT/21n)*exp (- W/kT), where nz is the mass of each initial elementary particle or molecule and W the mean value of the interaction energy between each molecule and all its neighbours in an aggregate. A more elaborated treatment including the shear break-up of the particles is now in progress.The case of flow aggregation and disaggregation of elongated particles leads to a more difficult mathematical derivation, the principle of which has been given before.35.39 These calculations will be published in full in a forth- coming paper. They give a good approximation for the order of magnitude of the experimental results of streaming birefringence and dynamic turbidity, and enable one to determine the values of the potential barrier and interaction energy between the aggregating particles and to compare them with the theoretical values when available.40 1 Smoluchowski, Z. physik. Chem., 1918, 92, 129. 3 Mason and Bartok, Brit. SOC. Rheology Meeting (Swansea, 1957). 2 Kuhn, Z.physik. Chem. A , 1932, 161, 1 and 427 ; Kolloid Z., 1933, 62, 269. 4 Joly, Bull. soc. Chim. biol., 1948, 30,404 ; 1949, 31, 108. 5 Joly, Biochim. Biophys. Acta, 1952, 8, 134, 245. 6 Clavier, Thesis (Paris, 1956). 7 Joly, Schapira and Dreyfus, Arch. Biochem. Biophys., 1955, 59, 165. 8 Greenstein and Edsall, J. Biol. Chem., 1940, 133, 397. 9 Mommaerts and Parrish, J. Biol. Chem., 1951, 188, 545. 10 Raeber, Schapira and Dreyfus, Compt. rend., 1955, 241, 1000. 11 Schapira, Broun, Dreyfus and Kruh, Compt. rend. SOC. Biol., 1956, 150, 944. 12 Szent-Gyorgyi, Arch. Biochem. Biophys., 1953, 42, 305. 13 Barbu and Joly, Faraday SOC. Discussions, 1953, 13, 77. 14 Rybak, Bull. SOC. Chim. biol., 1950, 32, 703. 15 Frey-Wyssling and Weber, Helv. chim. Acta, 1941, 24, 278. 16 Joly, in Techniques de Laboratoire (Loiseleur) (Masson, Paris, 1954), 1, 538. 17 Joly, Trans. Faraday SOC., 1952, 48, 279. 18 Barbu, Basset and Joly, Bull. SOC. Chim. biol., 1954, 36, 323, 19 Barbu and Joly, J. Chim. Physique, 1956, 53, 95 1. 20 Gans, Ann. Physik, 1928, 86, 628. 21 Scheraga, Edsall and Gadd, J. Chem. Physics, 1951, 19, 1101. 22 Paine, Kolloidchem. Beih, 1912, 4, 24. 23 Paine, Kolloid-Z., 1912, 11, 115. 24 Kahler, Lloyd and Eden, J. Physic. Chem., 1952, 56, 768. 25 Frenkel, Acta physicochim., 1944, 19, 51. 26 Joly and Barbu, Bull. SOC. Chim. biol., 1949, 31, 1642. 27 Barbu and Joly, Bull. SOC. Chim. biol., 1950, 32, 116. 28 Barbu and Joly, Bull. Soc. Chim. Belg., 1956, 65, 17. 29 Winsor, Trans. Faraday Soc., 1948, 44, 376. 30 Silberberg and Kuhn, Nature, 1952, 170, 450. 31 Silberberg and Kuhn, J. Polymer Sci., 1954, 13, 21. 32 Joly and Rybak, Compt. rend., 1950, 230, 1214. 33 Joly and Rybak, Bull. SOC. Chim. biol., 1950, 32, 894. 34 Joly, in Deformation and flow in biological systems (Frey-Wyssling), (North Holland 35 Joly, Kolloid-Z., 1956, 145, 65. 36 Blatz and Tobolsky, J. Physic. Chem., 1945, 49, 77. 37 Trevelyan and Mason, J. Colloid Sci., 1951, 6, 354. 38 Manley and Mason, J . Colloid Sci., 1952, 7, 354. 39 Joly, Kolloid-Z., 1952, 126, 77. 40 Isihara and Koyama, J. Physic. SOC. Japan, 1957, 12, 32. Pub. Co., Amsterdam, 1952), p. 51 1.

ISSN:0366-9033    

DOI:10.1039/DF9582500150

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

STRUCTURE, MOLECULAR FORCES AND AGGREGATION REACTIONS OF MACROMOLECULES OF COMPLEX PQLYMERS BY S. E. BRESLER Institute of High Molecular Compounds, Academy of Sciences of U.S.S.R., Leningrad Received 5th February, 1958 A study of the shape of protein macromolecules in mixed (water + dioxane) solvents showed that globulation forces, causing the tertiary structure of protein macromolecules are connected with interaction of nonpolar side groups. A study of the kinetics of iso- tropic exchange of globular proteins with water showed that some 20 hydrogen bonds are split simultaneously during the fusion of secondary protein structure. A study of hydrodynamical properties of serum albumin at different pH and its change under the influence of denaturing factors showed the importance of local chemical bonds between side groups of the protein for its rigidity. The laws of ionic adsorption of di- polar ions were elucidated and applied to the investigation of the topography of charged groups on the surface of globular proteins.Aggregation reactions of a number of pro- teins were studied and the local character of macromolecular linking was proved. GLOBULAR PROTEINS IN SOLUTION In 1944 in our work with Talmud 1 we considered a globular protein as an equilibrium shape for three kinds of molecular forces-electrostatic repulsion of the dissociated side-groups, van der Waals cohesion of the uncharged side-groups and hydrogen bonds between the peptide groups. In order to estimate the energy of these three types of bonds, we accepted that a coiled polypeptide main chain assumed the shape of a knot so that the hydrophobic side groups, formed a kind of hydrocarbon drop inside the knot.This hypothesis must now be changed to take regard of recent developments in the field. Hydrogen bonds between the peptide groups are the most significant. They force the main chain of regular polypeptides to coil into a very tight Pauling- Corey cc-helix. All the possible intramolecular hydrogen bonds are formed here. The qfiestion arises : how the a-helix is packed in the globules of the protein and what forces stimulate the formation of a knot. The single polypeptide chain of a serum protein forming a helix would look like an extended thread, not a globule (the molecular weight of serum albumin reaches 68,000. The length of the a-helix would be 600 x 147 = 850 A, diam.10 A). In water solutions the macro- molecules of serum proteins are almost symmetrical as follows from their hydro- dynamic properties. Doty et al.2 have developed a method for the quantitative estimation of the pitch of a-helix formation in the peptide chains of globular proteins by neasuring the optical rotation and its dispersion. Their results show that in water solutions the polypeptide chain of protein is partly (to 40-60 %) coiled into an a-helix but includes also some disordered sections. In our experiments with Frenkel we have tried to elucidate the forces twisting the molecular chain into a compact globule when an a-helix is already formed. We reckoned that the most important in this respect must be the interaction of the uncharged hydrophobic or slightly polar (e.g.hydroxyl-containing) side-groups which tend to gather into a hydrocarbon drop. To prove the connection between the globulation and the interaction of hydro- phobic groups (or with hydrogen bonds in the water itself producing a high surface 158S . E. BRESLER 159 energy on the hydrocarbon-water boundary) we changed gradually the nature of the solvent, replacing water by a much less polar dioxane or chlorethanol. In FIG. la.-Specific rotation-[cc]~(4), intrinsic viscosity of serum albumin solution [v] = lim r?] :?There c in is g/100 mI (2), axial ratio of the macromolecule ap- proximated as an ellipsoid a/b (3), intrinsic viscosity in the presence of neutral salt (0-33 M Na acetate) (l), plotted against the solvent composition (concentration of dioxane in water in volume per cent) at pH 10, r/2 = 0.01. 0 FIG.lb.-The same in water + chloroethanol solvent, pH 3.5, r/2 = 0.01. (1) intrinsic viscosity, (2) axial ratio, (3) specific rotation. these experiments we used serum albumin (human). Hydrodynamic character- istics-sedimentation constant and intrinsic viscosity-and optical rotation were measured simultaneously. The measurements were taken at several values of pH at different intervals from the isoelectric point. In fig. la the optical rotation160 MOLECULAR FORCES A N D AGGREGATION OF PROTEINS or serum albumin (compared to that in pure water) is plotted at pH 10 agzinst the composition of the solvent. We wish to stress that the hydrodynamic charac- teristics of the protein in dioxane 3- water solution are affected to some extent by the mode of preparing the solution.We added always first dioxane and then adjusted the pH from 7.5-8-5 to 10. If the solution is prepared first by addition of alkali the figures are changed and the effect (change of shape) is somewhat less pronounced. With chloroethanol these discrepancies are absent. The axial ratio of the serum albumin globules was calculated with the help of a formula for a rigid ellipsoid (Simha, Oncley), using data for the intrinsic viscosity and sedimentation constant. For the hydration ratio in these calculations we assumed w = 0.2 but changes from w from 0 to 0.3 do not alter the general picture. The axial ratio calculated from viscosity and sedimentation data are in fair agreement.We see that a serum albumin molecule turns into a stretched body with an a-helix practically unchanged ; these molecules behave like polyelectrolytes. Fig. 1 a demonstrates that the intrinsic viscosity of the protein falls by a factor of two with addition of salt (sodium acetate 0.33 M) on account of the increase of ionic strength, the usual effect found with polyelectrolytes. These experiments show that globular proteins consisting of peptide chains (primary structure according to Linderstrom-Lang 3) coiled into a-helices (secondary structure) form compact knots (tertiary structure) owing to the interaction of hydrophobic radicals ; the knots unroll in non-polar solvents with the a-helix unchanged. THE NATURE OF DENATURATION; THE EFFECT OF LOCAL CHEMICAL BONDS The term denaturation is used for all kinds of changes of the native macro- molecular protein structure.Consequently the concept is rather vague. The protein structure may be broken by different means : (i) by heating, i.e. thermal motion and chemical reactions initiated by heating ; (ii) by a large increase of the macromolecular charge, i.e. by addition of an acid or an alkali which changes the pH of the medium to a value far from the isoelectric point. Assuming a macromolecule to be an ellipsoid with its surface uniformly charged we can calculate the forces causing the stretching. The re- pulsion energy of surface charges of the macromolecule 4 is where e is an electron charge ; D, the dielectric constant of medium ; s, the surface area of the macromolecule; n, the number of elementary charges of a single macromolecule, and K, the constant of the Debye-Hiickel theory, or reciprocal value of the ionic atmosphere thickness.The axial ratio a/b of the ellipsoid being large (a/b = 4.0 for serum albumin) the surface area of the ellipsoid may be expressed in terms of its volume Vand length a : s = (d/2)(3/r)* V*a*. The force stretching the macromolecule as a result of the repulsion of the electrical charges with its volume remaining constant is F =- 3u/3a =- (aU/3s)(as/aa) ; (iii) by a high pressure. Forces arising here depend on the anisotropy of compressibility, i.e. on the intramolecular structure ; (iv) by a change of solvent. An addition of a second component (e.g. urea or guanidine) to a solvent sometimes produces a scission of nearly all the hydrogen bonds present in a macromolecule.In other cases, on the contrary, only theS. E. BRESLER 161 degradation of the tertiary structure is observed (e.g. by dioxane addition), the secondary structure remaining unchanged. Our experiments with Frenkel, Gorbacheva and Dmitrenko demonstrated that the stability of proteins against denaturation depended very much upon local chemical bonds which sometimes linked the distant units of the chain brought together with its coiling. Four types of local bonds are now firmly established : (a) disulphide bonds of cystine ; (6) ester bonds between carboxyl groups of dicarboxylic aminoacids and (c) diphosphate bridges characteristic of phosphoproteins ; (d) salt bridges formed by two- or polyvalent ions with carboxylic groups of phenolic hydroxyl of tyrosine ; the proteins.I 2.0 I .o 0'0 I 2 3 time (h) 4 5 b t FIG. 2.-Degree of isotopic exchange (weight per cent of exchanged hydrogen x) between serum albumin and water at pH 7 plotted against time (hours) : 2, the temperature of the solution 50°C ; 3, the temperature of the solution 70°C ; 4, the level of exchange after boiling at 100°C ; 1, the level of exchange at room temperature. We studied serum albumin, chymotrypsin and trypsin where only the local bonds of a and d types are essential. To examine the stability of proteins against denaturation we determined their hydrodynamic properties (sedimentation, diffu- sion, intrinsic viscosity) and kinetics of isotropic exchange with tritium-marked water.The kinetics of the isotopic exchange reveals only the primary disturbances of the secondary structure of proteins connected with scission of hydrogen bonds between peptide groups. The existence of hydrogen bonds prevents the imide hydrogen of CONH groups from isotopic exchange with the medium. Fig. 2 represents kinetic curves for isotopic exchange of serum albumin with tritium water. The exchange of 0.84 weight per cent of hydrogen takes place immeasurably fast at any temperature. This value corresponds to the calculated amount of hydrogen in the side chains capable of rapid exchange. At 100°C the protein exchanges 1.70 % of its hydrogen, in good agreement with the sum F162 MOLECULAR FORCES A N D AGGREGATION OF PROTEINS of 0.84 % and the imide hydrogen of the all peptide bonds (0.86 %).At 50°C the exchange rate of the additional hydrogen atoms is negligible; at 70°C the exchange occurs in 6min to the extent of 1.4 % and then practically stops; following this some hydrogen bonds (nearly 30 %) being more stable split only at the boiling point. We can make a rough estimate of the energy of activation for the scission of less stable hydrogen bonds from the two kinetic curves. We get a great value of 60,00Ocal/mole corresponding to the rupture of at least 20 hydrogen bonds. Similar estimations follow from the results of Linderstrom- Lang5 et nl. It means that the destruction of the hydrogen-bond system is a co-operative process. Doty suggested the formation of intramolecular a-helix to be called intramolecular crystallization.Then the simultaneous destruction of a considerable amount of hydrogen bonds may be termed intramolecular fusion. The importance of local chemical bonds for the stability of protein structure was demonstrated on serum albumin. In acidic medium considerable deformation cccurs, namely the stretching of serum albumin globules. In the presence of a small concentration of bivalent ions (Ca2+, Fez+, Cu2+) a stabilization of the native structure takes placc which is characteristic for neutral p1-i values (table 1). The ions " cure " the protein, forming salt bridges with carboxyls as often happens with acidic polyelectrolytes. Serum albumin contains 18 S-S bonds which stabilize the macromolecular structure. The S-S bridges may be broken by reducing substances, e.g.thio- glycolic acid or cystein. S-S bonds could be broken otherwise by boiling the protein in alkali medium (PH 9 or 10) on account of hydrolysis : 6 R1-S-S-CH2R2 + € I 2 0 + RlSH + RzCH2SOH. The last substance is unstable and dissociates to a carbonyl compound and hydrogen sulphide : R2CH2SOH -+ H2S + R2 - CHO. In urea solutions the protein macromolecule swells and S-S bonds are split at alkaline pH. High pressure causes the same process. The rupture of disulphide bridges destroys the regularity of the secondary protein structure, the shape of the protein becoming more symmetrical (table 1). It means that the existence of TABLE 1 .-HYDRODYNAMIC PROPERTIES AND SHAPE OF MACROMOLECULES OF SERUM ALBUMIN (HORSE) (a) Action of pH and bivalent ions alb t w = o w = 0.2 S(S) fN0 bivalent ions conc.(weight %) PH 5.0 0 4-5 1 -28 4 9 4-0 2.0 0 2 9 1.96 18.9 15.0 2.0 FeS04 or CuC12 4-36 1.32 6.1 5.0 2.0 CaC12 1-0 3.8 1-5 9.5 8-7 0.5 (b) Action of structure degradation (by means of high pressure) alb t 7.6 4.5 1-28 4.9 4.0 native 7.6 5.5 0.96 1.0 1-0 degraded * f/f~ was calculated from sedimentation and diffusion measurements. 7 The axial ratio a/b for an equivalent ellipsoid was calculated according to Svedberg and OncIey usingflfo for two values of hydration, w = 0 and w = 0.2.S . E. BRESLER 163 chemical intramolecular links stabilizes the structure which is not equilibrium when other molecular forces are considered. The native macromolecule of a protein can be strained because of chemical bridges connecting different parts of its main valency chain.TOPOGRAPHY OF THE ACIDIC AND BASIC GROUPS ARRANGEMENT ON THE SURFACE OF PROTEIN GLOBULAS The arrangement ef the functional groups on the surface of macromolecules must be studied by new methods. The investigation of the ionic sorption of dipolar ions by Samsonov, Kuznetsova and Ponomareva offered us such an oppor- tunity. The sorption of dipolar ions occurs in a peculiar way. Both oppositely charged groups, i.e. NH3* and COO-, being adjacent the corresponding amino acid and peptide are sorbed only on an H-cationites or OH-anionites. The sorp- tion of a dipolar ion on a cationite is not accompanied by the appearance of an hydrogen ion in solution because it simply neutralizes the second group of the dipolar i0n.7 The mechznism is expressed by the equation (for the exchange of a sulphoresin with a dipolar ion) : RSO3I-I + NH3+-R’-COO- + R-S03NH3-R’--COOH.Here the hydrogen ion of a sulpho group migrates from the resin to the carboxyl of the amino acid or peptide and screens it. So the sorption of the amine com- ponent of the dipolar ion suppresses the dissociation of its carboxylic component. The consequences of this mechanism are peculiar. (i) The sorption of dipolar ions occurs only on H-cationites. On the Na-, or any other salt form of cationite the sorption does not take place if both groups are near. This effect decreases when we take a dipeptide and tripeptide instead of an amino acid (table 2). TABLE 2.-sORPTION CAPACITY OF SULPHOCATIONITE FOR DIPOLAR IONS sorption capacity mg/g sorbed substance H form Na form glycine alanine gl ycyl-gl ycine leucyl-leucyl-glycine y-globulin serum albumin ovalbumin ACTH myogen insulin chymotrypsin 185 175 410 890 171 145 96 117 47 1 40 90 1.5 1 22 140 0 0 0 63 26 62 105 The sorption capzcity of different proteins on a sulphostyrene resin is repre- sented in the same table. The first three proteins behave apparently as an amino acid with adjacent acidic and basic groups; chymotrypsin is an example of a protein with distant oppositely charged groups.(ii) By electrostatic screening of the component of a dipolar ion similarly charged with the functional group of an ionite, the dipolar ion can be forced to sorb on the Na-form of a cationite. For this purpose we must increase the ionic strength of the solution, i.e.introduce some neutral salt. Fig. 3 represents a paradoxical increase of sorption of amino acids and some proteins on a cationite164 MOLECULAR FORCES AND AGGREGATION OF PROTEINS resin with the increase of the Na-ion concentration in solution. The capacity curve has a maximum at an ionic strength of approximately 0.15 and then decreases in consequence of the competition of the Na ions. The effect is observed only for substances weakly sorbed by the Na form but strongly by the H form of resins. (iii) The sorption of dipolar ions of amino acids and proteins with adjacent groups on the Na form of cationite may be intensified by addition of acetone to the medium (up to 40 %) ; the effect is due to suppression of carboxyl dissociation.t Cm FIG. 3.-The dependence of the ionic sorption of dipolar ions M (mg of sorbate per g of resin) on a Na form of a sulphostyrene resin on the ionic strength of the solution C 1, the curve for alanine ; 3, the curve for y-globulin. (changed by means of NaCl addition) ; C mole/l. 2, the curve for serum albumin ; THE AGGREGATION OF PROTEIN MACROMOLECULES The aggregation of macromolecules is one of the most characteristic features of proteins. The reactions of this type are most important in the formation of biological structures. Frenkel, Gorbacheva and Smirnova in our laboratory studied some cases of aggregation from the point of view of the molecular forces acting there. We paid attention to the initial stage of aggregation, i.e. the formation of oligomers of the basic macromolecule (in the papers of Barbu and Joly 9 were reported results of the studies of advanced aggregation leading to formation of big particles).The subject of aggregation as well as the internal equilibrium of protein globules includes all the main types of cohesion and repulsion forces which contribute to the general energy of interaction. But the local links formed by specific functional groups are the basis of aggregation. This point was proved by direct experiments. It is suggested also by the exceptional stability of the aggregates with a definite co-ordination number. The aggregation of serum albumin is especially inter- esting. Serum albumin aggregates under the influence of various agents breaking the internal structure of the macromolecule. When attacked by heat, urea or pressure in alkali medium the internal S-S bonds of the protein macromolecule can split forming sulphydryl groups (by reduction) or sulphydryl and carbonyl group (by hydrolysis).S.E. BRESLER 165 Secondary reactions are induced that might be the result of an exchange between sulphydryl and disulphide groups : 10 RlSH + R2-S-S-R3 -+ R2SH + R1--S-S-R3 or the reaction of the oxidative formation of S-S bonds from the sulphydryl groups or the reaction of sulphydryl groups and amino groups with aldehyde groups formed by the hydrolysis of the S-S bonds. Here is a number of possi- bilities for linking of macromolecules; the initial point is the scission of 18 S-S bonds existing in the macromolecule and the final effect is the formation of oligomeric globules and disorderly aggregation up to large macroscopic particles.The preservation of the proteins from aggregation in the presence of a number of specific agents is evidence of the chemical mechanism of aggregation. The action of monoiodacetate is most complete as the substance carboxymethylates SH as well as NH2 groups of the protein. The serum albumin treated by mono- iodacetate completely loses its aggregation ability.8 p-Chlormercuribenzoate is somewhat less effective as it blocks only the sulphydryl groups and does not affect the amino groups. Cysteine and thioglycolic acid at pH 9 reduce the disulphide bridges and prevent them from oxidation ; aggregation is also stopped. These facts concern the aggregation of serum albumin by denaturation in alkali media.In moderately acidic media, particularly near the isoelectric point, other groups (probably free NH2 groups) forming hydrogen bonds, are significant. Ascorbic acid (4 %) is the only effective stabilizer in this case. At acidic pH the disulphide bonds being not split, the ascorbic acid is supposed to affect the free amino groups of the protein. The chemical mechanism of stabilization appears already in the persistence of the ascorbic acid action after dialysis. The influence of ions of variable valency Fe2f and Cu2+ on the aggregation of serum albumin in acidic media is particularly interesting. These ions are catalysts of the exchange reaction 11 RI-S-S-R~ + R3-S-S-& + RI-S-S-R~ + Rz-S-S-R~ and accelerate very effectively the aggregation connected with disulphide groups.The bivalent ions of constant valency (Ca2+, Mg2f) are not active. Obviously the partly protecting action of complex-forming agents (ethylene diamintetra- acetic acid) on serum albumin against urea denaturation is a consequence of com- plexing with the transitional metals. The chemical nature of aggregation of serum albumin is apparent. In other cases studied, the nature of reacting groups is unknown but the local character of bonds is certain. The plant globulin, glycinine from soya-beans is capable of reversible aggregation depending on the pH and ionic strength of the solution.12 At pH lower than the isoelectric point of the protein (pH 5) an equi- librium of two components occurs-the main macromolecule with a sedimentation constant of S = 2.4 S and diffusion constant of D = 11-4 x 10-7 cm2/sec, hence the molecular weight amounts to M = 20,000 and asymmetry ajb = 1 (w = 0.2), and the symmetric hexamer with S = 8-0 S and M = 128,000, a/b = 1.Ap- parently the hexamer is a combination of six octahedrically packed glycinine globules. This is the only stable configuration apart from the monomer. The intermediate oligomers have not been observed, since at a pH higher than the iso- electric point aggregates form containing integral numbers of the stable hexamer. If the electrostatic interaction in solutions is diminished by means of screening the charges (high ionic strength) most of the globules are present as stable hexamers at any pH. This is an example of regular aggregation with a definite co-ordination number. The third case is connected with reversible aggregation of chymotrypsin. Two stable aggregates exist in this case-a dimer at pH 3-4 and a pentamer at pH 7.5 and an ionic strength lower than 0.01.The aggregation reaction takes place with the liberation of groups which are blocked in chymotrypsinogen. Hence166 MOLECULAR FORCES A N D AGGREGATION OF PROTEINS the local nature of bonds in aggregates is also clear. After deformation of the macromolecule by heat or pressure, the structure of the active centre of the enzyme is unchanged. The proteolysis stops reversibility and then the enzyme is cataly- tically active again. At the same time the reversible aggregation is abolished. After heating or by pressure increase, the equilibrium between the monomer and the dimer, which has taken place before the distortion of the internal structure of the protein, is frozen. 1 Bresler and Talmud, Compt. rend. U.S.S.R., 1944, 43, 326, 367. 2 Doty and Lundberg, Proc. Nat. h a d . Sci., 1957, 43, 213. Jen Tsi Yang and Doty, 3 Jacobsen and Linderstrom-Lang, Nature, 1941, 164,411. 4 Bresler, Biochim., U.S.S.R., 1949, 14, 180. 5 Linderstrom-Lang and Hvidt, Biophysica Biochim. Acta, 1954,14,547 ; 1955,16, 168. 6 Stauff, Kolloid-Z., 1956, 146, 48. 7 Samsonov and Kusnetsova, Compt. rend. U.S.S.R., 1957, 115, 351. 8 Gorbacheva, Bresler and Frenkel, Biochim., U.S.S.R., 1957, 22, 70. 9 Barbu and Joly, Favaday SOC. Discussions, 1953, 13, 77. 10 Hospelhorn and Jensen, J. Amer. Chem. Soc., 1954, 76, 2830. 11 Sanger, Nature, 1953, 171, 1025. 12 Kretovich, Smirnova and Frenkel, Biochim., U.S.S.R., 1956,21,842; 1958,23, 135. J. Amer. Chem. SOC., 1957, 79, 761. Krause and Linderstrom-Lang, Compt. rend. labor. Carlsberg, 1955, 29, 367, 386. Schoberl and Eck, Arm. Chem., 1936, 522, 97.

ISSN:0366-9033    

DOI:10.1039/DF9582500158

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

OPTICAL ROTATION AND INFRA-RED SPECTRA OF SOME POLYPEPTIDE AND PROTEIN FILMS BY A. ELLIOTT, W. E. HANBY AND B. R. MALCOLM Courtaulds Limited, Research Laboratory, Lower Cookham Road, Maidenhead, Berks Received 23rd January, 1958 Refinements of technique have enabled the dispersion of the optical rotation of solid films to be measured in the visible and near ultra-violet regions. Such measurements have been made for a series of polyalanines containing different proportions of D- and L- residues, for the sodium and potassium salts of poly-L-glutamic acid and also for some protein films. The infra-red spectra of these films have also been observed. The poly- alanine films show the characteristic dispersion of the a-helix, but the other materials do not. Since all these films have carbonyl absorption bands at ca.1660cm-1, it is evident that this frequency is associated with two or more configurations of the poly- peptide chain. With Bornbyx silk films cast from aqueous solution, a random coil appears possible. Infra-red spectra and X-ray diffraction patterns of polypeptides and fibrous proteins have chiefly been made on solid films, because the technique and inter- pretation is often more difficult if the material is in solution. On the other hand, measurements of optical rotation, which have recently yielded important informa- tion in connection with the a-helical configuration of polypeptides (Doty and Yang,l Moffitt and Yang,2 Elliott, Hanby and Malcolm,3 Yang and Doty,4 Moffitt, Fitts and Kirkwood,s Downie, Elliott, Hanby and Malcolm6) are much more easily made on liquids and recent published work has been restricted to solutions.Since the origins of the frequencies which characterize different polypeptide con- figurations have never received a satisfactory explanation and are empirical observations, it has seemed desirable to fill certain gaps in our knowledge by measuring the optical rotations of solid films on which observations of infra-red spectra and of X-ray diffraction patterns could be made. This has clarified a somewhat anomalous and unsatisfactory situation concerning the configuration of Bornbyx silk cast from an aqueous dispersion. EXPERIMENTAL METHODS FOR MEASURING OPTICAL ROTATION OF FILMS The peculiar difficulties which arise in measuring the optical rotation of films (as distinct from liquids) are caused by birefringence from strain, from local orientation of the polymer molecules or from birefringent foreign bodies in the film.Birefringence in the specimen produces an error in the se:ting of the analyzing polarizer and causes the field to be more or less brightly illuminated. This diminishes the sensitivity of a visual or a photo-electric polarimeter, since source fluctuations become troublesome when the field is bright. To prepare specimens which are sufficiently homogeneous, it is usually necessary to make a number of thin films rather than a few thick ones. In practice, reasonably accurate measurements can only be made with a suitable photo-electric polarimeter, and we have used one of our own design (Malcolm and Elliott 7).Since a small amount of specimen birefringence is usually present, it is desirable to rotate the specimens round the axis of the polarimeter tube in which they are housed. Initially this was done in steps by hand, and the average of a number of such readings was taken. Later a small motor was fitted to rotate the specimens at a high speed, and only one reading was then required. Careful filtering and drying in filtered air is needed to 167168 INFRA-RED SPECTRA OF POLYPEPTIDE AND PROTEIN FILMS produce clean films. When the number of films required is large (say twenty or more), or when the film thickness is irregular, it is advantageous to immerse the films in a suitable liquid to reduce reflection and distortion of the polarized beam. Edwards' silicone fluid 703 as used for diffusion pumps is suitable for some purposes.Its disadvantage lies in the presence of dissolved air, some of which may form a bubble which, owing to centri- fugal forces, remains on the axis of rotation just in the middk of the field of view. Fre- quent evacuation of the filled tube is needed to remove this air. Styrene is also suitable in some cases. Measurements of the dispersion of optical rotation were made initially with filtered light from a mercury lamp. Later a small monochromator was substituted for the filter, since it is difficult to get sufficient spectral purity and intensity with filters. The films were cast on small squares of thin sheet glass which were annealed after cutting. The weight per unit area was obtained by marking a small central square with a razor blade and subsequently weighing the film contained within this square.Since the total amount of this material was usually only a few milligrams, the principal source of error lies in this determination. It is not necessary to know the specific gravity of the polymer in order to calculate the specific rotation, for the latter quantity may be defined as the rotation produced by 100 g of material in a column of 1 sq. cm cross-section. The accuracy of measurement of optical rotation in films naturally falls short of that which can be obtained with solutions, for with the maximum number of films which can in practice be used, the rotation produced is small. In the work to be described, the angle of rotation varied from 0.04" to about 1.2" ; the polarimeter could be set to about 0-001".RESULTS POLY ALANINE Four polyalanines, copolymers containing different ratios of L- and D-alanine were examined as films. They were cast at room temperature from 5 % solutions in dichlor- acetic acid into an alcohol. To produce the best films, it was found desirable to use isobutyl alcohol for the D-polypeptide, n-propyl alcohol for the next two members of the series and methyl alcohol for the last one. D-residues were present in excess in these polymers, but to facilitate comparison with earlier results 3,6 they are recorded and plotted as if the L-component had been predominant. The films were well washed in ethyl or methyl alcohol and air-dried. Films of suitable thickness were cast on plates of thallium bromo-iodide under the same conditions to provide specimens for infra-red examination.Residue rotationsp corrected for an assumed refractive index of the polymer of 1.5 are shown in fig. 1 plotted against the fraction of L-residues for five different wavelengths. It will be seen that all the observed rotations for polyalanine films are negative, and by comparison with rotations observed for this polymer in solution it might be thought that the polyalanine films were in some form other than an a-helix. This, however, is not the case, for the dispersion is of the " anomalous " type associated with a-helices. As shown by Moffitt and Yang 2 the dispersion of the optical rotation produced by a-helices is of the form and on plotting [RVac](X2 - A@ against 1/(P - A@ a straight line of slope b& is ob- tained.The results here presented do not allow an independent determination of Ao, but when this constant is assumed to have the value 2120A, as found by Moffitt and Yang for poly-y-benzyl-L-glutamate, the curves shown in fig. 2 are obtained. For poly- alanine films whose L/(D + L) composition is 1.0, 0-9 and 0.8 the values of bo are re- spectively - 475, - 500 and - 505 deg. cm2 per decimole. These may be compared with - 630" for poly-y-benzyl-L-glutamate 2 and - 460" for polyleucine with L/(D + L) equal 6 to 0.875. When the optical rotation contains a contribution from an arrangement of a-helices, the plot of [R,,] against L/(D + L) gives a linear part of greater or less length which on extrapolation does not go through the origin.% 6 This is clearly the situation in fig.1. In this graph, for reasons discussed below, the linear relationship appears to be restricted to points for which L/(D + L) is 0-8 or more. Over this range, the linearity of the pIot and the approximate constancy of bo both show that one sense of helix is predominant. The plots extrapolate to intersect the [RvJ axis at points which represent the values of this quantity for a right-handed helix of meso composition. Although such extrapolationFIG. A. ELLIOTT, W. E. HANBY A N D B . R . MALCOLM 169 0.5 0.6 0-7 0.8 0.9 L D*L - 1 .-Optical rotations of solid films of polyalanine plotted against composition, different wavelengths : a, 5780 A ; b, 5461 8, ; c, 4358 A ; d, 4047 A ; e, 3663 FIG. 2. - Dispersion of optical rotation for various solid films.a, poly-L-alanine ; b, poly- alanine L/(D + L) = 0.90 ; c, polyalanine L/(D + L) = 0.80; d, polyalanine L/(D + L) = 0.67 ; e, Bom- byx mori silk (from aqueous solution) ; A Bornbyx mori silk (from dichloracetic acid) ; g , potassium salt of poly-L-glutamic acid ; h, lysozyme. I 08/(X2 -A02 ) 0 5 I 0 for A.170 INFRA-RED SPECTRA OF POLYPEPTIDE AND PROTEIN FILMS is very inexact, i t is interesting to note that the values are all positive, that they increase at first with diminishing wavelength and then become stationary as though passing through a maximum before diminishing again-exactly as has been observed with solutions of a-polypeptides. The value of bo for the meso polymer (subject of course to considerable uncertainty) is - 560".The points corresponding to L/(D + L) equal to 0.67 do not lie on the linear part of the curves, and on plotting [R,a,-](A' - A;) against 1/(P - A;) a graph of nearly zero slope is obtained (fig. 24. This shows that bo is nearly zero, and the anomalous dispersion of the helix is absent. The infra-red spectra of the polyalanine specimens shown in fig. 3 are in complete agreement with the deductions made from the optical rotation measurements. In the spectra of polymers of 1.0, 0.9 and 0.8 L/(D + L) composition, the bands at 893 and 906 cm-1, known to appear in or-poly-L-alanines are well-marked. The 906cm-1 band is particularly important, since it appears to be associated with a crystalline arrangement of or-helices. In the spectrum of polyalanine of 0.67 L/(D + L) composition, this band is absent.There is, however, a strong band at about 966 cm-1, which shows the presence - 9 0 0 I000 900 1000 1100 wave number, cm-1 FIG. 3.-Infra-red spectra of polyalanine films whose optical rotation is given in fig. 1. a, L/(D + L) = 1.0 ; d, L/(D + L) = 0-67. by L/(D + L) = 0.9 ; c, L/(D + L) = 0.8 ; of the /3 form of polyalanine. The carbonyl band in the spectrum of a thin film shows the sharp band at about 1630cm-1 characteristic of /I polypeptides, as well as a broad band centred at about 1655 cm-1. This coincides with the wave-number of the.carbony1 mode of simple synthetic polypeptides in the a-helix form, but as will be shown in the section on the structure of water-soluble silk, it also occurs in the spectrum of an amor- phous, possibly random coiled arrangement of polypeptide chains.Since the dispersion of the optical rotation shows conclusively that or-helices are absent in the film with 0-67 L/(D + L) compoyition, it appears likely that part of this polymer film is randomly coiled. It must be realized that the difference in behaviour between this polyalanine and the other three may not arise solely from the difference in the L/(D + L) ratio. The molecular weight may be different, and in addition it was found necessary to use a quick precipitant (methyl alcohol) to produce suitable films in the one case, whereas with the other three polymers a slower precipitant was used. These results may be compared with some which were obtained earlier for solutions of polyalanine.6 In chloroform containing 1 % dichloracetic acid the rotations are posi- tive for all values of L/(D + L) in excess of 0-5 and the form of the curve shows that helices are present.The fact that we find negative values for [Rva,] in the solid film shows the great effect which the environment has on the value of ao, an effect which has been observed (in a much smaller degree) by Yang and Doty in solutions of poly-y-benzyl-L-glutamate. 1 t is therefore particularly interesting that the values of bo should be similar to those found for other polypeptides. This confirms Moffitt and Yang's contention that bo is much more invariant than ao. The new results also extend the validity of a conclusion drawn from our earlier observations on poly-y-benzyl glutamate that solvent effects on the opticalA .ELLIOTT, W. E . HANBY AND B . R . MALCOLM 171 rotation of an a-helix of meso composition are small. Thus this quantity, given by the intercept on the y-axis of fig. 1 has the value 70" for light of wavelength 5780A, for the solid polymer. The corresponding value (for light of not greatly different wavelength 5893 A) for meso polyalanine in chloroform containing 1 % dichloracetic acid 6 is about 80". From comparison of the X-ray diffraction pattern of solid films of a-poly-L-alanine with the optical transform of the a-helix, it has been shown that the right-handed helix is the dominant one (Elliott and Malcolm 9. lo). It was at first believed that the negative value of bo found in measurements of optical dispersion in some synthetic polypeptides was evidence of this sense in solutions, but it appears doubtful whether this is a valid conc1usion.s However, the fact that similar values for bo are found in polyalanine fi!ms and in a number of solutions of L-synthetic polypeptides must surely mean that the right- handed form is the stable one for the L-enantiomorph. The linear part of the plot in fig.1 shows that for values of L/(D + L) of 0.8 and over, the left-handed form in solid polyalanine is not present to a significant extent. ALKALI SALTS OF POLY-L-GLUTAMIC ACID Films of the sodium and the potassium salts of poly-L-glutamic acid were made by dissolving the poly-acid in aqueous solution containing the stoichiometric amount of alkali, to give solutions of about 13 % concentration (w/v).The solutions, after filtering, were cast on glass plates at about 40°C in a dry air stream. The resulting films are very hygroscopic and it was found desirable to store them in a warm desiccator until a sufficient number had been made. They were then quickly transferred, still warm, to the polari- meter tube which contained phosphorus pentoxide. After measurement, the further manipulation for determining film thickness was done over a warm plate, and the films were well dried at 70°C before weighing in a closed bottle. Fig. 2(g) shows the results obtained for the potassium salt ; for the sodium salt almost identical rcsults were obtained. The small value of the slope of the line is indicative of the absence of any considerable fraction of the polymer in the a-helix configuration.Films of both polymer salts of suitable thickness for infra-red measurement were prepared under similar conditions to those described above, and measured. The carbonyl stretching mode was found at 1658 cm-1 in both cases, hence these results furnish a second example of a polypeptide (in this case of simple composition) in which a carbonyl band near 1660 cm-1 is not associ- ated with the helix form. WATER-SOLUBLE SILK Aqueous dispersions of Bombyx mori silk (made for instance by dissolving the silk in aqueous lithium bromide and dialysing out the salt) may be used to cast films which are soluble in water. Some years ago it was found that the spectra of these films have a carbonyl absorption band at 1660 cm-1 and since this same band is found in the spectra of synthetic a polypeptides it was suggested that water-soluble silk was in the cc form (Ambrose, Bamford, Elliott and Hanby 11).At this time, although the a-helix had been proposed by Pauling and Corey 12 its validity had not been established. The a-helix is now known to be a stable form of the synthetic polypeptides, but although good evidence of this form has been found in water-soluble Antherea nzylitta and Anaphe moloireyi silks (from X-ray diffraction rings in both materials, and from infra-red spectra in the former) it has not been found in films of water-soluble Bombyx silk (Elliott and Malcolm 139 14). The evidence against the a-helix form for this last silk was, however, negative in character, and it appeared that a more positive indication could be obtained from measurements of optical rotation. Dilute aqueous solutions of Boinbyx silk have been shown to be in a random coil form, and the a-helix form has been found in solutions in a suitable mixture of ethylene dichloride and dichloracetic acid.4 Films were cast by placing a few drops of the aqueous dispersion on gIass plates heated to about 70°C on a small rotating table, and drying in a stream of dried, filtered air.Such films are not usually completely soluble in water again after drying, and for good solubility the films should be dried on mercury at 100°C. This is not practicable for films required for optical rotation measurement. The infra-red spectra of silk films dried on a solid substrate at 70°C are almost identical with those of water-soluble films except that a very small shoulder at about 1630cm-1 shows the presence of a small amount of /3 material, which prevents the film from dispersing completely in water.The silk films were much too irregular in surface to use without an immersion medium, and for this purpose styrene was employed. The optical rotation was negative at all wavelengths, and dispersion was found to be of the normal type, which shows either that a-helices are not present or that there are equal numbers of right- and left-handed helices. In172 INFRA-RED SPECTRA OF POLYPEPTIDE AND PROTEIN FILMS view of what is known of the sense of the a-helix in simple polypeptides, this latter possi- bility can be excluded. The most sensitive test for the absence of " form " rotation is furnished by a plot of [Rvac](A* - A;) against 1/(A* - A;).This gives a line of zero slope as shown in fig. 2 when A0 is given the value 2120 A, hence the coefficient bo is also zero. This result is an important one, and shows convincingly that a carbonyl absorption band at 1660 cm-1 does not necessarily indicate an a-helix form, even in a material which can under some circumstances take this configuration. The zero value for bo shows absence of " form " optical rotation. Polypeptides in the extended /3 form may have zero boy as is shown by fig. 2(d) and (f). However, the amount of /3 material present in silk films prepared from aqueous solution is insignificant, and a random arrangement seems likely. In air-dried silk films, an appreciable amount of water is present and it is likely that many peptide groups are hydrogen-bonded to water molecules.However, on heating such silk films to 70°C in a closed cell containing P2O5 for a number of hours much of this water is removed. The infra-red spectrum shows no trace of a free NH band and the carbonyl absorption band remains at 1660 cm-1; hence it must be concluded that the hydrogen-bonding capacity of the silk is satisfied by intra- or inter-chain bonds. This presumably random-coiled form in the solid state must be considered as a stable form of silk, and one which may well be found in other dry proteins. Since it is now established that neither aqueous, dilute dispersions of Bornbyx mori silk nor the films cast under suitable conditions from such solutions contain a-helices, it is most unlikely that aqueous dispersions would have the helical form at some inter- mediate concentration.This conclusion casts considerable doubt on the claim to have established the existence of the a-helix as a major component in such dispersions by methods based on small-angle scattering (Kratky, Sekora and Pilz 15). LYSOZYME It was found possible to cast films of lysozyme from aqueous solution and to measure the dispersion of the optical rotation, and also the infra-red spectrum in the region of the carbonyl stretching mode on specimens prepared under as far as possible identical con- ditions. A rather broad band centred at 1660 cm-1 was observed, with no indication of any peak or shoulder at 1630cm-1. The plot of the optical rotation measurements is shown in fig.2(h), and gives no indication whatever of the kind of dispersion characteristic of a-helices. It is reasonable to conclude that the polypeptide chains in lysozyme films are neither in the extended (p) nor in the a-helix form. Lysozyme is capable of forming single crystals, but it by no means follows that in our films the polypeptide chains are in the same form as in crystals. CONCLUSION Since a polypeptide chain is capable of forming internal and external hydrogen bonds in a number of different folds, it is perhaps not surprising that polypeptide and protein films can be made in which neither the a-helix nor the extended /3 form are detectable, and this is what our experiments show. It is not known whether in such cases there is any degree of order in the films, and it may be that they are simply disordered states. Whatever the state of the polypeptide chains in these films, it is clear that they cannot be distinguished from a-helices by observations of the frequency of the C=O band alone. 1 Doty and Yang, J. Amer. Chem. SOC., 1956, 78,498. 2 Moffitt and Yang, Proc. Nat. Acad. Sci., 1956, 42, 596. 3 Elliott, Hanby and Malcolm, Nature, 1956, 178, 1170. 4 Yang and Doty, J. Amer. Chem. Soc., 1957, 79, 761. 5 Moffitt, Fitts and Kirkwood, Proc. Nat. Acad. Sci., 1957, 43, 723. 6 Downie, Elliott, Hanby and Malcolm, Proc. Roy. SOC. A, 1957, 242, 325. 7 Malcolm and Elliott, J. Sci. Instr., 1957, 34, 48. 8 Elliott, Proc. Roy. SOC. A, 1954, 226,408. 9 Elliott and Malcolm, Nature, 1956, 178, 912. 10 Elliott and Malcolm, to be published. 11 Ambrose, Bamford, Elliott and Hanby, Nature, 1951, 167, 264. 12 Pauling and Corey, Proc. Nat. Acad, Sci., 1951, 37, 235. 13 Elliott and Malcolm, Trans. Faruday SOC., 1956, 52, 528. 14 Elliott and Malcolm, Biochim. Biophys. Acta, 1956, 21,466. 15 Kratky, Sekora and Pilz, 2. Naturforsch., 1954, 9b, 803.

ISSN:0366-9033    

DOI:10.1039/DF9582500167

出版商:RSC    

年代:1958

数据来源: RSC