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.