Protein-Protein Interactions

Protein-ProteinInteractions

BY DAVID F. WAUGH Massachusetts Institute of Technology. Cambridge. Massachusetts

CONTENTS I . Introduction . . . . . . . . . . . . . . . . . . . . . ................................. I1. Group Interactions . . . . . . . . . . . . . . . .................... 1. Covalent Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Charged Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Page 326

331 333 4 . Van der Waals’ Forces (Electronic Dispersion Forces) . . . . . . . . . . . . . . . . . 334 ................................... 338 .................................... 338 7 Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 I11 The Native Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 341 1 Protein Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . A View of the Protein Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 3 . The Nature of the Internal Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 a . Electrostriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 b Enzymatic HydroIysis and Denaturation . . . . . . . . . . . . . . . . . . . . . . . . . 355 c . Side Chain Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 d . Spectroscopic Data . . . . . . . . . . ................................. 357 e . Size, Shape, Hydration . . . . . . ............................... 357 4 . The Internal Volume and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 IV . Specific Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 359 1. Antigen-Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................... 362 a . Urea and Other Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 3 . Plant Seed Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 4 . Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 a . Readily Reversible Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 b.’ Insulin Fibrils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 5 . Trypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 6 . Crystalline Soybean Trypsin Inhibitor. . . . . . . . . . . . . . . . . . . . . . . . . 379 7 . Pepsinogen and Pepsin . . . . . . . . . . . ....................... 379 8. Chymotrypsinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 9 Chymotrypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 10. Serum Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 a . Aggregation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 b . Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 c . Other Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 ............................

.

. .

.

.

325

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PROTEIN-PROTEIN INTERACTIONS

11. Hemoglobin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

. . . 392

.............................

b . Experiments with Incomplete Heme-Globin Dissociation. . . . . . . . . . . c. Experiments Involving a Heme-Globin Dissociation. . . . . . . . . . . . . . . . d . Urea., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................. ............................ ............................

a. Heat.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................

............................ .............................

394 395 396 398 398 399 400 400 401 402 403

...................

............................. V. Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................... ...................

408 414

....................

...................

............................. uring Agents.. . . . . . ............................ References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

422

428 429

I. INTRODUCTION Inevitably in a search for the features to be used in describing quantitatively the unique characteristics of different proteins, a n interest is developed in the interactions of proteins with each other, with ions, and with nonprotein materials such as lipids, nucleic acids, and carbohydrates. This is particularly so since the manipulations of covalent bonds in biological systems largely remain mediated by proteins. Increasingly in recent years, attention is being directed toward an analysis of protein-protein (and other) interactions in terms of the variety of bond types possible. These range from covalent bonds and electrostatic interactions to the weaker electronic van der Waals’ attractive forces characteristic of all atoms and groups of atoms. In view of the widespread interest in associating changes in physical, chemical, and biological properties with changes in the complexion of such forces, it is fruitful to examine the interactions of proteins, if not directly from this standpoint, at least with this objective in mind. For basic physical reasons, the interactions of proteins with each other in an aqueous environment may be divided into two categories: first, those which take place a t such distances that the average properties of the protein or its surface may be used in calculating interaction energies; and second, those in which the interacting molecules approach closely and thus bring into play local forces, the total of which depends t o a major extent on specific groups and group configurations.

DAVID F. WAUGH

327

Interactions a t distances may he further subdivided into those interactions, for example, which lead to a concentration dependence of osmotic pressure, sedimentation coefficient, etc. (Edsall, 1), and those long-range interactions which lead to more complex phenomena such as the separation of phases in suspensions of tobacco mosaic virus and bentonite (Oster, 2; Onsager, 3). In both cases one is concerned not so much with questions of molecularity or specific surface structure as with net charge, ionic strength, the dielectric constant of the medium, average electrostatic interactions, and long-range van der Waals’ forces. The addition of solvent alone is sufficient to alter rapidly the average distance of separation; thus without the intervention of an appreciable energy barrier. The frequency of occurrence of groups of particles which manifest themselves as an increase (or decrease) in the average concentration is calculated on the basis of statistical theory. Interactions of this type ( 4 , 5 , 6 ) are not of direct concern here. Where close approach and surface contact are an important part of interaction, a wide variety of results may be obtained. As in many salting out and crystallization procedures no noticeable physical or chemical change may accompany interaction. In other cases interaction may be limited as in association-dissociation phenomena. The conditions under which dissociation is observed may be such as to suggest that variations in electrostatic repulsion are mainly responsible. No general rule is immediately apparent, for dissociation may be favored by a shift in pH away from the isoelectric point and at the same time be favored by an increase in ionic strength. In other cases an interaction is observed only when the protein molecule is subject to a structural change which brings bonding groups into positions which allow them to interact. Structural changes of this type may be reversible or irreversible, and in the former case, reversibility may be observed most easily when the interaction velocity has been decreased t o a negligible level by the establishment of an energy barrier which prevents close approach, and by the use of low concentrations. Certain ephemeral interactions, particularly enzymatic reactions, generally lead to chemical or physical changes in one of the interactants to a much greater extent than the other. These are but a few instances. Discussions of many protein-protein interactions cannot be included here. They have been excluded for reasons of space limitation, because they are considered to require too extensive a treatment, or because it is judged the proteins involved have not yet been characterized sufficiently. Such systems include notably the muscle protein systems, the interactions of caseins, and the synthetic fibers which may be prepared by relatively drastic processes of coagulation, spinning, stretching, etc. The discussion will also be limited to corpuscular (globular) proteins, although clear connec-

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PROTEIN-PROTEIN INTERACTIONS

tions between these and fibrous proteins are indicated (Bear, 7; Waugli et al., 152; Barbu and Joly, 346). The wealth and complexity of information t o be obtained from a study of proteins suggest that other more complicated systems may be reserved for future analysis. For example, there can be isolated from tissues, combinations of protein with lipid, nucleic acid, or carbohydrate; combinations in which the interacting components are not only present in comparable amounts, but so far as the protein chemist is concerned the complex after isolation has the general characteristics of molecularity and stability. Experimentally, it has been found in these complexes that the components have been integrated with a structural precision and specificity which will not be easily duplicated by introducing the isolated components in systems in vitro. That this is so may be inferred from an examination of the difficulties which will attend the resynthesis from subunits of a riucleoproteiri such as tobacco mosaic virus or the plasma lipoprotein recently analyzed by Pedersen (8) and Oncley et al. (9). Although of enormous interest, these interactions of protein with other compounds, the complexes of which present evidence for specificity, will not be considered here. However, their stability must depend, to a large extent, on the same types of interactions as are observed in protein systems alone.

11. GROUPINTERACTIONS A brief inspection of the types of groups found to constitute the main and the side chains (Table 111) of a representative chain (-CONHCH-) group of proteins suggests that most of the known types of interactions may be expected to occur. Not only is this apparent, but the fact that the simplest of proteins, for example, lysozyme of molecular weight 14,000 t o 17,000 or insulin of molecular weight 6000 or 12,000, will contain a t least 50 to 140 amino acid residues, suggests that a given bond or interaction type will occur repeatedly throughout the structure of the complex molecule. The discussion which follows assumes that in the majority of cases proteins are examined in an aqueous environment and that only in exceptional cases, designed for specific studies, is water removed from the groups with which it can interact strongly. The bearing of group interactions on protein structure will be examined in a later section. The purpose of the present section is to enumerate such interactions and examine some of their properties. The covalent bond exists as such over a relatively short range of distance of separation of atomic centers, a range well below the sum of the van der Wads’ radii (Pauling, 10) characterizing the two interacting atoms. The other types of interactions (coulombic, hydrogen bonds, electronic van der

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DAVID F. WAWGH

Waals' forces, and other secondary valence forces) produce interaction over larger distances. The interaction energies corresponding to those forces which decrease with some high power of the distance will be negligible when the centers of the groups are separated by two or three times the sum of the van der Waals' radii (electronic van der Waals' forces). In other cases, such as coulombic interactions, the forces extend to distances considerably larger than those related to the size of the protein molecule. The fact that the protein molecule is large and may restrict the motion of numbers of charged groups may alter the extent of the interaction. The maximum interaction is obtained when the molecules or groups are in closest contact, a distance closely related again to the envelope of the van der Waals' radii of the constituent atoms. In general, the van der Waals' radii determine the shape of the group and the ways in which groups may be packed. Table IA lists van der Waals' radii. TABLEI A . V a n der Waals' Radii Atom or Group

Van der Waals' Radius

H C N 0 P S CHI

1.2 1.6 1.5 1.4 1.9 1.85 2.0

B . Covalent Bond Distances and Angles Bond

c-c c-0 C-N c=o C-H

c-0

C=N

d,(A.) 1.543 1.098 1.44 1.48 1.215 1.271 1.265

N-I3

1.014

0-H

0.96 1.81 1.334

c-s

H-S

Bond

c-c-c

C-N-C CN-H N-CN C-C-H

c=c-c

Angle

109" 28' 110" 108" 109" 28' 109" 28' 125" 16'

0

,/ -C '\ 0 c-s-c c-c-s

120" (all)

104" 109" 28'

330

PROTEIN-PROTEIN INTERACTIONS

1. Covalent Bonds Pauling (10) has treated extensively the properties of the covalent bond. Table IB lists the characteristics of a few important covalent bonds. D a ta have been taken from Pauling (10) and Huggins (11). The work of Fischer and of Hofmeister (see Low, 12) essentially showed that the most reasonable protein subunit was the amino acid. These units, held together by covalent bonds in polypeptide chains, introduce a repeating unit of -CO. CHR. NH-. As has been pointed out in the reference just cited, the occurrence of polypeptide chains in proteins has been established beyond any reasonable doubt. The mild conditions under which proteins are extracted and generally studied suggest that no extensive changes in the covalent bonds of the peptide link will occur. The high-energy barrier attending hydrolysis suggests that it may be assumed, in spite of the low energy associated with the introduction of an amino acid into the polypeptide chain and conversely the low energy associated with the disruption of such a bond, that the configurational changes preceding or attending certain types of protein-protein interactions are due to a redistribution of other bond types. The same argument would apply to those few situations where the extraction of water might form a covalent link between an a-amino or carboxyl group and a carboxyl or amino group on some side chain. In this connection the disulfide group of cystine, presumed able to join covalently two polypeptide chains, stands out as an exception often invoked to account for a number of situations in which protein molecules associate in stable configurations. The disulfide bond, of all covalent bonds in proteins, is most labile with respect to reducing agents and t o mild conditions of alkalinity. Reductions with a variety of agents can be carried out over a wide pH range (Herriott, 13), although most treatments are performed a t near neutral or slightly alkaline p H values. Even in the absence of reducing agents and when traces of atmospheric oxygen may well be present, it has frequently been assumed that breaking of disulfide bonds may occur with their subsequent reformation to accomplish intermolecular bonding. As will be seen later, such a rearrangement is expected only in specific instances. The labile SH and SS forms of sulfur in proteins may be augmented by another type of incorporation, as suggested by LinderstrGm-Lang and Jacobsen (14). 2-Methyl thiazoline (A) CHZ-S

CH,-SH

CHI-N-

C H*--NH co cH~ A

B

331

DAVID F. WAUOH

is unstable; under the influence of heat (60°C.) and acid it hydrolyzes to form a thiol group and a peptide bond, the latter capable of enzymatic cleavage (B). A similar shift may take place with serine as indicated by H2CCHzOH

I

R’-CO.NH.CH.

acid

0

\ /

___f

C0.NH.R

CH.CO.NH.R

I

alkali

HO

I

NH

R‘

LT

R’*CO*O.CHzCH(NHs+)CO*NH*R Elliott (15) finds that the major portion of serine in silk fibroin will undergo this rearrangement on being treated with concentrated acid. Earlier references are given here. Where the “acyl shift” proceeds to completion the chain length is increased and an unusual main chain bond, the ester bond, appears. The latter can be hydrolyzed easily. 2. Charged Groups

The interaction of two ions a and b with valences Z and charges 2.e and lead t o attraction or repulsion according to Coulombs’ law.

Zbe

where c is the dielectric constant of the intervening medium and Tab is the distance between centers. The change in potential energy is given by integrating this expression over the change in distance. The maximum interaction energy would be obtained by allowing the groups to approach from infinity t o the van der Waals’ contact distance. Pauling, Campbell, and Pressman (16) remark that the electrostatic attraction of a positive group such as a substituted ammonium ion and a negative group such as a carboxyl ion become significantly strong, with bond energy of 5 kcal. per mole or more, if the structure of the molecules containing the groups is such that they can come into juxtaposition. I n making calculations of the interaction energies of charged groups the . dielectric constant of the intervening medium (water) becomes of considerable importance. Ordinarily, water has a dielectric constant near 80. In the presence of a free charge, such as occur with the ionizing groups of proteins, the effective dielectric constant is decreased owing to the strong orientation of the water dipoles in the field around the charge. The dielectric constant becomes sensibly different from that of pure water at a

332

PROTEIN-PROTEIN INTERACTIONS

distance of approximately 15 A. from the center of the charged group. Pressman et aE. (17) use values obtained by approximating a function of Schwarzenbach (18), and given by e =

6r - 11.

(2)

This function is useful over the range of r between 5 and 10 A. The values so obtained are close to those calculated by Debye (see ref. 19) and used by Kossiakoff and Harker (20) in treating the dissociation constants of oxygen acids. Integration of Eq. 1, assuming that the groups approach from large distances, gives, per mole of interactions,

E = N Z. Za ez lo-'' eT

4.18

(3)

= 4.8 X 1 t 1 *e.s.u., t is in A., N = 6.03 X loz3,and e is constant. With rare exceptions, such as with phosphorylated proteins, the charged groups on proteins have unit valence. A further important consequence of the strong attractions between water dipoles and charged groups is a volume contraction on the part of the former. The contraction, termed electrostriction, may be as much as 18 to 20 cc. per mole of pairs of cationic and anionic groups (see refs. 1, 21). The water molecules closest to the charged groups show the greatest increase in density and associate with the ionic groups with the evolution of considerable heat. Dole and McLaren (22) calculate a liberated differential heat of -6 kcal. per mole water for collagen, although values for other proteins are in the range -3 to -4.5 kcal. per mole. These values refer to the first portions of water bound by the dried protein. Dole and McLaren point out that possibly the proline and hydroxy-proline residues separate protein layers to such an extent that the differential heat of hydration is large; that is, less heat has t o be supplied to separate the layers when water adsorption takes place, hence more is liberated. Such an effect is suggested also by the high heat data for regenerated cellulose. Edsall (1) and Cohn et al. (23) consider electrostriction in more detail. According to Pauling (24) the strongly bound water in proteins contributes one water molecule per polar group. The total water of hydration may be as high as two water molecules per residue. Salt linkages, i.e., interactions between unhydrated -COO- and -NH,+ groups, have occasionally been assumed t o occur. Jacobsen and Linderstrgm-Lang (25) present convincing evidence that in most cases they would not be expected to occur. One notable illustrative exception is the possi-

E will appear in kilocalories when e

333

DAVID F. WAUGH

TABLEI1 Lengths and Energies of Some Hydrogen Bonds Bond

Substance

OH...0 OH ...0 OH ...0 CH ...N NH ...N OH ...N NH ...F

HzO CHaOH (CHaC0OH)z (HCN)* NH3 Peptides NHS

Length, A. 2.76

3.38 2.79 2.63

Energy9 kcal. per mole

4.5 6.2 8.2 3.28 1.3 5.0

bility that water of hydration may be removed on the association of insulin monomers (see Insulin, Doty and Myers, 139). Other charge interactions are considered under long-range forces. 3. Hydrogen Bonds

A hydrogen bond results from the interaction of a hydrogen atom attached to one electronegative atom with the unshared electron pair of another electronegative atom. It is then essentially an electrostatic interaction between permanent dipoles. Pauling (10) has analyzed the occurrence of hydrogen bonds and summarized the evidence which shows them to be of great importance in stabilizing a variety of crystals. Table I1 lists a number of hydrogen bonds of possible biological importance, giving the bond distances andbond energies. It is apparent that there is an inverse correlation between bond energy and interatomic distance (10). Corey and Pauling (26) consider the hydrogen bond distances found in several amino acid and peptide crystals. They are interested in the bond as it exists between positively charged -NH3+ or =NH.;' groups and the oxygens of carboxyl or hydroxyl groups. The bond distances usually fall in the range 2.79 f 0.12 A. Rarely does the H----0 vector of the hydrogen bond deviate by more than 20" from the direction of the N-H bond. On the basis of the compressibility of the O-H----0 bond in ice, Pauling (27) estimates a strain energy of 0.1 kcal. per mole for a stretching or compression of the hydrogen bond by 0.09 A. It is also pointed out that changes of this order of magnitude may result from environmental changes such as electrostatic charge on the atoms involved or resonance effects. The formation of a hydrogen bond, for example, by the oxygen of an amide group increases the hydrogen bond forming power of the hydrogen atom of the same amide group. Pauling also estimates the strain energy of bending of the bond as 0.1

334

PROTEIN-PROTEIN INTERACTIONS

kcal. per mole for a change of 6" in the N-H and H----0directions (deviations from 180"). This estimate is based on the assumption that the strain energy of a lateral displacement is approximated by one half of the same displacement along the direction of the bond. Where a hydrogen bond is shielded from interaction with other hydrogen bond forming agents, for example, neighboring water in the case of proteins, an investment of the full bond energy would be required for dissociation. Where hydrogen bonds with water must first be broken in forming a hydrogen bond between two designated groups, the net change in energy is considerably decreased. In considering the serological properties of simple substances Pauling and Pressman (28) calculate a n energy difference of 0.4 t o 0.7 kcal. per mole for the hydrogen bond formed by the hapten with antibody and with water. The hydrogen bond is assumed to form between the azo nitrogen of an azo protein and an amino or similar group on the antibody.

4. V a n der Waals' Forces (Electronic Dispersion Forces) Whenever atoms, groups of atoms, or molecules are brought into proximity, there arises an attractive force which is the result of the polarization of each group in the rapidly fluctuating electrical field arising from the instantaneous configurations of the electrons and nuclei of adjacent groups. London (29) in 1930 obtained a general formula describing the energy of interaction of nonpolar molecules. Margenau (30) has examined a variety of van der Waals' forces (which he defines as those forces which give rise t o the constant a in the van der Waals' equation), including interactions between permanent dipoles, etc., as well as those between fluctuating dipoles referred to above. A useful approximation to London's general equation is

in which and aB are the polarizabilities of the two groups, I , and I , are the average energy differences for normal and excited states, and T A B is the distance between groups. I , and I, are approximately equal t o the ionization potentials of the molecules. The electron polarization is related to other quantities (31,Ch. 8) as

where R is the molar refraction, N is Avogadro's number, n is the refractive index (in theory measured at infinite wavelength but in practice use of the

335

DAVID F. WAUGH

sodium D line is sufficiently accurate), M is the molecular or group weight, and p the corresponding density. Pauling and Pressman note that ionization potentials for most atoms of concern in proteins are near 14.0 electron volts (28). Making appropriate substitutions gives R A R B

cal. per mole.

The limits of approach of atoms in molecules have been shown to be related to the distance from the atomic centers to the outer shells of electrons. As electronic shells are approximated and caused to overlap strong repulsive forces arise which vary with the inverse twelfth, thirteenth, or higher, power of the distance (31). A5 one group approaches another from a distance the energy of attraction evident from Eq. 6 is dominant. A t some distance, however, repulsive forces become appreciable. The net energy of attraction will then be less than that given by Eq. 6. At the equilibrium distance the attractive and repulsive forces will balance. Wave mechanics suggests that the repulsive energy should be of the form P(r) exp(-r/@),where P(r) is a polynomial functionof r and /3 is a constant for the given group. For many purposes the simple approximation B r n is used giving the expression

Pauling and Pressman (28) estimate the second term on the right t o be 0.30 to 0.50 of the first term. As a useful average they suggest 0.4, giving

In applying Eq. 8 to the interaction of group A on Protein A with group B on Protein B, allowance must be made for the fact that water, W, must first be removed from contact with A and B before they can interact. The net interaction is then

E = -23,000

('XB -RARw - RBRw -+ g) + f(HB),(9) 6

TAB6

TA W 6

rBW6

TWW

where H , is the energy of the water hydrogen bond and f is the number of new hydrogen bonds made when water is removed from A and B. As will be seen below, there is little doubt that, for example, hydrocarbon chains associate in aqueous solution with an energy corresponding to the heat of vaporization of the hydrocarbon. Such a n effect would not be predicted on the basis of differences in van der Waals' attractive energies

336

PROTEIN-PROTEIN INTERACTIONS

alone, since the van der Waals’ interactions between nonpolar groups themselves and between nonpolar groups and water would be quite similar. The high interaction energy is due to the formation of new water hydrogen bonds on the coalescence of the nonpolar chains which do not make hydrogen bonds. An estimate of the number of new hydrogen bonds may be made by calculating the amount of hydrocarbon-water interface lost during interaction. This will equal the amount of water-water interface formed, from which an estimate off may be made. Where differences in energy between the combination of a group A and B and A and B’ is sought, since van der Waals’ forces are additive, subtraction of interaction energies gives

If the distances r may be assumed essentially the same, Eq. 10 becomes

An examination a t this time of Table I11 reveals with the exception of collagen the variety of proteins listed has an occurrence frequency for nonpolar residues of 0.25 t o 0.49. The larger nonpolar residues include valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and tyrosine. The unique position of tyrosine will be discussed below. It appears fruitful therefore to examine the increase in association energy in an aqueous environment produced by one CH2 group per molecule. Debye (32), in an analysis of the critical micelle concentrations and the structures of the micelles in equilibrium, as found in detergent solutions, gives a “sketch” of the theory of the micelle. On the basis of previous evidence the micelle is considered to be a flat sandwich having the hydrocarbon tails oriented internally and a surface, consisting of polar groups, having a charge density determined by the space requirements of the hydrocarbon portion of the monomer. Energy is obtained when tails are removed from contact with water molecules and brought into contact with each other. At the same time energy is expended in approximating the charged end groups. Assuming the surface to be represented by a disc, Debye calculates that, in order to build a disc of n molecules, there must be supplied an electrical energy of W e = n3I2w,,

(12)

where we is a fundamental electrical energy. Since the forces between hydrocarbon tails are short range in character, the energy gained through

337

DAVID F. WAUGH

approximation is

,

W, = - nw,

(13)

where w, is a fundamental molecular (van der Waals') energy. The net energy W = W, Wmhas a minimum for a certain value of W = Woand n = no, work being required to increase or decrease the micelle size. Since dW/dn = 0, it follows that

+

and Wm Wo = -no -.

3

For the case of dodecyl amine hydrochloride, no was determined by light scattering to be 55, yielding w,/w, = 11.1. The free energy difference between the micelle and the equivalent number of free molecules is given by +RT In K . When the equilibrium concentration, Co , is expressed in mole fractions

K

=

CgnO-l

and

W O=

-no

W 9 X =(no - 1) kT In co.

3

Since no - 1 and noare nearly the same, it follows that W,

-3kT In co

.

(15)

For dodecylamine co = 1.311 X 1 e 2 mole per liter, giving W, = 25kT

and

W e = 2.2kT

(16)

or, at 300"K., the energy per mole is

NW, = 15 kcal. (17) This is compared with the heat of vaporization of the corresponding hydrocarbon, dodecane, which is 14.63 kcal. per mole. The energy of attraction per mole CH2groups is then 1.18 kcal. Debye makes a further calculation of W e ,assuming a disc covered with a constant density of electricity in a medium of dielectric constant e, as

338

PROTEIN-PROTElN INTERACTIONS

where A is the surface covered by one monomer and is taken as 27 A?. The dielectric constant is taken as the average of that between hydrocarbon and water. In this way We is calculated as 2.8kT to be compared with 2.2kT. Although there may be some doubt as t o the structure of the micelle as discussed by Halsey (33), the energy of hydrocarbon chain association is taken as approximately the heat of vaporization of the hydrocarbon as indicated above, namely, 1.18 kcal. per mole CH2 groups. According to Coulson and Davies (34) the interaction energies of conjugated groups of long-chain molecules vary not with r S but a t intermediate distances and where the groups are in close contact with some smaller power of the distance possibly as low as -3.5 (see also London, 35). 5 . Use

In 1945, Pauling and Pressman (28) developed and used Eq. 11 in accounting for the inhibition of antigen-antibody precipitation by free haptens. These authors give a number of values of R of use where interactions are to be calculated by this technique. Where the interactions of charged groups were likely, Pressman et al. (17) employed Eq. 3, using the free energy difference between the combinations of haptens with and without charges with antibodies to calculate an equilibrium distance between a positively charged group on the hapten and a negatively charged group presumed to be on the antibody. An equilibrium distance of 7.0 A. was obtained. These authors point out that this distance is interesting because it is close to the smallest distance, 4.9 A,, structurally possible for the trimethyl ammonium ion and the radius of the oxygen atom. Using essentially Eq. 3 and 10, Adams and Whittaker (36) have treated the bonds possibly involved in the relative affinities of plasma and erythrocyte cholinesterases for substrate malogs. The operation of a variety of bonds in < enzyme-small molecule substrate > interactions has been recognized by Neurath and collaborators (see Neurath and Schwert, 37) and Smith and collaborators (see Smith, Lumry, and Polglase, 38). 6. Forces at Long Range

Near the isoionic point in salt-free media Kirkwood and Shumaker (39) calculate that the fluctuations of mobile protons on a group of particles will affecteach other so that a net attractive force arises. This long-range attractive force decreases with the square of the distance. The ionic strength markedly affects the interaction. They point out, however, that the mutual interactions of charged groups could “freeze” a surface pattern which would, when spatial distribution is adequate, lead to a specificity of interaction.

DAVID F. WAUGH

339

The spontaneous two-phase formation by suspensions of tobacco mosaic virus, bentonite, etc., since its discovery, has excited interest from several standpoints, one of which concerns balance of forces responsible for its development. Evidently there must be an attractive force operating a t large distances, for the phase separation takes place a t concentrations near 2 % to 3 %. Also, a t relatively large distances the attractive force must be balanced by a long-range repulsive force. The equilibrium position of particles in the more concentrated phase can well be the result of a balance between repulsion due to the combined electrostatic effects a t smaller than equiIibrium distances and osmotic effects due t o the overlapping of ion atmospheres, and attraction due to long-range van der Waals’ forces or electrostatic attraction at larger than equilibrium distances. Although long-range effects are of considerable theoretical interest and are of importance in understanding the properties of specific systems, they are apparently of minor importance in determining the properties of systems dependent upon short-range interactions. These constitute the majority of systems to be considered here. For more extended discussion of longrange forces the reader is referred to Oster (2) and Onsager (3).

7. Urea Hopkins (206) in studies of the denaturing effectsof urea refers to earlier uses of high concentrations of this material. As will be evident from an examination of specific proteins, urea has been used extensively in investigation on proteins. It is of considerable importance to understand the multiple effects of this reagent. Mirsky and Pauling (57) in a consideration of protein denaturation pointed out that urea would be expected to rupture hydrogen bonds, stating “alcohol, urea, and salicylate are well known hydrogen bond forming substances. They form H-bonds with the protein side chains.” I n all considerations of protein denaturation, urea is assumed to act almost entirely as an H-bond breaking agent, although, as Pauling has pointed out more recently (27), the ability of urea to act in this fashion has not been tested adequately. Meyer and Klemm (40) state that a concentrated urea solution easily dissolves diketopiperasine, which is only slightly soluble in water. A number of materials such as the amino acids, thyroxine and cystine (Cohn et al., 41; Winnek and Schmidt, 42), and tartrates but not oxalate (Pedersen, 43) have increased solubilities in the presence of urea, which are qualitatively accounted for by the increased dielectric constant. The first indications that the effects of urea might be more subtle than simple hydrogen bond breaking came in a German patent application in 1940 (see Bengen and Schlenk, 44), where it was observed that urea could form crystalline complexes (adducts) with straight-chain hydrocarbons.

340

PROTEIN-PROTEIN INTERACTIONS

Since that time urea adducts have received considerable attention from Schlenk (45),Smith (46),Redlich et al. (47),and Zimmerchied et al. (48). The complexes form when the hydrocarbon is introduced into aqueous solutions of urea between about 5 M concentration and saturation, a concentration range frequently used with proteins. The urea molecules form a hydrogen-bonded, cagelike structure which extends in space in such a way as to utilize all of the hydrogen bond forming groups on each urea molecule. The urea molecules thus form a hexagonal network, the continuous channels defined by the urea hexagons being large enough to accommodate an extended normal hydrocarbon chain. The molal ratio of urea to reactant, M , for normal hydrocarbons as given by (46)is

0.688 (n - 1)

+ 2.181,

where n is the number of C atoms in the chain. For present purposes we are interested mainly in the nature and strength of the interaction between urea and hydrocarbon. Schlenk (45)has found that the heat of formation of the hydrocarbon-urea complex is 0.800 to 1.300 kcal. per mole urea. To explain the complex, an interaction energy of 2.8 kcal. per CH2 group is calculated by Schlenk to occur between urea and hydrocarbon. This would require unusually strong electronic van der Waals’ interaction. Smith (46)notes that half of the urea hydrogen bonds in the complex are slightly shorter than normal, and since there is an inverse relationship between bond strength and bond length, part of the heat of formation and stability of the complex could be accounted for in this way. As Smith notes, however, the shortening of the hydrogen bond in the complex is just beyond the experimental error, and the differences in packing between urea crystals, hydrocarbon, and urea-hydrocarbon crystals suggest a net increase in utilization of van der Waals’ interaction. Undoubtedly, both play an important part in establishing the complex. From the standpoint of the effects of urea on proteins, of most importance are the facts that complex formation is possible and that the complexes have stability. It must be remembered that the complexes of hydrocarbons with urea involve compounds having sufficiently long unbranched hydrocarbon chains. Small branched chains and aromatic rings do not form crystalline complexes with urea-a circumstance which is made the basis of hydrocarbon fractionation. The nonpolar side chains of proteins fall largely in the groups just named, which might suggest that little interaction is possible. It should be remembered, however, that the side chains are of varied character and that they are held in close proximity. The complexes of hydrocarbon with urea can only be taken as suggesting that the nonpolar residues may be involved. Further information is clearly necessary. Thiourea is of considerable interest in comparison with urea. Consider-

DAVID F. WAUGH

-

341

ablyless soluble than urea ( 9.5 g. per 100 ml. at 20°C.), this compound is capable of denaturing proteins, as shown by the appearance of insolubility. Hopkins (206), who describes denaturation by thiourea, observes that it displays little of the dispersive power that is characteristic of urea. At 17"C., 9 % (saturated) thiourea produces a precipitation of 4 % egg albumin solution which increases with time and is complete in about 48 hours. More recent uses of thiourea with protein are given by Chernyak and Pasynskii (49). It should be noted that a 9 % concentration of thiourea denatures proteins. The same concentration of urea often not only does not denature but enhances solubility, i.e., it displays a dispersive action. Thiourea differs from urea not only in solubility and in the fact that the S of thiourea should form much weaker hydrogen bonds than the 0 of urea (lo), but also in the complexes it forms with hydrocarbons. Whereas urea complexes with straight-chain hydrocarbons, thiourea complexes with small branched hydrocarbons and cycloaliphatic compounds. These compounds have recently been studied by Fetterly (50), Redlich et ul. (51), and Smith (52). As Redlich el al. point out, the stability of thioureahydrocarbon complexes is low compared to those of urea complexes, the heat of formation is low (for example, 4.4 kcal. per mole for 2,2-dimethylbutane), and there appears to be no relationship between the heat of formation, the free energy, and the molal ratio. Smith (52) suggests that the thioureahydrocarbon complexes are similar in structure to urea-hydrocarbon complexes. The presence of the larger S-atom results in a larger channel which can accommodate branched chains. In view of the prevalence of branched hydrocarbon side chains in proteins (Table 111)i t would be significant that (if generally the case) thiourea can denature a t a much lower concentration than urea. Cramer (53) and Zimmershied et al. (48) have recently reviewed the adducts formed by urea and thiourea. The interactions of urea (and thiourea) with proteins must be complex. It is quite possible that structural modifications are produced by a number of urea molecules making hydrogen bonds and van der Waals' interactions with each other and with side chain and main chain groups. Cagelike structures penetrating the internal volume (see below) and extending over the surface of the protein molecule might well result. 111. THENATIVE PROTEIN 1. Protein Composition

Before proceeding with a consideration of protein structure, it is reasonable t o examine in a series of proteins the occurrence of side chain groups. A few proteins are tabulated in Table 111. Most of the data given have been calculated from the amino acid compositions as tabulated by Tristram

TABLEI11 Part I Certain Ckavacteristics of Specific Proteins

-

8

Calf Human Bovine ChymoRabbit Ribotropo- nucle- Rabbit liver fibrin- serum trypsin- Edestin Collagen myosin ase myosin histone ogen albumin ogen .

1. M O ~ . m7.t. x 10-3 2. G r a m X 10-3 3. End groups 4. H per unit" 5. SH per u n i t " 6. SS. per unit 7. Av. CH, per 8. 8 . NPS per unitd 9. XPS, frequencye 10. Av. CHI per NPS 11. Max. -I- side chain charge 12. M a x . - side chain charge 13. Tyrosine per unit 14. Hisbidine per unit 15. Lysine pef unit 16. (GIycine, alanine, serine, threonine, methione'i per unit

100 1073 0 0 1.33 223 0.21 4.1 85 83 5.5 4.8 30.7 526.6 ~.

100 0 842

15

122 0.7 3.15 4 2.15 2.16 202 28 0.24 0.23 4.3 4.4 158 20 245 15 7 17.2 5.5 4 107.4 11 183.7 36

bumin

. _

.

.

Oval-

100 750 -

5.65 2.28

m

0.26

4.4 139 150 18.8 15.5 81.4 205.2

15.5

loo

845 3.3 9.5 2.31 2.29 35 250 0.29 0.30 4.1 4.6 27 121 12 125 3 30.4 2 17.1 12 63.0 -10 251.8

I19

_._.._...__________I.~..~~

69

24

46

44

.-

Horse myoglobin 16

n 1 588 215 401 387 113 1 3 2 3 2 14 3.5 2 1 0 2.39 2.37 2.33 2.29 2.37 I68 70 124 152 .I:, 0.29 0.31 0.34 0.29 0.33 1.8 4.3 4.3 4.3 4.4 101 19 64 42 29 121 43 76 65 23 3.5 12 9 2 19 I 0 18 2 8 60 13 8 20 15 126 85 92 122 43

-

~-

.. --

Part I1 Certain Characteristics of Specijic Proteins ~

@-Lacto- Sheep FSH globulin 1. M O ~wt. . x 10-3 2. Grams X 3. End groups 4. R per unitD 5. SH per unitb 6. SS per unit 7. Av. CH, per Sc 8. N P S per unitd 9. NPS, frequencye 10. Av. CH, per NPS 11. Max. side chain charge 12. Max. - side chain charge 13. Tyrosine per unit 14. Histidine per unit 15. Lysine per unit 16. (Glycine, alanine, serine, threonine, methionine) per unit

+

37 3 320 3 3.5 2.41 103 0.32 4.2 39

67 432

-

2.44 152 0.35 4.3 88

Human Rat OX sarcoma growth yhistone hormone globulin 15.6 132 0 . 0 2.47 49 0.37 4.1 30

47.3 344 2 2.57 127 0.37 4.5 56

156 839 5.8 9.95 2.54 324 0.39 4.3 100

Human Calf serum thymus histone a-Casein albumin

_

15.5 120 0 0 2.59 46 0.38 4.2 23

_

100

_

~

69

Horse hemoglobin

~

_

68 6 541 3 1.25 2.46 195 0.36 4.4 88

OX insulin

~

~

12 4 103

824 1.8 2.59 308 0.37 4.4 104

526 1 16 2.61 193 0.37 4.3 99 104

67

17

18 16 58 70

11 36 38 165.5

8 4 2 21

53

133

4

58

111

15

147

8 4 29 75

14 16 52 71

2 2 13 41

14 8 23 83

37.6 16.1 55.5 242.7

3 2 11 34

44.8 18.7 61 .O 190.9

0

6 2.52 42 0.43 4.3 8

The data given above have been calculated mainly from the tables of amino acid compositions compiled by Tristram (54). R

=

residue.

* The values for human and bovine albumin have been taken from Edelhoch et al. (173) and Edsall et al. (174) S = side chain.

e

NPS = larger nonpolar side chainb), which include valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and tyrosine. The occurrence frequency is the number of larger nonpdar side chains per unit divided by the total number of side chains per unit

__ p-Casein 100 857 2.77 404 0.47 4.1 84 98 17.7

20.0

44.6 173.8

344

PROTEIN-PROTEIN INTERACTIONS

TABLE IV Characteristics of Amino Acids

Amino acid Aspartic acid Asp. (amide) Glutamic acid Glu. (amide) Arginine Histidine Lysine Cystine W Cysteine Tyrosine

Length Vol- Equivmax., ume, alent A. A.8 CH2

5.0 5.1 6.3 6.4 8.8 6.5 7.7 2.9 4.3 7.7

58.4 65.4 85.5 92.5 125.7 89.0 121 .o 52.8 57.9 138.8

0 1

1

2 2 4 3 1 1 6

Amino acid Glycine Alanine Serine Threonine Methionine Valine Leucine Isoleucine Phenylalanine Tryptophene

Length Vol- Equivmax., ume, alent A. A.3 CHZ

1.5 2.8 3.8 4.0 6.9 4.0 5.3 5.3 6.9 8.1

5.1 32.2 36.0 63.1 112.1 86.3 113.4 113.4 136.6 175.5

0 1 1

2 1 3 4 4 6 8

-

(54) or have been taken from his tables. The first two lines give the unit considered, either the molecular weight or the unit of lo6 g. The third and fourth lines tabulate the number of amino end groups and number of residues per unit. The fifth and sixth lines give a similar tabulation for SH and SS groups. Lines 7, 8, 9, and 10 relate to the nonpolar characteristics of the protein as obtained from a consideration of the side chains. To obtain these data each side chain was assigned an equivalent value in terms of CH2 groups. These are tabulated for various side chains in Table IV. Line 7 of Table I11 gives the average equivalent number of CH2 groups per residue using all amino acids. Line 8 gives the number of larger nonpolar side chains per unit. These include side chains contributed by valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and tyrosine. Line 9 tabulates the average frequency with which the larger nonpolar side chains occur in the polypeptide chain, and line 10 gives the average CH2 equivalent of the larger nonpolar side chains. Lines 11 and 12 of Table I11 give the maximum charge, positive and negative, which can be contributed per unit by the side chain composition. These calculations include tyrosine, SH, and histidine groups and have been corrected for amide groups. A rather wide variation in charge is observed. Lines 13, 14, and 15 indicate per unit the numbers of side chains such as tyrosine, histidine, and lysine which have polar and nonpolar character. Line 16 gives the sum of the smaller side chains including glycine, alanine, serine, threonine, and methionine. Of these serine and threonine have polar character. It is a t once apparent from Table 111that a wide variation in structure

DAVID F. WAUGH

345

occurs, even as calculated according to the averaging techniques used. At this time, attention is directed specifically to the frequency with which the nonpolar side chains occur, a frequency which varies from 0.24 for tropomyosin to 0.47 for /3-casein. The proteins considered have been arranged according to the frequency of occurrence of nonpolar residues. Amides of glutamic and aspartic acid have not been included in the nonpolar category, mainly because of their hydrogen bond forming characteristics. Tyrosine has been included in the nonpolar category for two reasons. First, over a range in pH up to and beyond neutrality the side chain remains uncharged and will, as indicated by the properties of phenol, have considerable nonpolar character. Second, a portion of the tyrosine side chains is found to be unreactive in the native protein-a circumstance which suggests that the side chains be placed along with nonpolar residues in the internal volume (see below). In a section which follows in which specific protein-protein interactions are examined, it will be clear that the starting materials in all cases will be proteins prepared by a “mild” set of physical and chemical treatments. The products of such treatments are accepted generally as “native” proteins. It should be remembered that few physical or chemical measurements are made on proteins in vivo, a preparation suitable for examination usually being extracted from biological materials. Taylor (55) has examined the wealth of preparative procedures available for protein fractionation. It has always been apparent that the native state is not to be associated with the in vivo state without critical examination. Just as important as the preparative procedure in determining the acceptance of a protein as native are the results of techniques designed to establish molecularity. The term molecularity generally implies a uniformity of structure from one particle to the next. As Edsall (1) has pointed out, however, the size and complexity of the proteins suggest that limits be placed on uniformity rather than that one expect a complete structural coincidence. Indeed a t the present time a protein preparation homogeneous by a variety of criteria may actually consist of an association of subunits or of a family of molecular structures all readily interchangeable with each other. No one protein has been prepared which will satisfy in a strict sense all criteria for molecularity. Acceptance of a preparative procedure as leading to a native protein depends, as one would expect, on the sum total of evidence regarding the possible chemical or physical changes which might be induced by the separate steps of the procedure and on the lability of the resulting protein. Although there is considerable evidence that many large native protein molecules may be dissociated into subunits, there seems little reason to doubt that the smallest subunits which can be produced reversibly without

346

PROTEIN-PROTEIN INTERACTIONS

the rupture of covalent bonds may legitimately be considered molecules (see also Edsall, ref. 1). There exists the possibility (indeed in cases it seems highly probable) that two or more kinds of subunit molecules may he produced, all of which must be considered molecules whether or not they have been fractionated as yet into pure components. There are many strong reasons which suggest that, within as yet unknown limits generally believed to be narrow, protein particles conform to the definition of molecules. It is well known that any demonstrable physical-chemical alterations lead to polydispersity, loss of biological activity, crystallizability, etc. Certainly, in the theory of structure and denaturation proposed by Wu (56) molecularity wasI assumed. More recently Mirsky and Pauling (57) have defined denaturation, on the basis of the changes in properties produced, as the loss of the uniquely defined configuration. The universal occurrence of the peptide chain in proteins is now well accepted (see Low, ref. 12, for a summary of evidence). Each amino acid contributes, possibly to a major extent in a structurally uniform manner, the group of atoms COCHNH to the backbone chain plus any one of approximately 22 different side chain groups bonded with the central C atom. In certain proteins a single backbone chain may account for all of the amino acid residues (see Table 111). More frequently the atoms of the main chain are grouped into several chains having terminal a-amino and carboxyl groups. In all corpuscular proteins of any size there can be little doubt that the polypeptide chains, extended, would be consideIably longer than the lengths of the protein molecules. Therefore, the polypeptide chains must be coiled back upon themselves. Covalent bonding between main chains is thought largely to be limited to the disulfide bonds of cystine, since, with the exception of the possibility that terminal carboxyl or amino groups in a few instances may be covalently bonded t o side chain groups, the terminal groups and all ionizing side chain groups generally appear titratable and after mild unfolding quantitatively available to reagents with which they can combine chemically (58, 59, 60). The stabilization of the coiled structure of the main chain is then largely the result of the so-called weak interactions treated in Section I1 under “Group Interations,” namely electrostatic interactions, hydrogen bonds, and van der Waals’ forces. It seems clear that structural integration a t least in local regions of the native molecule must be such that the protein can manifest specific biological activity as, for example, is displayed in enzymatic action. In all probability, in different regions of the protein molecule, the side chain interactions vary in character and intensity. When taken together the properties of proteins suggest that each mole-

DAVID F. WAUGH

347

cule will have associated with it regions (or volumes) which can be viewed as surface, i.e., the regions responsible for intermolecular interaction (the surface volume), and internal regions (the internal volume) in which through close fitting side chain interactions are at a maximum. For reasons to be given below one would expect the specific nature of the side chain interactions in this “internal volume” to make a major contribution to protein stability. In this latter volume one would expect, for example, to find “masked” groups. Depending on the environmental conditions one would also expect the complexion of groups making up the surface and the internal volumes to change as one progresses from one member to the next of the group of structures which collectively describe the native protein or the denatured protein. 2. A View of the Protein Molecule The author is indebted to Mr. David A. Yphantis for his co-operation in preparing particularly this section and the section on urea. Within recent years studies of the X-ray diffraction patterns given by protein crystals and concentrated protein solutions have provided evidence which suggests that, within the protein molecule, there often occur nearly parallel rodlike regions of high electron density. Data for individual proteins are given for methemoglobin by Perutz (61) and Bragg, Howells, and Perutz (62); for myoglobin by Kendrew (63); for insulin by Low (64); for ribonuclease by Carlisle and Scouloudi (65) and by Carlisle, Scouloudi, and Spier (66); and for albumin by Riley and Arndt (67). Low (12) has recently examined critically the structure problem. The diverse nature of the polypeptide side chains suggests that they do not contribute materially to the diffraction pattern. Conversely, since the main chain presents repeating unit of six atoms (COCHNH) which can be arranged in a number of geometrical arrays having different repeating periods, it has been assumed that the characteristics of the X-ray diffraction patterns are due largely to the arrangement of the main chain. The most acceptable proposed arrangement of the main chain is that of a helix, a configuration proposed some years ago by Taylor (68) and by Huggins (69). More recently Pauling, Corey, and Branson have proposed a helical structure, the a-helix (70, 71, 72), which they believe is the most suitable arrangement for several corpuscular proteins. The a-helix is a result of the combination of (1) planarity of the group of atoms (CONH) as found i n a series of peptides (26, 73) and (2) stabilization through the formation of a maximal number of hydrogen bonds between GO and NH groups separated by intervening peptide groups along the main chain. The average hydrogen bond distance (2.79 A.) and bond directions for the interacting groups (CO and NH) are preserved within narrow limits. Of the recently pro-

348

PROTEIN-PROTEIN INTERACTIONS

b

a

FIG.1. The a-helix of L. Pauling, R. B. Corey and H. R. Branson (ref. 70, 71, 72) is shown in longitudinal aspect (a) and in plan view (b). The helix has 3.7 residues per turn, a translation of 1.7 A per residue, and 13 atoms in the hydrogen bonded ring. The dotted lines between CO and NH groups represent hydrogen bonds; one per amino acid residue. The density of t h e a-helix, not including side chains, is 1.69 (see text).

DAVID F. WAUGH

349

posed helices several offer possibilities, since within limits they can approximate the residue volume (density) of proteins. Three will be discussed here. Donohue (74) has recently described others. The first of these is the a-helix shown in Fig. 1 in longitudinal arid plane view. There are 3.7 amino acids per complete turn and a translation of 1.7 A. prr residue or a helix pitch of 5.44 A. There are 13 atoms in the hydrogen bonded ring, the CONH groups being almost parallel to the axis of the helix. The polypeptide chain plan used in the a-helix is shown in Fig. 2. The second, the ?r-helix of Low and Baybutt (75), is structurally similar to the a-helix. There are 4.4 residues per turn and a translation of 1.14 A. per residue or a helical pitch of 5 A. Pauling (72) suggests that the ?r-helix is less stable by 1 kcal. per mole residue than the a-helix, since it has a hole about 1 A. in diameter running down the center. The third, an 11 atom ring model, has been proposed by Huggins (76, 77). In this helix the CONH group is not coplanar. Although such a helix, in combination with the a-helix, would provide considerable structural flexibility, it is not accepted by Pauling and Corey (73) on the basis that the nonplanarity of the CONH group introduces a n unstability of 6 kcal. per mole residue. An understanding of the wide variety of interactions of protein molecules eventually requires a detailed analysis of the complete structures of the molecules involved. The author has felt it reasonable t o examine

-

/

'*

N h

r;

FIG.2. Polypeptide chain plan.

350

PROTEIN-PROTEIN INTERACTIONS

at this time, on the basis of available information concerning amino acid composition and proposed structures, the general problem of surface structure versus internal structure. The relative occurrence of possible bonding groups in the protein surface or in the protein “interior” is a t the basis, for example, of the alterations in protein-protein interaction observed during chemical or physical treatment. A preliminary examination of the significance of the terms surface structure and internal structure is, therefore, considered to be of importance. I n the discussion which follows the a-helix will be taken as an appropriate model. Caution has been advised (Wrinch, 78; Bamford et al., 79, 80) in accepting any structure so far proposed for corpuscular proteins. It may turn out that the a-helix describes the actual structure a t corpuscular proteins only in part. Whether or not the a-helix will turn out to describe exactly the structures of solubIe proteins, its current general acceptance (Riley and Arndt, 67; Pauling and Corey, 70, 71, 72; Linderstrom-Lang, 81; Kendrew, 82) prompts us to approximate the structures of proteins on this basis. To this end let us examine first a four-chain molecule. In this protein (insulin), as in others examined (see above), the center-to-center distance of rods of high electron density is 10 A., the rods in crystals occurring in eases in hexagonal or linear array. Figure 3A depicts the general situation on projection on a plane perpendicular to the axis of the a-helix. In Figure 3A solid-line circles have been inscribed around each helix center. We have chosen the areas of these circles to be equivalent to the projected area of the atoms of the main chain of the 3.6 residue per turn cr-helix as indicated in Fig. 4. It is assumed that the average area per turn is approximated by completing the inner-polygon by a line joining the center of the C atom of residue 1 with the projection of the corresponding atom of residue 3. The outer figure includes the assumed average van der Waals’ radius of

-

A FIa. 3. The internal helix and surface components of

B a four helix

protein: insulin.

DAVID F. WAUGH

351

1.4 A. Like the inner polygon, the outer figure is a projection on a plane perpendicular to the helix axis. In addition, the projected area is to be slightly increased by the hydrogen atoms joined to the main chain carbon atoms. These atoms increase the area as drawn in Fig. 4 by about 7%. The projected area thus formed is 36.7 A.2, eyuivaleiit to a circle of radius 3.41 A. The helix volume per turn is then 198 A.3. With this information and the respective atomic weights, the density of the main chain helix is calculated to be 1.69. Traube (83), Cohn et al. (23), and Cohn and Edsall (21, p. 157 et seq., p. 371 et seq.) give the volumes occupied by the various groups found in proteins. According to these figures the molal volume of the COCHNH group of atoms is 54.9 A.3. The volume for a 3.6 residue helix being 198 A.3, this leads again to a density of 1.69. The light circles in Fig. 3A have been drawn to conform to the average extended side chain length obtained from the amino acid composition of insulin. Table IV lists amino acids, maximum side chain lengths (including one half of the CC bond distance and the van der Waals’ radii of the terminal atoms), side chain volumes, and the number of CH2 groups to which the side chain is felt to be equivalent in nonpolar van der Waals’ interactions. Figure 3A allows us to make a preliminary differentiation between internal volume and surface structure. The wavy lines suggest planes of demarcation, which are drawn on the basis that an interaction between molecules is likely to involve mainly residues lying outside of this region. The average volume per turn not occupied by the main chain lying within the wavy lines of Fig. 3A is 611 A.3. This represents the internal volume for this particular model. Since the average side chain volume for insulin is 80 A.3 (Table V), this internal volume can accommodate 7.6 average residues. Figure 3B illustrates the assumptions which have been used in estimating

FIQ.4. Projection of a single turn of the a-helix on a plane perpendicular to the helix axis. Outer figure includes van der Waals’ radii.

352

PROTEIN-PROTEIN INTERACTIONS

TABLE V T h e Internal Volume Bovine IliboHorse myo- Bovine seruin nucleglobin insulin albumin ase 1. Average side chain length, A. 2. Average side chain volume, A.8 3. Average nonpolar side chain volume, A .a 4. Average neutral side chain volume, A.8 5. Internal volume per turn, A.3 6. Internal vol./av. side chain vol.

Probability that Average Side Chain 7. a . Av. max. side chain length 8. b. Half av. max. side chain length 9. c . Approximate min. length

IIorse hemoglobin

5.2 81 104

4.9 80 105

5.3 84 102

5.3 81 107

4.9 79 102

33

36

42

52

33

457 5.6

611 7.6

902 11.2

Will Occur in Internal Volume Based 0.21 0.33 0.37

0.40 0.48 0.49

on:

0.47 0.55 0.58

Number of Side Chains Contributed per Y u m by All Helices Based on: 10. a . Av. max. side chain length 11. 6. Half av. max. side chain length 12. c . Approximate min. length

For details see text.

3.1 4.7 5. 3

5.7 6.9 7.1

10.2 11.9 12.5

.

the probability that an average residue will be found in the internal volume. The distance O A extends from the helix center to the plane of demarcation. The probability of finding a residue in the internal voluine will be approximated by the ratio of the sum of the angles 8%to 27rn where n is the number of helices involved. The distance OA is to be the .average side chain extension from the center of the helix. It can be taken as the distance from the center of the helix t o a position midway between the a and p carbon atoms of an amino acid residue plus some fraction, f, of the average side chain length (Table IV). In Figure 3B, which refers to insulin, j has been taken as 0.5. Extreme values of f have been used in calculations. Table V gives calculations made for several proteins for which X-ray diffraction data and amino acid compositions are available. It lists average side chain length @),average side chain volume, average large nonpolar side chain volume, average neutral side chain volume, arid the internal volume per turn. Line 6 gives the number of average residues which can be accommodated in the internal volume. 'The lower part of the table lists the probabilities that an average residue will be found in the internal volume for f values of 1.0 and 0.5, and for f j of 2.1 A., the latter

353

DAVID F. WAUGH

TABLEVI Infinite &-Helices i n Hexagonal Array Interchain spacing, A. Internal volume per helix turn, A? Side chains in internal vol. per helix turn Max. av. vol. of aide chains contained in internal vol., A.3 Max. no. of 81 A.3 side chains in internal v01.~

9.5 224 3.6

10.0 270 3.6

10.5 318 3.6

62

75

88

2.8

3.3

3.9

11.0 368 3.6 102 4.5

The value 81 f A.3 is an average side chain volume for myoglobin, insulin, serum albumin, ribonudense, and hemoglobin.

value being somewhat less than that expected for alanine. The probabilities associated with f = 1.0 and 0.5 suggest that the choice off is not critical for condensed proteins. On the other hand, open arrays such a s myoglobin are more sensitive to the choice of f. The lower portion of Table V finally lists the number of side chains expected in the internal volume (probability X 3.6n). A comparison of the capacity of the internal volume t o accommodate side chains with expected numbers for different f values, suggests a t this point that the internal volume is close packed with approximately 10% of the space unoccupied by side chains. Certain proteins will have one helix almost entirely or entirely surrounded by other helices. In this case the calculated internal volume is less subject to the assumed position of the plane of demarcation and f. Table VI gives the packing characteristics for a-helices of infinite length arranged in hexagonal array. As the center-to-center distance increases from 9.5 to 11 A., the available volume for each side chain varies from 62 to 102 A.3. The average side chain volume of a number of proteins is 81 A.3. Sufficient space is provided when the above helices are 10 to 10.5 A. apart. Helix separations near 9.5 A. have been recorded for hemoglobin and ribonuclease. One concludes again that the side chains must be close packed in the internal volume. The difference between the calculated and observed separations is to be attributed to deviations from the model such as nonuniformity in side chain distribution, a deviation from the a-helix, distortions due to cross-linking SS bonds, and the end effects which occur when the helices exist in segments. Pauling and Corey (84) propose structures for synthetic polypeptides based on the data of C. H. Ramford, W. E. Hanby, and F. Happey. Calculations similar to those given ahove suggest that these also are structures in which the side chains are closely parked, as already suggested (84), to such an extent that in a number of polymers the pitch of the helix is significantly increased.

354

PROTEIN-PROTEIN INTERACTIONS

The calculations given above have been made on the basis of the average residue and an average occurrence in the internal volurne. Actually, the chains of insulin are not identical (Sanger et al., 85, SS), :and other proteins are not likely to have identical crystallographic chains. It seems a necessity to assume that where an exceptionally large residue side chain occurs on one helix (e.g., phenylalanine), a smaller than average residue must occur on a neighboring chain. There are reasons given below for feeling that certain large nonpolar residues must be predominantly in the internal volume. For these there must be compensating small residues; the average then may not be far from that calculated for the entire protein. For the proteins given, the average volume for nonpolar plus neutral residues is slightly less than the general average (76 vs. 81 A.7. The general agreement between the available internal volume and the volume of expected side chains is not fortuitous. There are a number of reasons for expecting that the internal volume in protein molecules is well packed with side chains, in solutions as well as in the crystalline or dry states. 3. The Nature of the Internal Volume Before proceeding with an examination of the internal volume it should be pointed out that, in solution, any residue occurring in the surface volume will be expected to be surrounded by water, to be accessible, and to be free to rotate, unless, of course, unusual interactions with the neighboring internal volume or neighboring residues of sufficient length make strong interaction possible. The reasons for expecting close packing in the internad volume are then summarized as : a. Electrostriction. McMeekin and Marshall (87) have noted that the sum of the residue volumes for a variety of proteins obtained from the amino acid compositons may be used to calculate partial specific volumes in excellent agreement, with those obtained experimentally. Edsall (1) has pointed out that electrostriction of water about polar groups of serum albumin should be about 3.5 % of the observed partial molal volume and that, presumably, all of the charge groups are free to interact with protons and, therefore, to bind water. The values determined by McMeekin and Marshall, as these authors point out, are expected to be about 3.5% too high. The explanation may be found in the unoccupied space occurring in the internal volume, i.e., space which is too small to acconnmodate water or other molecules. For a protein such as serum albumin the expected electrostriction suggests an unoccupied space of 1.8 1. per mole. On the same basis the unoccupied space in egg albumin would be 730 ml. per mole and in p-lactoglobulin 830 ml. per mole (2.2 % and 2.7 %, respectively, of the

DAVID F. WAUGH

355

apparent molal volumes). That such a space occurs and is of the correct magnitude may be inferred from the observations reported next, b. Enzymatic Hydrolysis and Denaturation. Some time ago it was found that the enzymatic cleavage of the first few peptide bonds produces a volume decrease much greater than that expected from electrostriction of water by the charge groups which appear. Linderstrgm-Lang (88) has summarized the pertinent evidence, which has come mainly from the Carlsberg Laboratories. After correcting for electrostriction, the volume change for p-lactoglobulin is a maximum of -700 ml. per mole. Similar excess volume decreases have been observed for other systems given in ref. 88 as chymotrypsin-8-lactoglobulin, chymotrypsin-insulin, pepsin-ovalbumin, and pepsin-8-lactoglobulin. Linderstrgm-Lang points out that there is here a structural principle that is common to all of these substrate proteins. He compares the enzymatic cleavage to the melting of ice and suggests that (similarly) the microcrystal structure of the protein molecule has some forced structure which makes it occupy more space than the unfolded elements. Slow alkaline denaturation at a pH of 7.83 and 30°C. leads also to a volume decrease of 250 ml. per mole protein. More recently Christensen (89) has observed a volume change of -612 ml. per mole protein in the urea denaturation of p-lactoglobulin. Kauzmann (see Simpson and Kauzmann, 187) has found a volume change of -300 ml. per mole protein concomitant with changes in optical rotation, in the urea denaturation of ovalbumin using 6 to 8 M urea at 30°C. Volume changes accompanying denaturation are seen to be less than the excess volume changes produced by enzymatic hydrolysis. c. Side Chain Groups. An important part of any consideration of denaturation is the set of phenomena associated with the masking in the native protein of the reactivities of groups such as sulfhydryl, disulfide, phenolic, and imidazole as compared with the appearance of reactivity on treating the proteins with reagents, physical or chemical, which swell and unfold the protein. The unmasking of reactive groups has received much critical attention, and the reader is referred for details to reviews on denaturation by Neurath et al. (58), Anson (60), and Putnam (59), and to a consideration of structure and activity by Porter (90). In most proteins the titration curves of native and denatured proteins are quite similar, indicating that so far as the dissociation of protons is concerned, such groups are in equilibrium with the solvent. However, Steinhardt and Zaiser (232) have found in the case of ferrihemoglobin, a difference between the instantaneous acid titration curve and the back titration curve, (at pH values below 4.0), the back titration curve being obtained after denaturation has occurred. The difference amounted to

-

356

PROTEIN-PROTEIN INTERACTIONS

36 groups per mole and could be due to the masking of carboxylate or imidazole groups. It is quite apparent that the unmasking of such group$ is accompanied by reversible structural changes (see also Section IV, 11, hemoglobin). When the availability of a group, such as the e-amino group of lysine or the sulfhydryl group of cysteine, is examined by a number of reagents, i t is found in instances to react in a graded series, some of the groups being available to all reagents, others to selected reagents, and still others unavailable in the native protein to the group of reagents. Results of this type suggest strongly that steric hindrances and interactions through secondary valences are responsible for masking, rather than that the group is exposed and masked through its participation in covalent bond formation. In all probability if methods were available for examining nonreactive groups, they too would be found masked. Aggregation of denatured protein or aggregation of native protein may serve to mask otherwise reactive groups. Thus Anson (60) reports that the SH groups of ovalbumin, heat-denatured in neutral solution containing no urea, guanidine, detergent, or the like, are readily oxidized by ferricyanide in the absence of aggregation. As aggregation progresses, however, there is a progressive failure to react with ferricyanide present in slight excess. Reaction occurs, however, with ferricyanide :in strong solution and with the stronger oxidizing agent porphyrindin. As will be seen below, the formation of insulin fibrils involves essentially the linkage of corpuscular protein molecules, fibril growth a t low temperatures suggesting that a small unfolding, if any, is involved in the process. Porter (151) has found a decreased reactivity of imidazole groups with DNFB to accompany insulin fibril linkage. The unaggregated protein has an average of 3.6 to 3.7 out of 4 reactive iminazole groups per molecule, the fibril 2.2 out of 4 groups per molecule. Other proteins have been noted which exhibit similar effects. The appearance of chemically reactive groups and cither evidences for structural change such as solubility, altered optical activity, loss of biological activity, and change in size and shape, appear progressively with time, some much sooner than others and as though a series of changes of progressively increasing magnitude were involved. That such is the case may be inferred also from comparisons of the reversibilities of the properties measured. Certain properties (such as the amount of protein in solution under chosen conditions) can easily revert toward or to normal whereas, a t the same time, only a partial return of a property such as optical activity may be observed, and a loss of the ability to crystallize may accompany the others. The data from denaturation studies, as is evident from current trends,

DAVID F. WAUGH

395

link is stable. It becomes unstable as the pH is decreased, forming completely the adsorption complex noted above a t pH 3.1. The fact that a denaturation may have been carried out at even, for example, a pH of less than 3.1 does not necessarily mean that complete dissociation of the hemeglobin link has taken place, for, as noted above, complete dissociation in even 0.1 N acid is a slow process. b. Experiments with Incomplete Heme-Globin Dissociation. Mirsky and Anson (211) took 1 cc. ox blood, added 8 cc. water and 1 cc. 0.2 N HCL, and heated at 80"C. for 3.5 minutes. While at 80"C., 3 cc. of a phosphate buffer was added to return the system to pH 6.8. After cooling the hemoglobin forms a brown precipitate which shows none of the solubility properties of hemoglobin. Mirsky and Anson assumed that the step of structural alteration proceeded at 80" C. and that it was largely independent of the subsequent dependent coagulation step. Reversal was accomplished by carrying the above procedure through the step of heating. Instead of adding phosphate buffer before cooling, the solution was cooled, 10 cc. 0.04 N NaOH were added to make the system faintly alkaline, a little NazSz04 was then added, and the mixture was allowed to stand for 2 to 3 minutes. After filtering, the filtrate containing the regenerated protein was oxygenated. The resulting protein could be crystallized. Horse hemoglobin was treated somewhat differently so as to avoid alkaline solutions. The protein was precipitated by heating an approximately 4.5% solution for 3.5 minutes at near 90" C. The precipitate was dissolved in dilute acid. Cyanide buffer and alkali are added to make the solution faintly alkaline. A brownish precipitate forms at this point, but this precipitate goes back into solution. The supernatant obtained after neutralizing and filtering may be reduced, converted to the COHb form, and crystallized. The properties of the regenerated horse hemoglobin are very close to those of the native protein in solubility, heat coagulation, crystallization, absorption band, and combination with oxygen. Crystalline yields of 30% were obtained on the above procedure and also from heat-denatured horse hemoglobin. In a somewhat later paper (221) Mirsky and Anson made modifications in'the above. The hemoglobin was denatured in "acid" (equal volumes of 10% horse hemoglobin and 0.07 N hydrochloric acid) at 0" C. After 3 minutes all protein could be precipitated on complete neutralization. Regeneration yields were found to be independent of time in the "acid" (up to 18 hours) and to be about 70 % for (1) slightly alkaline regeneration in the presence of cyanide (this gave cyanomethemoglobin,which could be converted to carbon monoxide hemoglobin and then crystallized) and (2) regeneration by neutralization just short of precipitation. Standing

358

PROTEIN-PROTEIN INTERACTIONS

anced factors, to draw conclusions as to the compactn.ess of proteins in solution. I n general, the evidence from direct physical measurements suggests that they are relatively compact. The evideince, too extensive to be included here, has been admirably examined by Edsall (I). 4. The Internal Volume and Composition

Attention is directed again to one of the salient points illustrated by Table 111. Here the amino acid compositions of a variety of proteins have been presented among other things in terms of the relative frequency of occurrence of larger nonpolar residues. Reference to the initial consideration of forces suggests that the coalescence of a number of previously unassociated nonpolar side chains will be attended by the liberation of 1.18 kcal. per mole CH2 groups. All of the proteins examined have relatively high occurrence frequencies for nonpolar side chains and yet are generally quite soluble, some a t or near their isoelectric points. Such evidence strongly suggests that the nonpolar side chains are not to be found, according to their occurrence frequencies, in the protein surface. The average number of CH2 groups per nonpolar side chain is 4.3, which suggests 5 kcal. per mole on complete interaction. that each will contribute Pauling and Corey, as given a t the start of this section, have used as one of their criteria for stable configurations of peptide chains the assumption that a maximum number of intra-chain hydrogen bonds should be formed. The properties of many corpuscular proteins suggest that a similar rule be applied to the nonpolar side chains, in which case a maximum of stabilization energy would be obtained by placing the majority of such side chains in the internal volume, with small side chains to compensate for the large volumes occupied by the large side chains of the nonpolar class. It is quite possible that many of the details of individual solubilities are related (among other things) to the efficiency with which nonpolar residues may be accommodated in the internal volume. A preliminary examination of the positions of the NPS of insulin, using the a-helix and residue positions assigned by Sanger and associates (85, SS), suggests that, they are grouped so that the NPS occur predominantly in the internal volume. On the basis of the above brief examination the internal volume is considered t o contain an unoccupied space of approximately lo%, a fraction of the side chains whose groups may be masked, suc’h as OH, SH, SS, imidazole, etc., and compensating numbers of nonpolar side chains and small side chains (Table 111). The internal volume is not expected to contain many groups capable of becoming charged. As already mentioned, the structure of the internal volume and surface is felt t o have an important bearing on protein-protein interactions.

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DAVID F. WAUGH

359

IV. SPECIFIC INTERACTIONS We have noted previously the central position occupied by the native protein in considerations of interaction. Its position here is similar to that occupied in considerations of “denaturation.” As is well known, the term denaturation covers a number of molecular-structural changes, many of which lead t o interaction. Indeed, the early definition of denaturation was based on the appearance of insolubility under conditions where the native protein is soluble. As will be evident after a presentation of specific proteins, the classification of protein-protein interactions is dependent to a considerable extent on an understanding of the series of changes which are involved in passing progressively from the native protein through the states of reversible denaturation to irreversible denaturation. The present limitations in our information about structural detail in proteins suggest that a classification of protein-protein interactions must remain indefinite. However, it is also quite apparent that the basis for classification must be dependent upon a consideration of the changes in internal structure which occur as a prelude to interaction. In general the proteins which follow have been arranged according to the series which ranges from small reversible changes in internal structure -+ large irreversible changes in structure. It will be evident also that a given protein may show interaction phenomena a t different levels in this series. I n fact, the exhibition of a spectrum of changes and interactions is one of the most interesting features of the structural problem with proteins. I n the discussions which follow, the term “association” will be used to refer to the formation of a product of limited size for which association constants in theory could be obtained. “Aggregation,” on the other hand, will refer t o polymers of unlimited size or to polymers having a heterogeneity which prevents obtaining thermodynamic data. A suggestion similar to this has been made by Johnson and Naismith (125).

1. Antigen-Antibody I n many cases to be discussed protein-protein interactions take place under specific environmental circumstances (such as acid or alkaline pH values, after or during urea treatment, and a t elevated temperatures) which might well produce an abnormal distribution of residues, and consequently their main chain counterparts, between the surface and the internal volume. Such could hardly be the case with the interactions of antigens and antibodies, where the process of mixing may be all that is required t o obtain interaction. Campbell and Bulman (94) have recently summarized current concepts. Only a few of the salient points will be covered here. A wide variety of native and denatured proteins have been used as anti-

360

PROTEIN-PROTEIN INTERACTIONS

gens for the induction of antibodies in laboratory animals. Since the latter have considerable specificity with respect to the former even in the presence of excesses of nonreactive protein, a complementariness of surface structure is evident, as has been emphasized by Landsteiner (95). Such a complementariness would involve the positioning, a t least in portions of the surface of each molecule of compatible groups such that only a precise fitting together will allow the realization of sufficient interaction energy to produce a stable link (AP - 5 to - 10 kcal. per mole). Much of the information which has led to the formu:lation of interaction in terms of the forces between groups of atoms has come from an examination of the interactions between antibodies directed against a specific group of atoms, the hapten, and the same hapten and hapten analogs. hntibodies, which occur in the y-globulin fractions of immunized animals, are prepared by injecting the animal with protein to which the hapten is attached; generally through azo linkage to the tyrosine side chain. Campbell and Bulmaii (94) calculate the sizes and shapes of antibodies as ranging in molecular weight from 90 to 920 X lo3 (mostly near 160 X lo3)with uniform widths near 37 A. and lengths ranging from 166 to 964 A. These authors point out that the dimensions so obtained are “essentially in agreement with closest packing arrangements of antigen-an tibody molecules in precipitates assuming a bivalent antibody and polyvttlent antigen molecules” (Pauling, 96). Pauling and collaborators (17, 28, 97, 98) have examined extensively the inhibition of the formation of antibody-haptenantigen precipitates by free hapten and hapten analogs. An early preliminary theory of precipitation (97) was later extended to take into account the heterogeneity of the antibody combining power (98). This theory was used extensively to calculate the free energy difference between the combina,tions of two hapten analogs with a given antibody. Depending upon the haptens used the authors have implicated hydrogen bonds, charged groups, and van der Waals’ interactions (17, 28). The general approach and the results obtained have been referred t o extensively in considering group interactions. Wherever van der Waals’ forces were basically involved it was clear that the steric fit was of major importance in determining the competition of haptens of varying structure for antibody sites. As Paiiling and Pressman conclude (28), “the structure of the antibody is such that the molecule can approximate itself closely not only to the various portions of the haptenic group but also the azo group or other substituent group; and this approximation is close enough to correspond to intermolecular ‘contact,’ as determined by the outer electron shells of the atoms. On the other hand, the antibody has sufficient elasticity of configuration to permit the insertion of a much larger hapten than that to which it was moIded, and yet it re-

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D.4VID F. WAUGH

361

tains the impress of the immunizing antigen sufficiently to cause combination t o occur more readily with haptens similar to the immunizing antigen than with others.” Haurowitz et al. (99) have determined the solubilities of washed precipitates of rabbit antibody complexes with arsanil-beef serum globulin and with sulfanil-ovalbumin. The authors state that the associations follow the reaction AB,-i

+B

AB,

where A = antigen and B = antibody, since the supernatants contain mainly antibody. The two complexes were quite similar. The temperature variation in equilibrium led, for sulfanil-ovalbumin, at 4°C. to A F = -8.6 kcal. per mole; A F a t 29°C. = -9.1 kcal. per mole; AH = -2.85 kcal. per mole; and A 8 = 21 cal. per mole per degree. Boyd et al. (loo), by a direct calorimetric measurement a t 31”C., found that 3300 kcal. per mole of antigen (Busycon cannaliculatum, mol. wt. = 6.8 X lo6) were evolved when the antigen reacted with horse antiserum. Estimates of the antigen-antibody ratio led to 40 kcal. per mole antibody from which, on the assumption that a free energy change of - 10 kcal. per mole antibody occurred on linking, they obtained A S = -100 cal. per mole per degree. Singer and Campbell (101) studied the sedimentation patterns of soluble antigen-antibody complexes of bovine serum albumin and rabbit antibovine serum albumin. They employed an equation of Goldberg (102) to estimate, a t various concentrations, the species which satisfied the equation A+ABeABA

For the reaction a free energy change of A F = -5.4 f 0.4 kcal. per mole was observed in veronal buffer a t p H 8.5, p = 0.1, and 0°C. The free energy changes so far obtained are in a range which would be expected from the interactions of a few groups interacting with weak forces; quite possibly hydrogen bonds and electronic van der Waals’ interactions. General electrostatic interactions would not be expected, since the pH is well above the isoelectric point of bovine serum albumin (pH 4.7) and somewhat above that of rabbit y-globulin. The studies of antigen-antibody interactions are of particular interest in estimating the extent to which a specificity of surface structure may be operating in establishing reversible (and irreversible) dissociable systems such as hemocyanin and arachin where a number of smaller particles may form. For example Pauling, Pressman, and Campbell (103) prepared an antiserum A to the hapten group X and an antiserum A to the hapten

362

PROTEIN-PROTEIN INTERACTIONS

group R. If a single organic molecule containing X and R were mixed with A or A alone, no precipitate resulted, but if A and AR were present together, a precipitate formed. Clearly a mechanism for limiting association is indicated by these observations. Another mechanism for the production of aggregates of limited size is also well illustrated by the region of antigen excess. Where antibodies are bivalent (Singer and Campbell, 104; Plescia et al., 105; Goldberg and Williams, 106), the combination of two antigen molecules and one antibody molecule produces, as has been seen above, a soluble complex. Further possibilities will be considered where association systems are discussed. 2. Hemocyanin

The hemocyanins may be taken as representative of a number of large protein molecules from different sources, plant and animal, which exhibit reversible (and irreversible) dissociation-association reactions. The dissociation of hemocyanin was first described in publications from the laboratory of T.Svedberg in the later 1920’s. Eriksson-Quensel and Svedberg (107) present comprehensive data for 22 species studied. They point out that one of the most striking characteristics is the perfect homogeneity of various hemocyanins with respect to molecular weight. Although the number of components is a minimum in a pH range including the isoelectric point, a single component is not always observed. Thirteen species have 1 component, seven have 2 components, one has 3, and one, 4 components a t their respective isoelectric points. These components may be members of different dissociation systems, as will be discussed. All dissociate in acid or alkali and a t times under the influence of a change in ionic strength to give subunits whose sedimentation constants can be placed, almost within the experimental error, in 10 classes, with the following approximate values of S = 135, 100, 62,56, 49,34,123.5, 16, 11, and 5. A given hemocyanin has subunits in only a few classes, but for any one species the molecular weights of all components are simple multiples of the smallest well-defined component. The components were found to be interconnected by reversible association-dissociation reactions. At certain critical pH values a profound change takes place in the distribution of components; often a decrease of a few tenths of a pH unit is all that is required to bring about a marked dissociation. Ericksson-Quensel and Svedberg point out that the forces holding the units together must be feeble-only, however, under the environmental conditions where extensive dissociation is observed. Some of the effects of electrolyte are pointed out by Svedberg (108) for hemocyanin. (The reversible dissociations of another interesting system, thyroglobulin, are mentioned here. See also Pedersen, 109.)

363

DAVID F. WAUGH 120

I

A

I

!

8

!

I

I I I

II F I I I

u

I

0 0

I

I

I

I

I I I I

I

1

3

I

5

,

7

I

9

1 1 :

4

!

I

I

6

8

1 0 1

?

PH

FIQ.6 . Dissociation curves for hemocyanins from three sources. Ordinates give sedimentation constants and abscissae, pH values. A . Helix pomatia; B. Helix nemoralis; C . Littorina littorea.

Within a pH range all of the dissociations are reversible. Figure 5 gives the pH dissociation patterns of Helix pomatia, Helix nemoralis, and Littorina Eittorea, patterns which are characteristic of a given species. Figure 5 plots the sedimentation constants of the components observed versus pH. The patterns are quite similar for closely related species but vary progressively and become quite divergent for different orders. Just outside of the p H range associated with reversibility the fragments which form do not reassociate completely. Such ranges would be p H 3 to 4 and 9 to 11 for several proteins (see 107, 110). More vigorous treatments lead t o insolubility. More recently Brohult (110, 111) has investigated the size and shape characteristics of the products of dissociation. The first of these references is an extensive review in which considerable new information is presented. It treats Helix pomatia hemocyanin almost exclusively. The second includes work on Paludinia vivipara. Table VII gives the molecular constants for two of the m a b dissociation products of each protein. In agreement with, and extending, the work of Svedberg and associates, Brohult finds that the dissociation pattern is sensitive to pH, protein concentration, salt concentration and the nature of the ions, and to nonelectrolytes such as glycerol and sugars. Where an increase in ionic strength effects dissociation (Helix), the valences of the ions may be used generally to place them in the order of effectiveness:

This is true for dilute salt solutions, more concentrated salt solutions having

364

PROTEIN-PROTEIN INTERACTIONS

TABLE VII Molecular Constants of Helix pomalia and Paludinia viuipa,ra Hernocyanins and Their Dissociation Products

~

~

~

~

Helix pomatia

103.0 65.7 19.7

1.07 1.41 1.77

0.738 0.738 0.738

8.91 X 106 4.31 X lo8 1.03 X lo6

1.45 1.40 1.79

1130 820 820

Paludiniauivipara

102.5 64.5 21.8

1.09

0.738 0.738 0.738

8.70 X lo6

1.43

1090

1.13 X loB

1.72

790

1.79

890 890 960

~~

* Length of t Length

the molecules from fffo. of the molecules from stream double refraction (Snellman and Bjcrstahl, 112).

characteristic and unpredictable effects (1). Dialyzing out the salt (Claesson, in Brohult, 110) greatly retards dissociation. As expected, dissociation is increased by decreasing the protein concentration. The dissociation produced by sugars and glycerol is quite marked a t 1 M concentration a t pH 5.5 to 6.0. Quite possibly the adsorption of such molecules onto the protein surfaces introduces steric effects which prevent the realization of short-range attractive forces. The effects of varying ionic strength are instructive. In dilute salt solutions, Helix hemocyanins are dissociated by increasing ionic strength but to a variable extent between species. Paludinia vivipara, on the other hand, exists as half and whole molecules a t p < 0.05 and pH 7.4 (the isoelectric point of the protein is 4.71, according to Pedersen, 109). Whole molecules are found between 0.2 < p < 1.0. At higher ionic strengths some dissociation again takes place, but some of the molecules associate further into “double” hemocyanin molecules of S 140 to 160. A limited dissociation of Helix pomalia hemocyanin of 75 % is produced by salt. From this observation, Brohult (111) deduced that two kinds of molecules were present. That this is Bo was proved by (1) separation by centrifugation, and (2) by fractionating the hemocyanin solution with ammonium sulfate. It could be shown that products having quite different degrees of salt dissociation were produced, for example complete dissociation vs. 40 % dissociation. In addition, if the mixed molecules were dissociated into eights by changing the pH, and the eighths were then reassociated into wholes, the “wholes” thus obtained were now found to dissociate to the entent of 88% instead of 75% on addition of salt and to have an altered electrophoretic mobility. Thus, in spite of the fact that size and shape characteristics are the same within the ex.perimenta1 error,

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365

DAVID F. WAUGH

the integration of subunits into wholes is such that two different kinds of molecules arc present initially. Apparently from the experiment of Rrohult just mentioned there is a tendency for the one eighth size molecules to sort themselves out again into the two different kinds of initial molecules. The specificity involved in subunit association is clearly indicated in the experiments of Tiselius and Horsfall (113) in which they compare, using electrophoretic and ultracentrifugal techniques, the association products produced by reassociating subunits of Helix pomatia alone and in combination with those of Helix nemoralis and Littorina littorea. Dissociation a t pH 8.5 (eighth molecules produced) and reassociation a t pH 6.8 of H . pomatia and H . nemoralis in combination produces molecules of the original size, but an electrophoretic analysis of these reveals a series of three overlapping components of mobilities varying between that of H . pomatia (-3.08) and H . nemoralis (-5.27) alone. Clearly a number of mixed hemocyanin molecules have been produced. Dissociation a t p H 3.8 (in each case the protein is partly dissociated into half molecules) and reassociation at pH 6.8 produces mainly the original molecules with a few mixed molecules. Dissociation of H . pomatia and L. littorea a t p H 8.5, the latter partly dissociated a t this pH into fractions one half and one eighth the original size, and recombination at pH 6.8 produces four distinct electrophoretic components. Of major importance is the fact that the ultracentrifuge pattern also reveals several components with S varying between 90 and 16. Apparently some association can take place between the subunits from each original protein, but the packing is no longer sufficiently precise to allow the building up of the original particles. Tiselius and Horsfall (113) remark that, “It does not seem unlikely that the dissociation of hemocyanins and similar proteins into simple sub-multiples would indicate an ordered crystal-like structure of the individual particle or molecule. The cross reactions studied here would then have their analogy in the formation of mixed crystals between substances of similar chemical structure.,’ These same authors previously pointed out the analogy between the behavior of the subunits of hemocyanins and antigens and antibodies. a. Urea and Other Agents. Burk (114) from osmotic pressure measurements determined the molecular weight of Limulus hemocyanin a t its isoelectric point of p H 6.2 to 6.4 as 565 X lo3. In the presence of 6.66 M urea the molecule dissociates into subunits of mol. wt. 142 X lo3. The hemocyanin is decolorized in the process and denatured, for it is no longer soluble in dilute isoelectric salt solutions. Burk also presents evidence which suggests that hemocyanin oxidizes its own SH groups in urea solution. If the hemocyanin be first treated with acid a t p H 3, it dissociates further in isoelectric 6.66 M urea to giveunits of mol. wt. 69 X lo3. Acid-

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--

366

PROTEIN-PROTEIN INTERACTIONS

treated and normal hemocyanin are both more sensitive to p H dissociatioii in the presence of 6.66 M urea than native hemocyanin is in the absence of urea. Brohult (110) details a number of agents which split the hemocyanin of Helix pomatia. The splitting reactions produce subunits but the splitting is irreversible, for the subunits will no longer recombine under normal circumstances. Thus, the cavitation effects of ultrasonic treatment, ultraviolet light, and a-particles generally split the protein into halves. Of interest from the standpoint of interaction, if the ultraviolet splitting treatment is prolonged, a series of irreversible aggregation products appear. Presumably, other splitting treatments will produce the same effect. In discussing the phenomena Brohult suggests that no decision can be made between the breaking of a few specific bonds vs. the “a,ctivation” of the molecule as a whole.

3 . Plant Seed Proteins Proteins such as Amandin (almond), Excelsin (Brazil nut), Arachin (groundnut), and Edestin (hemp seed) are usually extracted from the seed meal with 3 % t o 10% salt solutions and are purified by salting out, dilution, or cooling procedures. As shown in Table VIII such proteins have relatively high molecular weights. Stability is shown only in a pH range near the isoelectric point. Depending upon the particular protein, dissociation is shown by dilution, a t low or high pH, and in the presence of urea. Thus in 16.66 M urea solution and a t pH 6.1 and 6.31, which correspond to the isoelectric points in this solution, Burk (115) has found that the molecular weight of amandin becomes 30.3 X lo3 and that of excelsin becomes 35.7 >(:los. A dissociation into one sixth molecules is evident. In each case the test for SH becomes positive in the presence of urea. Fifty per cent glycerol has no effect on excelsin. The dissociation of excelsin is not reversible, for dilution produces a precipitate no longer soluble in salt solution. TABLEV I I I Plant Seed Proteins Protein

Extraction

Mol. wt. X 10-3 Method

Amandin 10% NaCl 206 3% (NHdas01 214 Excelsin 330 Arachin 6-10% NaCl Edestin 5% NaCl 309 10% NaCl Variable Conarachin (O.P. = osmotic pressure; S = sedimentation; D

Reference

02. 0.I’.

s, 13 s, 11 S

= diffusion.)

115 115 118 116 125

367

DAVID F. WAUGH

Bailey (ll6),who refers to earlier work, has examined the acid splitting of edestin in salt-free solution to form edestan. On acidifying a suspension, the protein goes into solution at pH 4.9. If after some time the solution is brought to p H 5.5 (the isoelectric point), a precipitate as expected will form, but some of this precipitate remains insoluble on the addition of salt over a wide pH range. That attractive groups have been liberated is also indicated by the fact that the addition of salt to a material dissociated in acid produces an insoluble precipitate. It was found that edestan formation is slow a t pH 5.0 and very fast a t pH 4.0. None of the curves was first order. Edestan, the dissociation product, has a molecular weight of 17,000 (S = 2.63) (see also Adair and Adair, 117). The dissociation is retarded by divalent anions such as sulfate and oxalate, suggesting that the repulsion of positive charges is intimately involved in the dissociation process. Edestin is stable, on the other hand, in alkaline solutions u p to p H 9 to 9.5. At pH 9.8,Bailey points out, the excess negative charge is approximately the same as the excess positive charge a t p H value near 3.9. Bailey calls attention t o the fact that the remarkable insolubility which results from the dissociation procedure (and may be connected with dissociation itself) is symptomatic of a profound alteration in physical properties. Arachin, as studied by Johnson and Shooter (118), shows a reversible dissociation with respect to pH. The protein prepared in salt solution has a molecular weight of 330 x 103. If such a solution be diluted and adjusted from pH 6.0 to pH 5.0, two components are found to be present having properties given in Table IX. The data of Table IX show that the molecules are related by Az F! 2A

(19)

the axial ratios suggesting that either two equivalent rodlets or platelets are being separated to give subunits of twice the parent asymmetry. The reversibility suggested by Eq. 19 was tested and found to exist. TABLEI X Proverties o f Parent and Dissociated Molecules of Arachin Parent molecule (cm.z/sec.) S"20

Mol. wt.

f/fo

a / b (prolate) a/b (oblate)

3.86 x 10-7 14.6 330,000 1.216 2.6 0.37

Dissociation product 4 . 2 x 10-7 9.0 180,000 1.352 4.6 0.25

368

PROTEIN-PROTEIN INTERACTIONS

The arachin system differs significantly from other dissociation systems. In 6 % NaCl and a t pH 6.0 the parent molecule (dimer) is quite stable. In the presence of considerable salt, varying the pH produces little or no effect, but a t low ionic strength ( p = 0.04) the material becomes low in solubility over a pH range from the isoelectrir point (pH 5 where it is quite insoluble, see rcf. 120) to pH 8 to 8.5. The precipitate whirh forms a t pH 5.0 is found to be composed of a mixture of A2 and A. A homogeneous preparation of A, may be made by salting out the initial pH 6 extract. An A a t various p H examination of the properties of A2 and mixtures of A2 values revealed: (1) that the equilibrium is pH sensitive, low or high p H promoting dissociation, (2) that salt prevents dissociation, (3) that a t low ionic strength the rate of dissociation is fast a t pH 5.0 but that both the rates of dissociation and association are slow (many hours) a t pH values above 6.98. Ammonium sulfate (1 % of saturation) was found to decrease the rates markedly. Johnson et al. (119) confirmed by electrophoretic analysis several of the findings described above. The fact, that the rates of dissociation or association are slow suggests that some alterations of internal structure may be necessary for appropriate interaction to occur. The A unit of mol. wt. = 180,000 is not the smallest unit. Johnson (120) finds that small polydisperseunits of mol. wt. < 80,000 are produced a t pH 11 and in 4 A4 urea a t pH 8. One would expect that the A%or A units could not be re-established from these smaller units. In initial extracts Goring and Johnson (121) and Danielsson (122) report the presence of a high molecular weight component of Soto 20. The former authors report S = 21.1 and state that the component represents 6.3% of the protein. The conversion of S = 21.1 into S = 13.3 is apparently irreversible, suggesting that the S 21.1 component may be the natural form. In unfractionated arachin Eirich and Rideal (123) have found components of molecular weight 600, 300 (S 3.8), 150 ( S 4.2), and 40 X lo3. Arthur (124) reviews these and other properties of arachin. Johnson and Naismith (125) present evidence which suggests that conarachin is also a n association-dissociation system. I n this case the presence of salt increases dissociation, as does increasing pH. Electrophoretic separation of fractions is not possible. Depending upon pH and ionic strength, a series of components is observed having approximate molecular weights in the series ( X lof3),19, 190,290,380, 569, 1100,2200. Equilibria are established rapidly. The dissociations are attributed largely to the alterations in charge pattern which are produced by changing the pH and the effects of ionic strength in altering electrostatic interactions, although the authors note that van der Waals’ forces and hydrogen bonds may also be involved.

-

+

N

-

-

-

DAVID F. WAUGH

369

4. Insulin As information concerning the interactions of proteins accumulates, it is becoming increasingly evident that, exclusive of salting out, isoelectric precipitation, and the like, a given protein may interact with itself and with other proteins in several ways. Insulin serves as an excellent example of a protein capable of exhibiting a spectrum of interactions. Two circumstances stemming directly from interaction studies serve to heighten our interest in this protein: first, it is capable of exhibiting different modes of interaction under seemingly identical environmental conditions, and, second, although several of the aggregation products exhibit considerable stability, “native” protein may be regenerated from them. I n addition, of course, there is strong evidence that the sequence of amino acids in the insulin chains is known from the extensive work of Sanger and associates (85, 86). The minimum molecular weight of insulin has been in question for some time and may still be so. Edsall (1) has reviewed the problems involved, and they need not be reiterated here other than to examine the way in which the eventual accepted minimum molecular weight may affect views of molecular interaction. There can be no doubt that insulin dissociates a t low ionic strength below p H 2.0 into units of molecular weight 12,000, as has been evident for some time. Recent references covering earlier examinations by the same authors are Gutfreund (126), Fredericq (127), Tietze and Neurath (128, 129), and Oncley et al. (130). Some of the preliminary evidence for dissociation is contained in the work of Sjogren and Svedberg (131) using the ultracentrifuge and in the X-ray diffraction analysis of Crowfoot (132). There is still considerable evidence which suggests that the true minimum molecular weight, as found by Neurath and associates (see 129), is 6000. Thus Harfenist and Craig (133) have found evidence for this molecular weight from counter-current distribution of chemically labeled insulin. In addition Kupke and Linderstrgm-Lang (134) have reported that osmotic pressure measurements show that guanidine hydrochloride can dissociate the 12,000 unit into two 6000 units. Whatever the outcome, it is quite clear that the dissociation into units below 12,000 is achieved only with difficulty. To all intents and purposes, therefore, the minimum molecular weight will not be of definitive importance in most of the association-dissociation experiments to be described. The situation becomes otherwise when structural problems are concerned, for the fact that only SS bonds and no SH bonds occur in insulin presents problems of inter and intra chain bonding (see Robinson, 135; Low, 136). Likewise, kinetic studies in which molecular weight enters as a n important variable must take this possibility into account. Fortunately, for most of the studies to be described, insulin is a notor-

370

PROTEIN-PROTEIN INTERACTIONS

iously hardy protein in acidic media, although its stability in alkali is poor (150). Also of considerable importance, as will be seen, is the fact that many of the reactive groups of insulin may be chemically modified without necessarily producing an inactive protein or producing more than the expected physical alteration in size and shape. As might be expected, chemical alteration profoundly affects the association and aggregation reactions to be discussed. a. Readily Reversible Associations. The insulin monomer, which for present purposes may be assumed to have a molecular weight of 12,000, is observed a t pH values below 2.0 and a t low ionic strengths, for example < 0.1. If the pH be raised or the ionic strength be increased, there is a progressive association of monomers with, eventually, the formation of the extraordinarily insoluble isoelectric (pH 5.3 to 6.1) precipitate of insulin. Along the way there appear stable uniform aggregates, the uniform aggregate of mol. wt. 36,000 observed by Sjogren and Svedberg (131) being one of these. Other intermediates have been observed by careful manipulation of the environment. Oncley and Ellenbogen (130) and Moody (see ref. 130) were the first to make a study of the association process. Oncley and Ellenbogen suggest that the association equilibrium is the result of an electrostatic repulsive force the magnitude of which is mainly sensitive to ionic strength, dielectric constant, and the net charge on the monomer, and a ccmtant attractive force due t o short-range forces of some kind (which they suggest may be due to lipophilic groups). Their analytical data came from observations with the ultracentrifuge--a circumstance which complicates the problem, since (I) sedimentation velocity is being observed through a boundary whose concentration varies from 0 to that used to fill the cell (corrected for sedimentation in a sector-shaped cell) and (2) the association-dissociation reactions rapidly attain equilibrium, as found by Doty and his associates (see ref. 138). They make the simplifying assumption that the equilibrium is between monomers and trimers according to

-

311

13

and derive expressions which relate the equilibrium constant for this reaction to the average sedimentation constant and the weight fraction of insulin in the trimer form. Equilibrium constants are used to ohtain the free energy change for the reaction. The electrostatic work which must be done in associating three monomers is obtained by calculating the difference in energy involved in placing charges on the surfaces of spheres of volume equal to those of the monomer and trimer, respectively, using

DAVID F. WAUGH

371

where N is avogadro's number, e is the electronic charge, zi is the charge on the insulin particle, c is the dielectric constant, bi is the radius of the insulin particle, K is the Debye-Huckel parameter (0.329 X 1082/cLin water a t 25"C.),and ai is the sum of the radii of the insulin ion and chloride ion (assuming the latter to be the main gegenion). Equation 20 is given by Scatchard (137). For the monomer bi and aiwere taken as 15.6 A. and 18.1 A. and for the trimer as 22.5 A. and 25 A. At the maximum charge of 12 the electrostatic work involved in making the trimer is about 17 kcal. a t p = 0.1 and 12.5 kcal. a t p = 0.3. Equation 20 suggests that the electrostatic work will decrease rapidly with decreasing net charge. The sum of the attractive energy and electrostatic energy should equal the free energy change on association. The attractive energy was found to vary from -21 kcal. ( p = 0.1) to -18,000 kcal. ( p = 0.3). Qualitative agreement between calculated and observed sedimentation constants was obtained. Trimerization was considered to result in the formation of three common faces, leading to values of -6 to -7 kcal. per common face. Oncley and Ellenbogen point out that the accumulated evidence suggests that dimers and tetramers, etc., may be of importance. Doty, Gellert, and Rabinovitch (138), Doty and Myers (139), and Steiner (140, 141) have examined the association reaction of insulin using data obtained from light-scattering measurements. Doty et al. (138) concluded that below pH 2.2 association involved a monomer-dimer system only. The recent publications of Doty and Myers (139) and more recently Steiner (141) examine the association over a sufficiently wide concentration range so that thermodynamic constants for trimerization may be obtained. These authors are in essential agreement both as to methods and results. The equilibrium equations are

+

21 F? It

13

I2

+ I * It

+ I F?

(21)

14,

etc.

Let the equilibrium constants for each of those be ki related to the reaction Ii.l I d Ii. Let M be the monomer molecular weight, M , be the weight average molecular weight, x be the mole fraction of monomer existing as monomer, and c be the concentration in grams per milliliter; then the weight average molecular weight is related to the concentration by

+

M

- 1 + -

M, -

d In x d In c

372

PROTEIN-PROTEIN INTERACTIONS

and

By means of graphical integration x may be determined from the experimental data as a function of c. The data in this form may now be used in

(a

Plotting the quotient on the left against 2 - should give a line of intercept

4k2 and initial slope 9lc~lc3. As is remarked (139), the appearance of cur-

vature would indicate the existence of polymers high-er than the trimer which, if precise data were available, would permit the determination of higher equilibrium constants. Steiner (141) lists certain assumptions made in addition t o those already given in this section. Free energies and heats of association are calculateld in the usual way from AFi = -RT In lci and AHi = -R d In k i / d ( l / T ) . Table X combines data as indicated. The free energy changes with pH are in accord with a shift in average electrostatic repulsion due to charge and the differences between salts (Doty and Myer’s, 139) are attributed to an increased anion binding. These same authors note that the observed entropy of association would not be expected on the basis of the association of two structureless cylinders, which would give a value of -122 E.U. The entropy increase necessary to compensate for the decrease just cited, according t o Doty and Myers, might come from the simultaneous freeing of water molecules on association, the information being consistent with the reaction 2(I X 12Hz0) F1 Iz

+ 24H20

(24)

As few as three anionic groups on each monomer would be sufficient. The freeing of 24 water molecules is, as,evident from earlier considerations of electrostriction, a process requiring considerable energy.. Presumably, the energy for this step would be supplied partly by a number of short-range attractive forces. Steiner (141) also calculates a large part of tlhe free energy change on increasing pH as due to a decrease in electrostatic relsulsion. It is suggested here that the total entropy change is the sum of ( 1 ) the intrinsic entropy change due to the diminution in the numher of kinetic units (calculable as suggested by Doty and Myers), (2) the entropy change due to

TABLEX Thermodynamic Data for the Association Reactions of Insulin

PH

Salt

(Doty and Myers) 1. 9 NaHzPOl 2.6 NaH2P04 NaCl 2.0 2.6 NaCl (Steiner) 3.07 KC1 2.48 KCI 2.06 KC1

A F ~kcal. , per mole -4.32 -4.69 -4.93 -5.19

f .1

f .1 f .1

f .I

-5.6 f 0.1 -5.02 -4.94

UZ, kcal.

per mole

-5.2 -4.9 -8.1 -7.7

f .4

f .5 f .6 f 1.0

-6.7 f 1.1 -8.4 f 1.4 -7.7 f 1.3

A & , cal. per

mole per degree

-3.0 -0.7 -12.1 -9.0

AF3

AH3

AS3

-4.05 f .1 -4.77 f .1

-8.7 f 1.0 -8.1 f 1.1

15.4 f 5 19.2 f 14

-5.27 f 0.2 -4.58 -4.76

-7.5 f 2.6 -5.6 =t1.8

f2

f2 f 2.4 f5

-3.8 f 3 -11.6 f 3 -9.6 f 3

(It should be noted that Doty and Myers, 139, record data for the dissociation reactions of which the above equilibrium constants are the reciprocals.)

w -a w

374

PROTEIN-PROTEIN INTERACTIONS

rearrangements in the ion atmospheres about each monomer (not yet calculable), and (3) the entropy changes due to differences in solvent electrostriction in the dissociated and associated states. The last-named change may be of primary importance, as suggested by Doty and Myers (139). Tt is interesting that the AF and AH changes in dimerization and in the association of a monomer with the dimer are so closely the same. If all faces are equivalent, the trimer picture of Oncley and Ellenbogen would require the AF on trimerization to be about twice that on dimerization. Steiner provisionally suggests a semilinear configuration on the basis that the AFs is actually slightly less than p F2 . A decision must await a n accurate determination of A S a . As suggested in the discussion, the faces may not be identical. b. Insulin Fibrils. Under the conditions where insulin monomers predominate, e.g., a t low p and pH 2, insulin is quite stable. Indeed, under these conditions it is reported to have maximum stability. It was found, a t the time that relatively pure insulin preparations were becoming available, that heating insulin in acid produced a precipitate. Precipitation under these conditions constitutes an exception to general behavior, as is readily apparent from an examination of the other proteins treated here. The phenomenon, studied by several groups (Blathenvick et al., 142; du Vigneaud and associates, 143, 144; Gerlough and Bates, 145), was known t o have the elements of reversibility, for, as shown by Cierlough and Bates (145) and du Vigneaud et al. (144), activity and solubility could be recovered from the inactive precipitate. Langmuir and Waugh (146)) interested primarily in surface films of proteins, heated acidic insulin solutions but obtained generally gels which, because they exhibited flow double refraction on dilution, were shown to consist of anisometric micelles. These were later shown to be highly asymmetric fibrils (Wauglh, 147). As shown by Waugh (148), the heat precipitate and insulin fibril Eire directly related. The former consists of sphaero crystals consisting of radially oriented fibrils. As might be expected, spherite formation is promoted by agents such as divalent anions and an increase in ionic strength which suppress electrostatic repulsion between the growing fibrils. 'When electrostatic repulsion is kept maximal (pH 2.0 and low p ) , gelation is favored. The insulin fibril has been examined in electron micrographs by Waugh (147) and by Farraiit and Mercer (149), the latter showing the electron micrographs themselves. A population of fibrils in which the larger fibrils account for the majority of insulin is apparent. These larger fibrils are approximately 200 A. in diameter and have lengths of tens of thousands of A. An examination of the regeneration of insulin from insulin fibrils by the action of alkali has been made (Waugh, 150). Completely fibrous gels may be given an appropriate alkaline treatment and 80 % to 90 % of the insulin

-

DAVID F. WAUQH

375

recovered in crystalline form. It is interesting that the data suggest that disaggregation occurs mainly at the fibril ends. Thus rapid freezing and thawing, which produces fibril segments, accelerated alkaline regeneration. Porter (151) has recently examined the reartivity of the imidazole ring in proteins including native insulin and insulin fibrils. He finds that 1:2 :4 fluorodinitrobenzene will reart with 3.6 to 3.7 out of 4 imidazole residues in native crystalline insulin but will react with only 2.2 out of 4 when the insulin is in the form of the fibril. Apparently the approximation of the insulin molecules, as will become apparent from the discussion which follows, is sufficient to introduce steric factors which prevent the sizable reagent molecule from approaching the reactive groups. Both native and certain chemically modified insulins are capable of giving rise to a remarkably wide variety of insulin fibrils, as reported by Waugh et al. (152). The particular type of insulin fibril obtained depends upon the conditions of fibril formation as well as the type of insulin used. The set of insulin fibrils represent structures of varying stability, certain of which may be reverted as described above to give insulin. The communication examines the occurrence of nucleation, growth, and, to a lesser extent, reversion of insulin fibrils under chosen sets of experimental conditions. Fibrils from four types of insulin are considered: native insulin (FN), esterified insulin (FE),aeetylated insulin (FA), and diazotized insulin (FD). It should be noted that in each case chemical modification was extensive. For what they may contribute to a more general understanding of aggregation, the circumstances of fibril growth are important. The considerable stability of solutions of insulin in acid has been noted above. If, once having been formed, the insulin fibril is introduced by seeding into ti solution of insulin, at room temperature, for example, the fibril will grow and progressively remove native insulin from solution. A population of a few fibrils, as shown by Waugh, Thompson, and Weimer (153), can remove insulin quantitatively, even so far as the sensitive biological assay can tell. If one seeds 2 mg. per milliliter of insulin fibril segments into a solution which yields finally 20 mg. per milliliter native insulin, over 90% of the native insulin will be removed in 2 to 3 days a t 25'C. and in less than 3 minutes at 90°C. The extensive regeneration of crystalline insulin from insulin fibrils, the low temperature growth reaction, and the structural identity of fibrils of different types to be described below suggest quite strongly that the insulin molecule enters the fibril association with little distortion, certainly that the molecule is not extensively unfolded. The over-all and growth reactions in insulin fibril formation (152) suggest that the initiation of a fibril (nucleation reaction) requires the simultaneous interaction of several insulin monomers. The degree to which the monomers are prepared for bonding and are approximated is temperature

376

PROTEIN-PROTEIN INTERACTIONS

sensitive. On this basis nucleation was expected, and has been found, to vary with some power of the insulin conrentration, the exponent (as high as four) being a function of the minimal number of monomer unit,.: required to produce a stahle nurleus. Oncci having been formed thc surfwe of the fibril presents u configuratioii whirh is a mmposite of t tic properties ot the individual moleculw. During growth, cvllision betwren a single insulin monomer and the surface of the fibril hrings the moiiorner into assoviation with the requisite number of neighbors to produce a stable linkage. Thus the growth of a fibril was expected, and has been found, to be a function of the surface area of the fibril population and the concentration of insulin in solution. n’ucleation and growth have been found to vary differently with respect t o changes in ionic. strength, pH, etc. The occurrence of nucleation and growth with different kinds of chemically modified insulin and under different experimental conditions has been examined (152). Fibril growth a t low temperatures, regeneration of insulin from insulin fibrils, the fact that insulins having :z variety of blocked chemical groupings will form fibrils (FA, FE, FD), and the fact that the SS bonds must remain intact effectively eliminate covalent bond formation as being responsible for interinsulin linkage. For much these same reasons and the fact that fibril nucleation and growth occur a t low pH values electrostatic interactions are also effectively eliminated. The authors cwisider the possible significance of hydrogen bonds and van der ‘Waals’ forces. Hydrogen bonds appear unlikely on the hasis that fibril growth (*antake place and will proceed to completion in the presence of 8.3 M aretic acid, of other organic acids, or of 5.3 to 6.0 M urea a t p H 1.6 or pH ’7.0,with the above chemically modified insulins, and in solutions containing high roncentrations of ethyl alcohol. It appears likely that the inteiactions of nonpolar residues, a type of linkage proposed earlier (147), are mainly responsihle for the stability of the fibril. AdditionaI evidence for this view comes from the observations that: (1) the introduction of nonpolar groups through azo coupling produces a fibril which depolymerizes only with great difficulty in alkali (as do fibrils from esterified insulin), and (2) an increased tendency to reversible association in acid is obtained when insulin is so treated. Thus, the introduction of 10 azo-tolyl groups per molecule (154) makes insulin rompletely insoluble. With 5 groups, the insulin is soluble in 0.1 N HC1 but has a sedimentation constant of S 10.0, suggesting the association of 20 molecules. On dilution, a component S 2.4 appears, suggesting a depolymerization into monomeric units. This association-dissociation system is receiving further attention. The transformation of one type of insulin fibril into another becomes of particular interest in view of the structural studies to be described next. A system of fibrils is first prepared in acid using native insulin (FN). If

- -

DAVID F. WAUGH

377

this system be treated with 10%phenol (pH 1.6, GOT., 15 minutes) fibril formation is reversed and over 90 % of the insulin may be obtained in crystalline form. If the reversion time in phenol he extended from 15 minutes to about 1 hour, a new system of insulin fibrils develops which the authors (152) designate FN-FN(P). These fibrils are particularly resistant to reversion in alkali and are therefore clearly different from FN in this respect. Kolturi ef al. (155) by means of X-ray diffraction have examined essentially the group of insulin fibrils given above. The patterns obtained are somewhat diffuse, but, within these limits, the diffraction patterns given by FN, FA, FE, FD, FN-FN(P), and fibrils obtained by seeding a t low temperature are identical. The rather large differences in structure which must be represented by native insulin and insulin carrying 11 phenylazo groups per molecule are to be accommodated within the same basic molecular arrangement. A comparison is made of the spacings observed for insulin fibrils and those obtained by Low (12) for insulin sulfate crystals, which show, after air-drying, in addition to the crystal pattern a superimposed fiber pattern. A general correspondence in monomer packing is suggested. A fiber period of 48.5 A. is calculated from spacings of 16.1, 12.0, and 8.2 A. These and the fiber period suggest that in the fibril neighboring molecules are displaced progressively one third of the molecular length. Chains are believed to run parallel to the fibril axis. The structural aspects of the fibril are consistent with the kinetic requirement that several insulin molecules must co-operate in establishing a stable interinsulin linkage. The diffuseness of diffraction patterns, part of which is certainly due to irregularities in monomer packing, could be accounted for on the basis that the interaction of nonpolar side chains requires contact over an area as contrasted to the point-to-point approximation demanded, for example, by covalent bonds. A slight shifting of bonding regions relative to each other would be sufficient t o introduce irregularity. Additional surface groups could be accommodated by local modifications in side chain packing without requiring a new type of monomer arrangement. 5. Trypsin Pace (156), Northrop (151), Anson and Mirsky (157), and Kunitz and Northrop (158) have studied in detail the behavior of trypsin. Their criteria for denaturation were insohbility in 0.5 M salt sohtion at p H 2.0 and for the more recent investigations the loss of tryptic activity as measured by the hemoglobin methods of Anson and Mirsky (159). I n the latter procedure reactivation is suppressed by working a t optimal levels of pH, buffer, and urea concentrations. Reversal to the active state was carried out a t pH 2.0 in salt-free solution a t about 20°C.

378

PROTEIN-PROTEIN INTERACTIONS

Between pH 2.0 and pH 9.0 a t 30°C. irreversible inactivation is due largely t o srlf digestion and follows second-order kinetics. Deviations below pH 2.0 are accompanied by an increasingly rapid inactivation, following first-order kinetics, which is made irreversible a t higher temperatures. These two sources of irreversible inactivation are both small a t pH 2.3, a t which pH a progressive increase in temperature causes a progressive shift in the equilibrium between native and reversibly denatured protein, the latter having the insolubility characteristics described above. A similar situation occurs at p H values above 9.0 at 0°C. Between pH 9 and 12.0 there is a decreasing rate of self hydrolysis and an increasing level of reversible denaturation. Above p H 13.0 a transformation to a new irreversibly inactive protein occurs, more rapidly as the pH is increased. Irreversible effects are a t a minimum near pH 13.0, a t which pH all of the protein is “reversibly denatured.” That it is reversibly denatured is shown by the fact that a complete return in solubility and activity is obtained on standing in salt-free solution a t pH 2.0. Only a few minutes are required to reach equilibrium levels of reversible denaturation. Prolonged standing a t even the optimum conditions for demonstrating reversible denaturation will eventually produce irreversible denaturation, the structural changes leading to insolubility and loss of activity being fixed within the molecule. Stearn (180) has calculated thermodynamic constants from the data of Anson and Mirsky (157). These are given partially in Table XI. From the data of Pace (156) the reactions leading t o denaturation and reactivation have a t a temperature of 323.1’K. the following: Denaturation Reactivation

AH$

AF$

AS$

40.2 -27.4

25.7 27

44.7 -168.4

Trypsin is inhibited when it combines with another protein, soybean trypsin inhibitor, which is discussed next. Dobry an’d Sturtevant (161) TABLEXI Thermodynamic Data f o r the Denaturation of T r y p s i n AF

T

(kcal. per mole)

315.1 317.1 318.1 321.1 323.1

0.447 0 -0.190 -0.901 -1.269

AH (kcal. per mole)

t I

67.6

I

AS

(cal. per degree per moIe) 213.1 213.2 213.1 213.3 213.1

DAVID F. WAUGH

379

have measured the heat of reaction a t 25°C. and find it to be 1 kcal. per mole trypsin reacting. The fact that the trypsin-trypsin inhibitor complex is stable even a t high dilution suggests a large decrease in free energy which, in combination with the heat of reaction just given, suggests a rather large increase in entropy. Green and Work (162) find that the combination of trypsin and pancreatic trypsin inhibitor (mol. wt. = 9000) is not affected by denatured trypsin but is retarded by urea. The former observation suggests a specificity of interaction. The dissociation constant of the trypsin inhibitor complex was found to be 2 X I@1° M a t p H 7. The rate of attainment of equilibrium is rapid. 6. Crystalline Soybean T r y p s i n Inhibitor

Kunitz (163) has examined trypsin inhibitor thoroughly. Normally this protein, a globulin of molecular weight 25,000 and isoelectric point 4.5, is soluble in dilute acid or alkali and in dilute salt solution a t the isoelectric point. It is stable over a wide range in p H a t 30°C. Susceptible to heat denaturation in acid or alkali it remains soluble like many other proteins until brought to the isoelectric point, when it forms a precipitate under conditions where the native protein is soluble. When a solution of the protein in 0.0025 N hydrochloric acid (pH 3.0) is heated it becomes progressively denatured until a t 60°C. it is largely in the denatured form. The test for denaturation is insolubility in dilute buffer solution a t pH 4.5. Compared with other reversible denaturations that of trypsin inhibitor is relatively slow. At 45"C., where denaturation is 50% complete, 8 to 14 hours are required to achieve equilibrium. The denatured protein, in addition to being insoluble, will not crystallize, will not inhibit trypsin, and is more digestible by enzymes. Kunitz suggests that disulfide, sulfhydryl, and phenolic groups will be found more reactive. Reversal of denaturation was accomplished by lowering the temperature and storing for two days a t 25°C. Whatever the structural change involved, the kinetics for denaturation and reversal of denaturation were both first order at different protein concentrations, indicating purely intramolecular changes. If heating is prolonged, irreversible denaturation takes place. At 90°C. the irreversibly denatured protein rises from 5 % to 64 % between 2 and 60 minutes. Equilibrium levels of denaturation at different temperatures yielded the thermodynamic data given in Table XII.

7. Pepsinogen and Pepsin Herriott (164) found that pepsinogen (mol. wt. = 15,100, isoelectric point pH 3.7, see ref. 1) could be heated to 100°C. a t pH 7.0 without form-

380

PROTEIN-PROTEIN INTERACTIONS

TABLE XI1 Thermodynamic Data f o r the Denaturation of Crystalline Soybean Trypsin Znhibitor

AH = 57 kcal. per mole A S = 180 cal. pe-r degree per mole AHS1 = 55 kcal. ,per mole A S t 1 = 95 cal. per degree per mole For reversal of denaturation (activation AH$z = -1.9 kcal. per mole ASSa = -84 cal. per mole per degree step) Changes in heat of reaction Change in entropy of reaction For denaturation (activation step)

ing a precipitate. I n this case denaturation was demonstrated as an insolubility in 1 M magnesium sulfate and an absence of activity after a standard acid activation treatment. Denaturation was reversed in 0.05 N hydrochloric acid by standing for 5 minutes at 35°C. A gradual shift in activity occurred over the range 50°C. (97 % active) to 70°C. (3 % active). The heat of the reaction was calculated as 31 kcal. per mole pepsinogen. Heating at 100°C. gradually converts, according to a first-order reaction, reversibly denatured protein into irreversibly denatured protein. A similar equilibrium occurs over the pH range 8.5 to 10.5. Reversal of denaturation is obtained a t pH 8.5. It is of interest to note that alkaline denaturation becomes irreversible at higher protein concentrations; that acetylation of amino groups has no detectable effect on alkaline inactivation; arid that prolonged standing causes the appearance of irreversibly denatured protein (also indicated by enzymatic assay). Northrop (165) had previously described similar effects with the protein pepsin. This enzyme was completely denatured at pH 10.5 but was found to recover solubility and activity if titrated to pH 5.4 and allowed to stand for 24 to 48 hours a t 22°C. In addition to recovering activity and solubility the reactivated material could be crystallized. Sturtevant (166) has recently examined the pH dependence of the heat content change occurring in the denaturation of pepsin. The maximum in the AH vs. pH curve is abnormally sharp and requires the assumption of a triggered meehanism. The difficulties encountered in interpreting heat data are pointed out. For example, the heat data a t 38°C. lead to an ionization involving 5 protons, whereas the variation in the rate of heat absorption with pH points toward an average of 1.4 protons. The interpretation of Sturtevant is similar to that of Steinhardt and Zaiser (231, see “Hemoglobin”). 8. Chymotrypsinogen

Eisenberg and Schwert (167) chose this protein since its denaturation could be studied without the complications which result from proteolysis.

DAVID F. WAUGH

381

Normally (1) chymotrypsinogen has, according to the most recent determinations, a molecular weight of 22 to 23 X lo3and a frictional ratio, f/fo , of 1.13 to 1.19. I n their examinations of reversible denaturation, Eisenberg and Schwert define the native protein as that which is soluble in 0.083 M glycine buffer containing 1 M sodium chloride at p H 3.0. Denatured protein is insoluble in the same buffer. Reversal of denaturation was carried out by allowing the solutions to stand for 24 hours a t room temperature. -4series of preliminary experiments a t 50°C. and lOO"C., with heating carried out for 15 minutes or 2 minutes, showed that reversal of denaturation could be accomplished in the pH range 2 to 3. Protein solutions, about 0.25 %, were made in salt-free hydrochloric acid. At pH values between 4 and 6.5 no denaturation was observed a t the lower temperature, whereas considerable irreversible denaturation occurred a t 100°C. Between pH 6.5 and 9.4 the protein coagulated during heating a t 100°C., whereas a t p K values higher than 9.4 irreversible denaturation was not accompanied by a precipitate. Between pH 2.0 and 3.0 reversible equilibria were set up in the temperature range 40 to 50°C. These equilibria were found to be independent of the protein concentration in the range 0.25 % to 0.75 %. At 50"C., a plot of the logarithm of the equilibrium constant versus pH yielded a straight line of slope 3.16. The intercept suggested a pK, of 2.5. Thus, a decrease in pH of 1 unit causes the equilibrium to shift from completely native protein to almost completely denatured protein. Criteria such as crystallizability, solubility, activation, sedimentation rate, and behavior on reheating suggest a close identity between native and renatured chymotrypsinogen. A series of equilibria between native and denatured protein were studied and were analyzed on the reasonable basis that the foreward and backward reactions would follow first-order kinetics, thus

-4selection of data is summarized in Table XIII.

Eisenberg and Schwert are inclined to attribute the reversible denaturation to dehydration on the basis that the low heat of dissociation of carboxyl groups can account for only a small part of the over-all heat change. Arguments in support of this view are: (1) reaction rates are high, (2) the behavior of solutions of reversibly denatured chymotrypsinogen toward dilute salt solutions is parallel to the behavior of lyophobic sols in the presence of salt, (3) no definite change could be detected in the properties of the reversibly denatured proteins in comparison with the native protein, (4) the large entropy change is in accord with the view that water of hydration (assumed t o be similar in structure to ice) is removed from polar groups.

w

M

ts

TABLE XI11 The Reversible Denaturation of Chymotrypsinogen

p H 9.0

Temp., "C.

A H , kcal./mole A F , kcal ./mole A S , kcal/mole/"C.

99.6

43.6 -0.3

45.5 -.914

p H 3.0 46.4

47.2

-1.20

-1.44

202 -15.7

0.427

57.4 0.162

58.2

59.2

60.1

-.a7

-.6i3

-1.04

432

316 84.5

56.7 143

20.7 21.0

20.4 21.3

20.2 21.4

19.8 21.3

80.2 178 -63.7

21.7

21.5

21.4

21.2

21.1

21.3

21.3

21.6

21.8

22.1

DAVID F. WAUGH

383

On this basis 69 moles of water must be removed in the denaturation of 1 mole of chymotrypsinogen at pH 2.0 and 99 moles at pH 3.0 (the removal of about one fourth of the average hydration of proteins of 0.3 gm. water per gram protein). Suppression of carboxyl group ionization, with the consequent decrease in bound water, might well account for the smaller amount of water assumed to be removed at the lower pH. The differences between the reversible denaturation reactions at pH 2.0 and pH 3.0 (Table XIII) have not been accounted for in any quantitative way. Eisenberg and Schwert point out that the failure to do so may lie in the fact that the reversibly denatured protein formed at pH 2.0 is not identical with that formed at pH 3.0-a possibility for which other evidence is presented. Eisenberg and Schwert (167) in connection with denaturation describe another phenomenon of considerable interest. They added 0.002 to 0.02 M sodium chloride to a 0.224% protein solution at pH 2.0. On heating at 43°C. instead of observing an equilibrium they observed a progressive disappearance of native protein with time. The precipitable protein, recovered after cooling and standing 24 hours, was found to be soluble in 0.001 N hydrochloric acid on heating for a few minutes in a boiling bath. This solution, on being cooled, behaved identically with native chymotrypsinogen under conditions which lead to reversibility on heating and cooling. Ultracentrifugal examination of a similar solution (0.442% protein, 0.05 M glycine buffer, pH 2.0, 10 minutes at 52" C.) revealed, in addition to the normal component, one having Szo = 16.3 or roughly a molecular weight of 5 X lo6. As with the other proteins examined, prolonged heating at the higher equilibrium temperatures and pH 3.0 leads to some irreversible denaturation. Irreversible denaturation has a high concentration dependence, indicative of aggregation. This was verified in the ultracentrifuge on examining a 0.88% protein solution which had been heated a t pH 3 and 56" C. for 45 minutes. 9. Chymotrypsin

Kunitz and Northrop (168) some time ago reported a reversible denaturation of chymotrypsin similar to that detailed above for chymotrypsinogen. Schwert (169) and Schwert and Kaufman (170) have examined the interesting monomer-dimer association of a-chymotrypsin. Gladner and Neurath (171) give details of the activation of T , 6, and a-chymotrypsin. Chymotrypsinogen (172) has a sedimentation constant, SZO , extrapolated to infinite dilution, of S = 2.7. The variety of experimental conditions used, pH 3.86 to 6.20 and p 0.2 to 0.5, shows that the molecule

384

PROTEIN-PROTEIN INTERACTIONS

is stable in this range. Its molecular weight was calculated to be 23,000, and its frictional ratio to be 1.18 (viscosity and S-Dvalues). A molecule of low asymmetry is indicated, since a 30% hydration would give a frictional ratio of 1.13. On being activated (171) to form a-chymotrypsin, the closed polypeptide ring of chymotrypsinogen is opened and a basic peptide of mol. wt. 1000 is split off. (I-Terminal groups of leucine and tyrosine remain. In the same pH and ionic strength range as given above (pH 6.2 and = 0.2), a-chymotrypsin exhibits a concentration-dependent association. A recent extensive examination (170), based on viscosity measurements and determinations of S and D with respect to concentration, is analyzed in terms of the association of the two monomeric units to give a dimer. In an earlier paper (169), Schwert reported that y-chymotrypsin likewise associated.

-

10. Serum Albumin

The interactions of human serum mercaptalbumin in the presence of mercurials (Edelhoch et al., 173; Edsall et al., 174) present recent evidence not only of interest in defining the conditions under which interaction may be observed but in understanding the complications to be surmounted in making a physical interpretation of thermodynamic data. The albumin molecule has a molecular weight near 69 x l o 3 and n frictional ratio of flfo = 1.28 (1). Mercaptalbumin, denoted here as ASH, which constitutes the major fraction of serum albumin ( w 70%), has one free SH group and interacts rapidly with mercuric salts, HgXz , where X is chloride or acetate, to give ASHgX. If free albumin is present a slower reaction takes place giving ASHg+

+ -SA

@

ASHgSA

(25)

A similar series of reactions takes place with an organic mercurial XHgRHgX: XHgRHgX

=

/

0-CHI

XHgCHzHC.

giving ASHgRHg+

\

+ -SA

CHz-0

\

/

CH. CHzHgX

F? ASHgRHgSA

(26)

The reactions represented by Eqs. 25 and 26 may be inhibited by an excess of mercury salt, which converts all ASH present, to ASHgX. The interactions given above are so favored that interaction of the mercury

385

DAVID F. WAUGH

salts with other reactive groups normally present may be neglected, provided that the molar ratio of total mercury to total sulfhydryl sulfur is less than or equal to unity. This permits a calculation of equilibrium constants and reaction velocities. The former are actually obtained by examining the inhibition of interaction by a competing ion such as CN. The ratio of the equilibrium constants for the two reactions (I(,= 101a.6 and Kz = 1018.2in acetate buffer a t pH 4.75, ionic strength 0.05, and 25°C) is 5 X lo4, which leads to a molar free energy change of 6.4 kcal. per mole in bringing the albumin molecules 10 A. closer (175), the latter value being calculated as the difference in the distances between the sulfur atoms in the two dimers ASHgSA and ASHgRHgSA. The difference in equilibrium constant may well be due to repulsion and steric factors. The velocity constant for the reaction ASHgRHgX ASH -% ASHgH+ X- is about 2000 times as fast as that for the reaction RHgSA ASHgX ASH --k ASHgSA H+ X- . Both velocity constants decrease with increasing pH and in the same proportion, since parallel lines are obtained when log kz is plotted against pH between the pH limits 4.8 and 6.0. These changes in velocity constant have been interpreted in terms of the changes in electrostatic work involved in bringing the two molecules together (173). The equilibrium constant K’ = (ASHgSA)/(ASHgX) (ASH) is relatively independent of pH over the same range, although the equation for the reaction as written above indicates that it should decrease as (H+) - and also (X-) - increases. The observed result is probably due to the different effects of increasing net charge on the relative activity coefficients of monomer and dimer. The energy of activation, obtained from the temperature coefficients of the reactions, is approximately 18 to 22 kcal. per mole in both cases. Edsall et al. (174) have used these values and the observed velocity constant to calculate the entropy of activation for dimer formation with the 14 cal. per degree per mole a t organic mercurial, and have obtained A S pH 5.5 and 26 E.U. at pH 6.0. Edelhoch et al. (173) calculated the entropy of activation for formation of the mercury dimer as being 0 to 2 E.U. As was mentioned in connection with insulin, one would expect large negative changes in entropy on association, these being due to decreases in the translational and rotational degrees of freedom which attend linking. Edsall and collaborators suggest two possible mechanisms to explain the discrepancy. The first assumes that for steric reasons a configurational change must take place to make linking possible. If such a change were akin to denaturation (reversible), a large positive change in entropy would be expected. An examination of optical rotation, however, did not indicate a structural change on association (see Kauzmann and Simpson, 204, for optical changes in urea), suggesting that if such changes took

+ +

+

+

+ +

N

386

PROTEIN-PROTEIN INTERACTIONS

place they were confined to the active complex. The second possibility is similar to that proposed by Doty and Myers (139) to account for similar entropy changes on the association of insulin, as discussed under this protein. The removal of water from charged groups would provide an entropy increase. It is pointed out (174) that this explanation is difficult to accept for the dimerization of albumin. The entropy change is considerably larger and more positive for the organic mercurial dimer than for the mercury dimer. The mercury dimer, however, requires the closer approach, and one might expect surface interactions leading to positive entropy changes to be larger in this case. It should be remembered that the entropy changes discussed here are those associated with activation. Crystalline serum albumin per mole normally contains about 3 moles of a fatty acid, as described by Cohn et al. (175). EdEiall (176) describes experiments of R. H. Maybury in which it was found that fatty acid-free albumin not only reacted more slowly to form the mercury dimer, but the reaction curves were more complex. A marked increaue was obtained on adding 3 moles oleate ion per mole albumin. The changes in rate cannot be due to net charge, for a similar change in this variable produced by pH variation, decreases the rate.2 Edsall (176) also refers to observations by H. M. Dintzis and J. I,. Oncley on crystallized bovine mercaptalbumin, prepared practically free of fatty acid. The equilibrium constant of dimerization is larger and the rate of dimerization is considerably increased as compared with human mercaptalbumin. The bovine material appears to be rnore homogeneous. Straessle (177) has taken the mercury dimer ASHgSA, and oxidized it in the solid state with iodine a t -8 to -18" C. He obtains a 75% to 80% yield of the disulfide dimer ASSA, which does not di,ssociate on adding excess mercury. It can be returned to the monomeric form by careful reduction with cysteine. a. Aggregation. Using solubility as their criterion, Anson and Mirsky (178) make reference to the extremely rapid reversal of acid denatured serum albumin to the native state; indeed in those early experiments denaturation was difficult to establish. The authors (178) denatured serum albumin in 0.023 N acid at 100" C. A t this acidity precipitation does not occur at the higher temperature. The precipitate which forms as a result of neutralization at a lower temperature can now be dissolved in 0.1 N hydrochloric acid and the fraction of this material soluble in halfsaturated ammonium sulfate (yield 65 %) can be crystallized. Gutfreurid and Sturtevarit (179) report heat absorption as the pH of an albumin

* It is possible that this change is due to an interaction of the fatty acid molecule with the internal volume which allows the protein more easily LO assume the correct bonding configuration. Effects of this type will be discussed below.

387

DAVID F. WAUGH

solution is lowered from 4.5 to 3.4, the reaction absorbing 3.1 kcal. per mole at 25" C. The reaction follows first-order kinetics, is not an association or dissociation, and is reversible. Between the stated pH limits equilibria are established. The ionic strength used was 0.1. In a similar pH range (pH 4.4 to 2.0) Bjornholm et al. (180) report an increase in viscosity relatively independent of the temperature below 30" C. (see also Aten et al., 181). The last reference cited is an example of the several lines of evidence which suggest that in acid at ordinary temperatures, serum albumin (and other proteins to be discussed) undergo an unfolding unaccompanied by aggregation. Additional evidence comes from studies of optical rotation (Aten et al., 181 ; Jirgensons, 182), fluorescence polarizationa (Weber, 183), titration (Tanford, 184) and from combined studies of viscosity and optical notation (Yang and Foster, 185). In each case serum albumin (horse or bovine) is relatively constant in the property examined from pH 4. Between pH 4.0 and pH 2.0 values above neutrality to a pH of each property changes, its maximum change occurring at the latter pH. If aggregation has been avoided, a considerable degree of reversibility is evident, for neutralization is sufficient to restore nearly normal properties. For example, Cooper and Neurath (186) in an examination of the effect of heat treatment on solutions of horse serum albumin found that aggregation occurred at pH values 7.6 and 4.2 (70"C., 0.5% protein, p 0.2, and heating times up to 120 minutes). No differences in viscosity and diffusion coefficient were observed between heated and unheated protein solutions when heating was conducted at pH 3.6, although as seen above a t this pH certain of the intrinsic properties have started to change and the effect of increased temperature would be to displace such changes in the direction given by lowering the pH. It is reported that the protein heat treated a t pH 3.6 had an electrophoretic mobility 20% higher than that of the unheated protein. Apparently the additional positive charge has stabilized the protein against aggregation, but, as in the experiments of Anson and Mirsky cited above, the irreversible changes in structure which can lead to drastic changes in shape and solubility properties have not occurred. When very sensitive properties such as optical rotation are under consideration (see Simpson and Kauzmann, 187), it seems clear that reversal is not quantitative. As will be seen below, the unfolding or swelling of a

-

-

-

a It is interesting that Weber's (183) data on polarization of fluorescence suggest that theserum albuminmoleculedissociatesin acid solution. This is opposite to many other lines of evidence which suggest that the serum albumin molecule is quite stable so far as size is concerned. Yang and Foster (185) have suggested that Weber's data may be interpreted in terms of an increase in intramolecular freedom, due to unfolding, without the need of postulating an actual dissociation of the whole molecule.

388

PROTEIN-PROTEIN INTERACTIONS

protein should not be expected to be reversed in the quantitative sense. In molecules having the structural complexities of proteins, experimental manipulation requires that limits be placed on reversibility, rather than that a complete and absolute reversibility be expected. Although aggregation can be observed in serum albumin solutions at pH values below 2 to 3 (or above 8, see ref. 190), the necessary molecular change is usually induced at a higher temperature (60 to 100” C.) and visible aggregates caused to form by cooling or more often by neutralization. Heating albumin solutions between pH 4.0 and 8.0, on the other hand, can lead directly to the formation of aggregates (Spiegel-Adolph 188; Joly and Barbu, 189; Cooper and Neurath, 186) or of superaggregates, as evidenced by the formation of a gel or a precipitate (Jensen et al., 190; Jaggi and Waugh, 191). The physical properties of the aggregates are sensitive to the conditions leading to their formation, mainly, pH, ionic strength, protein concentration, temperature, etc. (186, 189, 190, 191). Between pH 5.0 and 6.0 (190) very low concentrations of protein lead to dense aggregates even at low ionic strengths. Below pl1 5 , a t low ionic strengths, clear gels are produced, whereas a t higher ionic strength visible aggregates result. Above pH 6.0 clear gels again result at low ionic strengths. In all cases, as the pH deviates progressively below 5 or above 6, the rates of gelation decrease and the “minimum” amounts of protein required for gelation increase (e.g., from 0.2% a t pH 4.8 to 3.2% a t pH 4.5, both in 0.1 M acetate buffer). The results of Cooper and Neurath (86), in which aggregation is obtained a t pH 4.2 or pH 7.6, and of Joly and Barbu (189), who studed aggregation of bovine serum albumin a t pH’s between 4.2 and 7.6, excluding the range pH 5 to pH 6, show that the disappearance of gelation in the experiments of Jensen and co-workers is due, first, to an insufficient number of fibrils of high enough axial ratio to form a continuous space filling network and, second, to a decrease in the number and strengths of the bonds responsible for cross linking. The clear gels have been shown (189, 191) to be composed of fibrils of approximate lengths of 3000 A. and diameters of 200 A., the segment distance given being approximately that between cross links. As has been indicated above there is little doubt that the physical changes attending denaturation in the absence of aggregation can be largely reversed. The possibility of reversing fibrous aggregates has been examined by Spiegel-Adolph (188) and by Waugh and Appel (192). Spiegel-Adolph noted a return in solubility of heat precipitates of dialyzed serum albumin treated with alkali. The regenerated albumin had a heat precipitability similar to the starting material. In the examination of TVaugh and Appel serum albumin fibrils prepared a t pH 4.1 and ionic strength 0.07 were

DAVID F. WAUGH

-

389

dissolved in alkali at pH 12 and 0°C. The protein soluble under the same conditions as native albumin (yield 35%) was found to have the same sedimentation constant, diffusion constant, and electrophoretic mobility as native albumin. It differed in its capacity to form crystals in that it gave aggregates which resembled crystals with rounded edges and corners. Jensen et al. (190) found that the addition of reagents which react with free sulfhydryl groups profoundly affects the course of coagulation and the nature of the coagulum. Thus in the pH range 6.9 to 7.4, thermal coagulation produces a soft opaque gel. Firm, clear clots are promoted by the addition, a t levels of about 20 equivalents per mole albumin, of sulfhydryl reagents such as iodoacetamide, iodine, p-chloromercuribenzoate, silver nitrate, mercuric nitrate, and hydrogen peroxide. In contrast sodium iodoacetate is a marked inhibitor. The authors point out that this reagent introduces additional charges (carboxyl ions) into the protein molecule. More opaque clots of less rigidity are given by cysteine, sodium cyanide, and propyl mercaptan. Interestingly, acetylation of amino groups imparts a marked resistance to coagulation. The possibility that the status of a single SH group may produce these far-reaching effects is noted. Haurowitz and Kennedy (193) report that bovine serum albumin, dissolved in 1.0 M sodium thioglycollate at pH 8.5, will polymerize extensively. The extensive polymerization (gelation) of bovine serum albumin (and ovalbumin and globulin) treated with 0.2 M sodium thioglycollate or monothioglycol in 0.1 M phosphate buffer at pH 7.4 and 37" C. was reported by Huggins et al. (194). Gelation took place in about 20 hours. The requirement for reducing agent decreased from 0.34 M to 0.08 M as the protein concentration increased from 1.2% to 5%. At 0" C. gelation was not detectable in 72 hours. A number of similar reagents produced gelation. Gelation was found to be favored by an increase in pH, above 7.4. With decreasing pH values the gel structure is replaced by a clear solution at pH values between 5 and 6.6 (0.2 M thioglycollic acid), while at pH 4.5 a synerizing precipitate is formed. Urea in 5 M concentration will dissolve these gels, although, as will be seen below, urea can in itself induce gelation (the urea gels, as pointed out by Huggins and associates, are different in character, i.e., more cohesive). Huggins et al. (194) conclude from the solubility of gels in urea that hydrogen bonds are responsible for gelation. That the linkages involved in coagulation may alter with time, even in a precipitate, is shown by Kertesz (195), who produced precipitates by heating a 0.5% bovine albumin solution a t 100" C. in 0.05 M phosphate buffer at pH 6.8. The solubility of the precipitated protein in 75 % acetic acid was examined. It was found that under these conditions serum albumin precipitates completely in 0.5 minute. At the start this precipi-

390

PROTEIN-PROTEIN INTERACTIONS

tate is entirely soluble in 75% acetic acid, but after heating for 4 to 8 minutes it becomes entirely insoluble. Similar results were found for y-globulin and egg albumin. b. Urea. There is little doubt that treatment of proteins with urea (or guanidine salts) may produce profound changes in structure. Parallel to the observations made above, conditions may be chosen with serum albumin so that these changes are largely reversible, certainly reversible with respect to all grosser aspects of structure. Burk (196) found that this protein may be treated with 6.66 M urea a t the isoelectric point without dissociating the molecule. Neurath and co-workers (197, 198, 199, 200) made a study of the general phenomenon. The albumin was denatured a t room temperature and pH 5.0 in 0.023 M acetate buffer containing 0.2 M sodium chloride and the requisite urea or guanidine. Reversal of denaturation was accomplished by dialyzing away the urea a t 4 to 5" C. with running water. The irreversibly denatured protein, which remains soluble or in suspension under these conditions, was removed by adjusting the suspension t o pH 5.25 (with 0.2 N sulfuric acid) and heating a t 41" C. for 30 minutes (198). At this time it was found that the reversed supernatant protein had properties similar to native albumin but that it differed somewhat in respect to crystallization, solubility, and electrophoretic behavior. The reversed protein was easily digested with trypsin (199). I n the denatured state the molecules exhibited radically altered shapes, as indicated by measurements of diffusion constants and viscosities. The magnitude of the change is a function of urea concentration, and, as has been pointed out (Neurath et al., 58), the greater the change induced in each molecule, the less likely it is that the molecule can return to the nearly "native" state in which it shows solubility. Neurath and coworkers calculated the changes in diffusion constant and viscosity in terms of a change in shape. They obtained a change in axial ratio from about 5/1 in the native protein to 20/1 in the presence of area. A return to the former shape was obtained after dialyzing out the urea. Scheraga and Mandelkern (201) point out that the experimental data (197) can be accounted for also by an increase in volume due to swelling. Doty and Katz (202) cgme to the conclusion that nearly isotropic swelling was involved. Bresler (203) also reports a combination of serum albumin with 360 moles urea per mole albumin. Aten et al. (181) observed that the customary increase in the lev0 rotation observed in urea (or after heat treatment) could be reversed by dilution (at low temperatures), suggesting a fairly precise restoration of the native distributions of the side chains and other groups. Although after urea treatment there is an increase in rotational freedom, as indicated by studies of the depolarization of fluorescence (Weber, 183), no changes in molecular weight are indicated.

391

DAVID F. WAUGH

Kauzmann and Simpson (204) also note the immediate increase in lev0 rotation which is reversible a t all urea concentrations used (up to 11 M ) a t temperatures below 40°C. Above 40" C. irreversible changes in rotation are observed, for after cooling the rotation is always increased above the original values. The kinetics of the changes in optical rotation are complex (204, 187). Frensdorff et al. (205) have studied the viscosities of urea solutions of serum albumin. At low protein concentrations (1% to 1.5%) and pH values near 5.0 the rapid initial increase in viscosity in the presence of urea is largely reversible on removing the urea, suggesting, again, a reversible change in shape and volume. Interaction of protein molecules may accompany and may of course be dependent upon the structural changes indicated above. Hopkins (206) found that horse or sheep serum albumin treated at pH 5 to 7 at 22" C. was markedly resistant, as compared with egg albumin, to the insolubilizing effects of urea. He noted that a jelly would form if a concentrated solution of protein were saturated with urea. Urethane had a more pronounced effect. On prolonged exposure to saturated urea the solubility characteristics are altered, since the albumin becomes insoluble if immediately diluted in the presence of salt. Huggins ei al. (207) have extended observations on the gelation of serum albumin by urea using as conditions 37" C., 0.05 M phosphate buffer a t pH 8.0, and 8 M urea. They note that high protein concentrations are necessary (3.4% serum albumin, 3.2 % egg albumin, and 5.6 % y-globulin) and that acetylated albumin does not gel in urea. Under the conditions used, strong evidence is presented that a mercaptan exchange reaction, leading to the formation of interS z S 2-+ S I S z SZH),can account molecular disulfide groups (SIH for gelation. This evidence is based on the following facts: (1) with respect t o albumin, equimolar concentrations of reagents that destroy -SH will inhibit coagulation, (2) the rate of gelation increases with pH, (3) gelation may be restored in inhibited systems by the addition of S H compounds, and (4) gels may be dissolved in higher (0.1 to 0.2 M ) concentrations of mercaptans. Caution should be exercised in extrapolating these concludions to gelation under other conditions, particularly to syetems which gel a t much lower pH values. Gelation in the presence of urea a t pH values near 10.1 has been further examined by Frensdorff et al. (205). Increasingly with increase in pH and urea concentration an aggregation takes place which is not reversible on dilution or neutralization. They agree with Huggins et al. (207) that blocking of S H groups retards gelation, that S H reagents such as cysteine accelerate gelation, and that a mercaptan exchange reaction is probably involved. The effects of sulfite (205) on aggregation present anomalies. It acts as an electrolyte which should suppress electrostatic

+

+

392

PROTEIN-PROTEIN INTERACTIONS

repulsion and, therefore, promote aggregation (as does sulfate) arid as an agent which breaks -S-Sbonds and should therefore suppress aggregation. A t 2 % protein concentrations 0.02 M sodium sulfite produces a leveling off of viscosity at a level much lower than that given in the presence of sulfate. Significantly at 1 % protein concentrtition there is no leveling off, the viscosity achieving abnormally high values. The authors suggest that a balance between the effects of sulfite on inter and intramolecular SS-bonds is involved. It would seem that an inhibition at the lower concentration would be expected and that, therefore, bonds other than disulfide bonds are also involved in intermol.ecular linkage. c. Other Interaction. Certain other interactions of serum albumin, although not as extensively studied as those given above, are instructive. Thus, Carroll et al. (208) found that subjection of air-sahurated solutions of serum albumin to 20 X lo3 - 15 x lo6 R of X-rays produced little change in reactive groups, no degradation products, and an increase in 300,000 had appeared in suspension extinction. Aggregates of mol. wt which, though remaining “soluble” at the isoelectric point, were much more sensitive to heat. Sulfhydryl groups have not been implicated in these changes; rather, they are attributed to the formation of groups such as biphenyl. Geiduschek and Doty (209) examined the interaction of bovine serum albumin and deoxyribonucleate. Strong interaction was found to occur only when the albumin had been denatured. Steiner (210) finds an interaction between serum albumin and lysozyme when the particles are oppositely charged. The reaction is readily reversible on increasing the ionic strength. Bresler (203) reports an informative reaction of sohtions of serum albumin in phosphate buffer of 0.03 M and pH 6.5. On saturation with ether (6.6 %) and on the addition of acetone (5 % and 10 %) the sedimentation behavior indicated a swelling in each case of 18.7, 8, and 15.9%, respectively. On saturation with benzene two peaks were observed, one due to serum albumin and the other apparently due to a dimer.

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11. Hemoglobin

Hemoglobins may dissociate or associate, may denature reversibly, and may denature irreversibly depending upon the species of hemoglobin used, its chemical state, whether it is a naturally occurring, normal, or abnormal protein, as well as the precise experimental conditions. As with most other proteins, it is relatively easy to convert hemoglobin to a form which shows a greatly altered solubility, the agents which produce such a change being, characteristically, heat, acid, alkali, and treatment with urea, guanidine, salicylate, alcohol, etc. In most instances if the agent is applied

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with sufficient intensity, the change in solubility with respect to the native protein is found to be irreversible. Of considerable importance from the standpoint of protein-protein interactions are those instances where reversible changes occur. This is even more the case because of the presence of heme groups, and the characteristics of the latter when associated with the globin moiety. The structure of the hemoglobin molecule, previously referred to (see Section 111, The Native Protein), is of particular interest in examining the various interactions of this molecule. The horse hemoglobin molecule, according to data obtained from X-ray diffraction, may be taken as a structure 65 A. long and 55 X 56 A. in the other directions (hydrated) or 57 A. long and 40 to 50 A. in the other directions. On the basis of the proposed model the a-helices run in the 57 A. direction. There are four heme groups per molecule and, according to Perutz (211), these are oriented perpendicular to the directions of the main chains (helices). The combining power of hemoglobin with alkyl isocyanates, compared with the combining power of heme, suggests that the heme groups are surrounded by protein in such a way that the oxygen, which attaches to the first heme and encounters the highest energy barrier, causes a structural change in the protein moiety which allows additional oxygens to attach progressively more easily to the other hemes (St. George and Pauling, 212). Pauling (see 213) suggests that the hemes are inserted into slits between layers of globin. Figure 6, taken from ref. 213, illustrates the mode of heme attachment proposed by Pauling. The figure also illustrates the changes in

FIQ.6. A drawing indicating some of the features of the structure of the hemoglobin molecule, and the postulated mechanism of sickling of sickle-cell-anemia erythrocytes. The four hemes are indicated t o be contained within slits i n the hemoglobin molecule, their planes being perpendicular t o the axes of the helical rods in the protein. At the right the molecules of sickle-cell-anemia hemoglobin, without oxygen attached t o the hemes, are shown as having self-complementary configurations, which permit them t o aggregate into long strings of molecules. At the left, the addition of oxygen or other ligand t o the hemes is shown as swelling them enough t o destroy t h e self-complementariness of the molecules, thus interfering with the formation of t h e aggregates.

394

PROTEIN-PROTEIN INTERACTIONS

structure presumed responsible for the sickling of cells on the removal of oxygen-a subject which will be returned to below. It must be remembered that the denaturation changes observed with hemoglobins may or may not be accompanied by the splitting of the hemeglobin association. Drabkin (214) has noted that the dissociation of this link in alkali and even in acid of 0.1 M concentration is a relatively slow process. Acid converts methemoglobin, as noted by Keilin (215), into an acid hematin, a substance in which the relationship between the pigment and the protein is probably that of an adsorption complex rat'her than of chemical combination, the globin functioning as a protective colloid. According t o Holden (216), the conversion to the acid hematin form is initiated a t pH 3.9 and is complete as pH 3.0. I n instances to be described below no immediate and obvious decision can be made regarding the heme-globin link, for there must be a number of intermediate stages between hemoglobin, acid hematin, and globin and hemin crystals. An additional factor of considerable importance in estimating the relative forces involved in all of the dissociation reactions to be described is the finding of Tiselius and Gross (217) that dilution alone (below 0.66%) is sufficient to cause dissociation of COHb, dissociation being extensive a t 0.17% (see also Svedberg and Pedersen, 218). In view of this it is not surprising, for example, that urea may dissociate hemoglobin without changing some of its sensitive characteristic properties. Lewis (219) was among the first to examine the denaturation of bovine oxyhemoglobin near neutrality. Hemoglobin solutions of 1 % concentration were heated a t temperatures between 60 and 70" C., and the denatured protein was taken as that appearing as a precipitate during heating or immediately on cooling. The rate of denaturation wals examined as a function of pH, temperature, and salt concentration. According to this type of determination, denaturation had a sharp rate minimum near pH 6.7, rising rapidly on either side. Lewis notes that this is not the isoelectric point of the protein but the neutrality point for water, and notes also that the activation energy (77.5 kcal. per mole) is probably a composite term. In a study of the effect of ammonium sulfate, :Lewis found that low concentrations (0.5 %) displace the rate curve toward lower pH values. High concentrations (10%) have a similar effect but also depress the minimum rate, and, therefore, have a marked retarding effect a t this and lower pH values. a. Acid. The interactions on the acid side of the isoelectric point may be conveniently divided into those in which a complete dissociation of the heme-globin link does not take place and those in which such a dissociation is clearly made. Above a pH of 3.9 (Holden, 216, 220) the heme-globin

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link is stable. It becomes unstable as the pH is decreased, forming completely the adsorption complex noted above a t pH 3.1. The fact that a denaturation may have been carried out at even, for example, a pH of less than 3.1 does not necessarily mean that complete dissociation of the hemeglobin link has taken place, for, as noted above, complete dissociation in even 0.1 N acid is a slow process. b. Experiments with Incomplete Heme-Globin Dissociation. Mirsky and Anson (211) took 1 cc. ox blood, added 8 cc. water and 1 cc. 0.2 N HCL, and heated at 80"C. for 3.5 minutes. While at 80"C., 3 cc. of a phosphate buffer was added to return the system to pH 6.8. After cooling the hemoglobin forms a brown precipitate which shows none of the solubility properties of hemoglobin. Mirsky and Anson assumed that the step of structural alteration proceeded at 80" C. and that it was largely independent of the subsequent dependent coagulation step. Reversal was accomplished by carrying the above procedure through the step of heating. Instead of adding phosphate buffer before cooling, the solution was cooled, 10 cc. 0.04 N NaOH were added to make the system faintly alkaline, a little NazSz04 was then added, and the mixture was allowed to stand for 2 to 3 minutes. After filtering, the filtrate containing the regenerated protein was oxygenated. The resulting protein could be crystallized. Horse hemoglobin was treated somewhat differently so as to avoid alkaline solutions. The protein was precipitated by heating an approximately 4.5% solution for 3.5 minutes at near 90" C. The precipitate was dissolved in dilute acid. Cyanide buffer and alkali are added to make the solution faintly alkaline. A brownish precipitate forms at this point, but this precipitate goes back into solution. The supernatant obtained after neutralizing and filtering may be reduced, converted to the COHb form, and crystallized. The properties of the regenerated horse hemoglobin are very close to those of the native protein in solubility, heat coagulation, crystallization, absorption band, and combination with oxygen. Crystalline yields of 30% were obtained on the above procedure and also from heat-denatured horse hemoglobin. In a somewhat later paper (221) Mirsky and Anson made modifications in'the above. The hemoglobin was denatured in "acid" (equal volumes of 10% horse hemoglobin and 0.07 N hydrochloric acid) at 0" C. After 3 minutes all protein could be precipitated on complete neutralization. Regeneration yields were found to be independent of time in the "acid" (up to 18 hours) and to be about 70 % for (1) slightly alkaline regeneration in the presence of cyanide (this gave cyanomethemoglobin,which could be converted to carbon monoxide hemoglobin and then crystallized) and (2) regeneration by neutralization just short of precipitation. Standing

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under such conditions permitted the regeneration of "native" protein. Cubin (222) has examined acid denaturation between pH 4.08 and 4.77 and temperatures between 18 and 45" C. Denaturation was carried out in the presence of 0.5 % ammonium sulfate, which greatly aided precipitation of any denatured protein at the isoelectric point (pH 7). Significantly, the activation energy, which averages 12 kcal. per mole, decreases with decreasing pH. Apparently the exposure of groups leading to interaction requires the formation of COOH groups from COO- groups. Cubin suggests that electrostatic interactions are involved. Holden (216, 220) notes some interesting differences between oxyhemoglobin, hemoglobin, and methemoglobin. The first two are more easily denatured than the latter, and oxyhemoglobin cannot be renatured beyond about 70%. If hemoglobin is denatured in the absence of oxygen by acid or alcohol, its renaturation compares favorably with that of methemoglobin as judged by its absorption spectrum. Incomplete renaturation of oxyhemoglobin is felt to be due to combination of the globin moiety with oxygen, the globin being protected in methemoglobin. G. Experiments Involving a Heme-Globin Dissociation. Anson and Mirsky (223) by using a milder acid-splitting procedu:re than had previously been used were able to show that globin and heme could be reconstituted to give a product remarkably like hemoglobin, at the same time showing a reversible acid denaturation on the part of the globin. An approximately 15 % (ox or horse) solution of hemoglobin is mixed at 0" C. with an equal volume of 0.1 N HC1 and 200 ml. acetone containing 2 CC. of 1 N HC1 (not cold) is added. The heme remains in -the filtrate and the globin precipitates. The precipitated protein, which is in the form of the hydrochloride, dissolves readily in water but will precipitate quantitatively on rapid neutralization to the isoelectric point. If it is neutralized in two steps, the first to the point of turbidity followed after 10 minutes or so by complete neutralization, a fraction remains soluble on 0.4 saturation with ammonium sulfate. A yield of 65% is obtained for hoirse globin. In the denatured state globin liberates S H groups which disappear on renaturation (224). The preparation and solubility of synthetic: horse hemoglobin are given in Anson and Mirsky (225); the solubility compares favorably with that of native hemoglobin (and regenerated hemoglobin to be discussed). Globin and the resynthesis of hemoglobin from globin and heme have received considerable further attention. Roche et al. (226) determined the molecular weight of horse globin by osmotic methods as being 29,000 and that of ox globin as being 37,000. They also report polymeric paraglobins with particle weights close to hemoglobin. Holden (216, 220) has prepared a regenerate from acid-treated (split) carbonyl hemoglobin and

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heme. This protein has the same molecular kinetic properties as hemoglobin and is devoid of SH. It differs in the shape of the oxygen dissociation curve, in its susceptibility to dissociation by alkali, and in electrophoretic mobility (221, 227). Also Gralen (228), using essentially the methods of Anson and Mirsky, studied horse globin and the recombination of this protein with heme. Their soluble globin preparations were freed of paraglobin using 0.4 saturation with ammonium sulfate, according to Roche et al. (226). They noted that their globin solutions were unstable, became turbid (polymerized) with time, and precipitated on electrodialysis. Combined measurements of sedimentation and diffusion constants gave a molecular weight of 37,000 for globin and a molecular weight of 69,000 for synthetic methemoglobin. Frictional ratios of 1.47 and 1.23 were obtained for globin and synthetic hemoglobin, these data suggesting that splitting gives rise to halves of twice the parent asymmetry. The synthetic hemoglobin of Gralen was homogeneous in the ultracentrifuge. The carbon monoxide derivative of the product of recombination had the same light absorption characteristics as COHb, but differed somewhat in cases in electrophoretic behavior. Although the three samples were electrophoretically homogeneous, two gave mobility curves that were displaced about 0.2 pH unit toward the acid side from hemoglobin. Similar results have been obtained by Roche et al. (229, 230). According to Steinhardt and Zaiser (231), the exposure of horse carboxy hemoglobin to dilute acid (pH < 4) initiates a time-dependent reaction which liberates 36 acid-binding groups. The liberation appears as an all-or-none phenomenon dependent upon three critical proton associations. Similar masked acid-binding groups have been found by examining ferrihemoglobin (232), a more suitable material since its acid denaturation is entirely reversible. As above, denatured hemoglobin is hemoglobin insoluble at the isoelectric point in salt solutions. Between pH 4.2 and 3.6 the fraction of denatured hemoglobin varies from 0 to 1.00. The attainment of the equilibrium between native protein (N) and denatured protein (D) is slower than the attainment of acidic equilibria. The reactions for ferrihemoglobin are given as N

+ %H++ NH, 4 DH,

DH,

- ZH + D 2 N

The reactions involving proton association or dissociation are almost instantaneous. The transformations which follow between native and denatured protein, as suggested by the rate constants Icl and kz , are considerably slower. The value of z is the number of protons in the trigger mechanism and is about 2.5; the basic groups liberated by the trigger, 36

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PROTEIN-PROTEIN INTERACTIONS

in number, are one-half of the total basic groups combining with protons in the range below pH 4.0. Steinhardt considers the molecule of hemoglobin to consist of a singly folded, flat, molecular fabric with the prosthetic groups on the inner surface of the fold. Combination of H+ with 2.5 COOper unit causes an opening which allows the additional groups on the inner surface to become available, After such combination, the changes in structure leading to denaturation take place. If the changes in the reverse direction are carried out slowly, a return to the native structure is observed. If rapidly, the structure of the denatured protein is such that precipitation occurs on complete neutralization. d. Urea. Concentrated urea will not decrease the molecular weight of sheep and dog hemoglobin at the isoelectric point (Wu and Yang, 233). Horse and beef hemoglobins, as found by Huang and Wu (234), Burk and Greenberg (235), and Steinhardt (236), will dissociate in 4 to 6 M urea into halves each having two hemes giving molecular weights in the range 36,000 to 39,000. Measurements of osmotic pressure, diffusion coefficient, and sedimentation coefficient have been made. Steinhardt found that extensive reassociation occurred on dialyzing away the urea, some of the protein forming aggregates and a precipitate. Immediate dilution and salt addition will cause a complete precipitation of the protein showing that urea has denatured it. Denaturation is also indicated by the fact that the presence of 5 to 6 M urea greatly increases the rate of digestion by trypsin, as shown by Lineweaver and Hoover (237). A t the same time that these changes are observed, the heme groups are not split off in the dissociation process as they are in acid dissociation. According to Steinhardt (236, 238) the dissociated molecules retain many characteristic properties such as their absorption spectra and capacity for combining with oxygen, although the latter (Taylor and Hastings, 239) is somewhat diminished. Sulfhydryl groups do not appear in urea denaturation (Mirsky and Anson, 224). e. Salicylate. Anson and Mirsky (240) found that salicylate at pH approximately 7.0 will, in the range 0.1 M to 0.5 M , cause a progressive denaturation of a 1% solution of methemoglobin at 25" C. Denaturation may be measured as a loss in solubility or, as was used here, a change in absorption spectrum. They felt that the combination was stoichiometric and complete at the higher salicylate concentration, since spectral changes were complete. The denaturation could be completely reversed by removing the salicylate. The phenomenon has been further examined with similar results by Holden (216, 220) and by Roberts (241). The latter noted that the heat of reaction of salicylate with met'hemoglobin had a sharp discontinuity in the region 0.2 to 0.28 M , where the protein is insoluble, and rose thereafter steadily at least up to 0.8 M , the concentration

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limit used. The total reaction of salicylate with methemoglobin is therefore not stoichiometric, although, of course, that portion leading to spectral change and insolubility might well depend upon a definite number of salicylate combinations. Roberts also noted that benzoate, with a negligible heat of reaction, produces the same spectral changes as salicylate. The reaction is attributed in part to hydrogen bond formation. f. Sickle-Cell Hemoglobin. One of the most interesting protein-protein interactions is that shown by the hemoglobin of red cells capable of sickling on the removal of oxygen. The hemoglobin of such cells shows abnormal properties which have been detailed largely by Pauling, Itano, and others in Pasadena, and by Perutz and associates in Cambridge, England. Pauling (213) has recently described the nature of the sickling process in which, on having oxygen removed, the normally shaped, flexible, red cell is distorted into a rigid crescent-shaped cell. The change in shape is reversible. The altered properties of the sickle-cell hemoglobin are of greatest concern here. As compared to normal hemoglobin, at a pH of 6.9 and ionic strength of 0.1 in phosphate buffer, normal hemoglobin is negatively charged, whereas sickle-cell hemoglobin is positively charged, the difference being about 0.2 pH unit in isoelectric point and corresponding to about three charges per molecule (Pauling et al., 242). The globin prepared from sickle-cell hemoglobin is similarly different from normal globin, although on denaturation the hemoglobins and globins have the same mobility (see ref. 157). Sickle-cell hemoglobin and normal hemoglobin do not differ significantly in amino acid composition (Schroeder et al., 243) nor in the five or six N-terminal groups (see 213). It thus appears that the differences between the two hemoglobins are structural and are due to -differences in the way in which the polypeptide chains are folded, the difference in isoelectric point being due to a shift in the pK values of certain groups. In their initial observations on sickling, Pauling et al. (242) proposed that the globins of normal and sickle-cell hemoglobins were different in such a way that the sickle-cell hemoglobin molecules had surface regions which were complementary, as in antigen-antibody interactions, and which produced interaction with at least partial alignment in the cell, thus accounting for the appearance of double refraction on sickling. The addition of oxygen or CO was considered to reverse these effects by disrupting some of the weaker bonds between hemoglobin molecules. Later (212) the explanation for the oxygen-combining curve of hemoglobin, in which combination with oxygen was deduced to cause a structural change in the globin moiety, suggested a parallel explanation for the decreased association of sickle-cell hemoglobin molecules with each other on oxygenation. Thus the combination with oxygens alters the structures of the comple-

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PROTEIN-PROTEIN INTERACTIONS

mentary combining regions, introducing a steric hindrance which prevents their combination. More specifically, the steric hindrance may result from a forcing apart of layers of protein, as illustrated in Fig. 6. The remarkable aggregating properties of sickle-cell hemoglobin have been observed by Harris (244). Sickle-cell oxyhemoglobin in over 10% solution showed, on removing oxygen, a progressive increase in viscosity, leading in more concentrated solutions t o a gel or semisolid state. All such changes were reversible on adding oxygen. It was also shown that spindle-shaped tactoids of parallel rodlets (nematic fluid crystals) were formed. These were also reversible. Long associatians of hemoglobin molecules were evident. Perutz and Mitchison (245) have found, from studies of pleochroism, that the heme groups are oriented perpendicularly to the long direction of the sickle cell. It would appear that the polypeptide chains or helices are oriented parallel to this direction. Other types of abnormal hemoglobin exist and have been reviewed by Pauling (213).

12. 0-Lactoglobulin Sedimentation and diffusion measurements on 0-laatoglobulin, summarized by Edsall (1) give mol. wt. = 41.5 or 38 X lo3 and flfo = 1.26 to 1.3. Osmotic pressure studies and X-ray measurements on the crystals give a somewhat lower value of molecular weight, near 36 X lo3. Its isoelectric point is pH 5.2. a. Heat. LinderstrZm-Lang et al. (246) examined the digestion of 0-lactoglobulin by trypsin. After heating a 2 % solution of 0-lactoglobulin at pH 7.0 for 2 minutes a t lOO"C., there is a marked decrease in the rate of digestion on comparing rates immediately after cooling and 20 hours later. A return of the protein to a structure more closely resembling that of the native protein is indicated. Briggs and Hull (247) have studied the heat aggregation of P-lactoglobulin a t pH 7.0 and p = 0.1 using measurements of mobility and sedimentation and diffusion constants. Their data suggest that two internal rearrangements take place, the net effect depending upon the characteristics of each with respect to temperature. First, increasingly a t temperatures between 65" C. and 99" C. there is a reaction which does not change the electrophoretic mobility but which leads to a limited fciurfold increase in particle weight and an increased frictional ratio (1.36 -+ 1.95). Assuming first-order kinetics leads to an activation energy of 48 kcal. per mole. A second process, dependent upon the first, proceeds a t temperatures lower than those required to initiate the first reaction and is repressed a t temperatures above 75" C. The product of this reaction, apparently a

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DAVID F. WAUGH

second-order reaction, are unlimited aggregates which have a mobility greater than that of the native protein. This second unlimited aggregation is suppressed by increasing the pH or decreasing the ionic strength. Thus, heating a 0.5 % protein solution at 99" C. for 30 minutes complete8 the changes described for the first reaction, but the second reaction is obtained only on cooling, for example to 70 to 75"C. If heating is carried out a t 75°C.) both reactions occur in series. It is interesting that the second reaction is not reversed by allowing it to occur at 70" C. and then raising the temperature to 99" C. The behavior of the second reaction conforms to a general pattern shown by many proteins in which inherently insoluble protein is retained in solution or suspension until, primarily but not entirely (see below), an energy barrier whose components are electrostatic repulsion and hydration is decreased (by salt addition or neutralization), at which point a largely irreversible aggregation takes place. Larson and Jenness (248) report that heat causes a liberation of hidden S H groups according to a first-order reaction having, between 64 and 75" C., A H $ = 80 kcal. per mole; A F $ = 25 kcal. per mole (74.6"C,); and A S$ = 158 E.U. A comparison of the rates of appearance of those SH groups quite sensitive to oxidation by dissolved oxygen and the product of the first reaction of Briggs and Hull cannot be made. There is certainly no direct correlaticm between the appearance of SH and the product of the second reaction of Briggs and Hull. Groves, Hipp, and McMeekin (249) report that P-lactoglobulin denatured by adjusting the pH to values between 8 and 9.5 (3" C. and 25" C,) cannot be renatured by dissolving in dilute acid, alkali, or guanidine salts. The rate of denaturation increases with increasing pH (inversely with [HI'.') but, surprisingly, is the same at 3" C. and 25"C. The sensitivity to pH is felt to be in accord with the importance of proton dissociations as formulated by Steinhardt and extended by Levy and Benaglia (250). The general induction of insolubility by pH values far removed from the isoelectric points will be discussed below. Groves et al. (249) also found that the presence of 2 moles of dodecyl sulfate per mole lactoglobulin would stabilize the molecule, since the rate 1 pH of denaturation a t a given pH was decreased (curves displaced unit). Serum albumin and p-lactoglobulin behave in a similar way in this respect, and they will be treated further in the discussion. b. Urea. Jacobsen and Christensen (251) have demonstrated a marked reversibility in the urea denaturation of p-lactoglobulin. 2.0 % protein a t pH 5.17 is treated with 38% urea. The denatured protein is that precipitated by diluting with 10 volumes of a solution containing 0.8 M acetic acid, 0.4 M sodium acetate, and 0.4 M magnesium sulfate. At 0°C. denaturation is rapid, being essentially a t an equilibrium level of 85% in

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402

PROTEIN-PROTEIN INTERACTIONS

3 hours. At 37°C. the rate is much decreased; thus the temperature coefficient is negative. Reversion of denaturation was shown by allowing a system to react for 6 minutes at 0' C. (70 % denatuiration). The temperature was now raised to 37.4"C. and held for 10 minutes, after which the addition of diluent now precipitated only 3 % of the protein. The remainder of the protein on the addition of ammonium sulfate gave typical crystals. Groves, Hipp, and McMeekin (249) have considered the effects of several variables on the alterations of &lactoglobulin. They note that the optical rotation in 5 M guanidine hydrochloride changes very rapidly, being complete in minutes, and that the appearance of insoluble protein takes place over periods of many hours. In each case the fraction soluble at pH 5.2 after removing guanidine had properties identical with those of native protein in crystal form, specific rotation, solubility, electrophoretic properties, and antigenic properties. Quite clearly a series of changes take place in the protein molecule. Johansen (252) has called attention to the fact that the exclusion of oxygen retards significantly the formation of irreversibly denatured protein. Here the conditions were 20% protein, 464 mg./ml. urea, and 0.1 M sodium chloride. Renaturation was allowed to take place by diluting with twice the volume of 1 M sodium chloride.

13. Tropmyosin

-

-

The properties of tropomyosin are listed in (1): mol. wt. = 92,700 (53,000),f/fo = 3.1, asymmetry 30, and I.E.P. 5 (54). The particle of molecular weight 53,000 appears to be the fundamental unit, but this has a strong tendency to aggregate. If muscle fiber is treated with ethanol and ether and then extracted with strong salt solution, a protein, tropomyosin, is obtained (Bailey, 253). The reversible and irreversible aggregations of this protein are of considerable interest in view of the fact that Table I11 suggests tropomyosin to be a more "polar" protein than the others, being low in large nonpolar residues and high in charged groups (of the total residues 45 % can become charged and 27 % are anionic, see ref. 254). Tsao and Bailey (254) have discussed a number of reactions of tropomyosin. A certain amount of salt is required to keep the protein in solution. If salt is removed, however, the solutions become highly viscous as a result of extensive endwise aggregation. Electron rnicrographs reveal fibrils 3,000 to 6,000 A. long and 200 to 300 A. wide. The addition of salt produces an immediate drop in viscosity, suggesting bot'h that electrostatic interactions predominate and that the aggregation is reversible. The monomeric form is not approximated until an ionic strength of 0.4 to 0.6

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403

is reached, which convincingly shows that only small nonelectrostatic attractive forces are present. The molecule as a monomer is observed at pH 2 or in the presence of neutral urea; thus tropomyosin is not dissociated by the latter reagent (Bailey et al., 255). If, however, the urea is dialyzed away, like many other proteins tropomyosin now will aggregate to a greater extent than is customary, but the protein is unusual in that it does not precipitate. Quite possibly this behavior is related also to its low “nonpolar’) complement of residues. 14. Egg Albumin Within recent years egg albumin has been examined extensively from a variety of standpoints. The structural alterations leading to interaction and insolubility are largely irreversible. The few reports of experiments in which reversibility was examined clearly suggest the limits of reversibility. Thus Simpson and Kauzmann (187) report a limited reversal of the optical rotation of ovalbumin denatured in different ways; Tongur (256) reports that pressure (2000 atm. for 24 hours) results in a return of solubility in ovalbumin (and conalbumin) solutions denatured by heating at 62” C. for 8 hours; and Rothen (257) reports that if heat denaturation is accomplished in the absence of salt, refolding occurs in such a way as to give rise to a homogeneous material of S = 7 in 1 % salt which corresponds to a molecule of approximately twice the molecular weight of ovalbumin. The behavior of ovalbumin is in marked contrast to that of the many other proteins which show, in varying degree and yet some quite extensively, a return to the properties of the native protein. The classical experiments of Chick and Martin (258) on egg albumin and hemoglobin have remained excellent treatments of the sequences of events occurring in denaturation and coagulation and of related factors such as acidity and salt concentration. Chick and Martin examined egg albumin at acidities and alkalinities corresponding to the pH ranges 3 to 4 and 9 to 11 and in the presence of 0.26 % and 0.5 % ammonium sulfate, respectively. They fully appreciated the fact that certain structural alterations first occurred (denaturation), which were then followed by the process of aggregation either directly, as in their experiments in acid, or after neutralization, as in their experiments in alkali. Chick and Martin made measurements between 50 and 65°C. and were inclined toward the view that denaturation was a first-order process, that is, that each molecule altered independently of the others. Their interpretations were not accepted by some. Neurath et al. (58) have reviewed the problems of interpreting the kinetic data. Chick and Martin point out the importance of charge in keeping denatured protein in solution at acidities or alkalinities

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removed from the isoelectric point. Lewis (259) has examined the effects of pH on the loss of solubility of egg albumin. His conditions were 65 to 70.2"C., unbuffered albumin, and a pH range from 5 to '7.8. He observed a pronounced rate minimum a t pH 6.7 and calculated an activation energy of 130 kcal. per mole. In the native protein SH groups are masked. They appear on subjecting the protein to heat, acid, alkali, urea, and detergents (Hopkins, 206; Anson, 260; Greenstein and Wyman, 261). Likewise the phenolic groups of tyrosine are nonreactive in the native protein (Mirsky and Anson, 262; Herriott, 263). Crammer and Neuberger (91), as indicated previously under Section 111, 3, d, found a spectral shift at high pH which also suggests that tyrosine groups were bound in some special way in the native protein, but released irreversibly from this binding above pH 12. Fevold (264) has recently reviewed these and other properties of egg proteins. More recently various techniques have been used to determine the properties of the denatured protein before and after aggregation using different denaturing techniques. Many workers have observed the ease with which egg albumin aggregates. MacPherson and Heidelberger (265) examined the properties of ovalbumin denatured by acid (pH 1.5 to 2.0, 25"C.), alkali (2.4 mg. albumin N per ml. and 0.04 N riodium hydroxide, 25" C.) and heat (76 to 100" C., pH 7). Insolubility in 0.1 M acetate buffer at pH 5.0 was taken as the criterion for denaturation. In all cases, although first- or second-order kinetics seemed to describe denaturation equally well, extensive aggregation was quite evident. An aging process was noted in which initial gels or precipitates brought immediately to the isoelectric point exhibited a decrease in their turbidities. If the initial gels, etc., were aged 16 hours before bringing to the isoelectric point, the decreases just noted were considerably less. Evidently time is required to fix all of the changes induced by denaturation; a slow rearrangement of bonds is evident. In a second paper MacPherson, Heidelberger, and Moore (266) examined the aging process further. Ovalbumin denatured by acid, alkali, or heat, purified at the isoelectric point, and then aged t o constant properties, was found to consist of aggregates of 5 to 20 molecules. In the case of acid denaturation, the decrease in viscosity on isoelectric aging was shown to be due to partial disaggregation. I n a third paper (267), studies of immunological response indicated that denatured ovalbumin has not been randomized in structure; on the contrary, all denatured forms retain sufficient common structural features to give complete precipitation in the region of antibody excess. Some differences were noted in the region of antigen excess. Fredericq (268) reports that denaturation at 100" C. in 0.08 and 0.12 N HC1 for 30 minutes produces particles 950 and 2300 A. long and of axial ratios 15 and 30, respectively. On adjustment

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to pH 8.0 the longer particle shortens irreversibly to 1800 A. These results were obtained by measurements of streaming double refraction. Foster and his associates (269, 270) have used streaming double refraction and light scattering to analyze the events occurring during denaturation and aggregation of 0.9 % ovalbumin heated for 15 minutes at 100" C. Certain solutions heated contained 0.1 M glycine buffer, others were salt free, and some contained 85% glycerol. In the absence of salt two pronounced minima in aggregation are observed, a narrow minimum near pH 2.5 and another broader minimum whose center is at pH 9.5. Between pH 4 and pH 7 aggregation is very extensive. Below pH 2.0 aggregation again becomes extensive under the conditions given above, there being evidence for aggregation at this pH in unheated solutions. Light-scattering studies suggest that the molecular weights at these pH minima are close to the molecular weights of the native protein-a circumstance which suggests that aggregation does not occur to any appreciable extent. The results of measurements of streaming double refraction suggest particle lengths of 350 A. (pH 2.5) and 350 to 450 A. (pH 10.3). The length for pH 10.3 is largely independent of protein concentration and heating time. These lengths are to be compared with a value of approximately 50 A. for the native protein, as calculated roughly from asymmetries and molecular weights (Edsall, 1). An extensive unfolding is indicated, provided that no aggregation has taken place. Aggregation during heat is sensitive to salt, the pH ranges for minimum aggregation becoming smaller, but surprisingly over the range used (0.01 to 0.03 M salt), the molecular weight at the minimum does not appreciably change. It appears that in spite of the unfolding indicated a t these, the pH values corresponding to minimum aggregation, the exposure of bonding groups is a minimum and aggregation does not take place. This point will be discussed further. If a solution is denatured at pH 2.5 and 100"C. and salt is added after cooling, aggregation will now proceed. Cooling has not returned the protein to a "native" state so far as solubility is concerned. Bier and Nord (271), using light scattering, observe aggregation at pH 4.2 at temperatures of 25 to 40" C. No reaction order could be determined, but these observations may be taken as indicating the inherent instability of ovalbumin. In later publications Gibbs, Bier, and Nord (272) and Gibbs (273) have studied denaturation at the much lower pH values 0.9 to 3.43, at temperatures of 25 to 44" C. Denatured protein was precipitated at pH 4.8 in 0.6 M salt. Under these conditions first-order kinetics are observed over a wide range in protein concentration and with four different protein preparations. The variation in rate with pH is not linear, the authors finding that a t pH values above approximately 2.0 the rate varies

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TABLEXIV Therrrmdunamic Data for Two Possible Denaturation lteactions of Ovalbuinin

A H $ kcal. per mole AFS kcal. per mole A S S (cal. per degree)

36.7 22.2 48.5

50.0 20.3 99.6

directly with the hydrogen ion activity (kl), whereas below pH 2.0 the rate varied with an additional term involving the product of .B constant ICq and the square of the hydrogen ion activity. The authors suggest two denaturation processes having the characteristics for the activation reaction given in Table XIV. Gibbs (273) suggests, from an analysis of data from several sources, that denaturation occurs at different rates for different states of the protein, the rates depending upon the relative stability of each. He suggests that reactions other than those immediately connected with the interactions of hydrogen ions play an important role. The heat of activation is, in this view as in others, a composite value for many reactions of which only the final one is the actual denaturation step. We have previously noted the disappearance of exposed SH groups when ovalbumin is allowed to aggregate (60, 274; see Section 111, 3, c). The addition of reducing agents such as sodium thioglycollate in 0.2 M concentration at pH 7.4 will produce a slow gelation of solutions of ovalbumin (Huggins et al., 194). The conditions and results have been detailed more extensively for serum albumin and will not be repeated here. a. Urea. Some of the first observations of the effects of concentrated urea on ovalbumin were made by Anson and Mirsky (4175) and Hopkins (206). The protein was described as becoming insoluble on dilution or dialysis. The fact that gels and viscous solutions develop at room temperature was noted as well as the fact that the temperature coefficient of urea denaturation is negative. The appearance of SH groups in a reactive form was also observed. At about the same time, Burk (196) and Huang and Wu (234) determined that urea produced no significant change in molecular weight. Their values were obtained by extraplolating to infinite dilution, under which circumstances aggregation should be sufficiently slow or absent. Bull (276) has examined the viscosities of native ovalbumin and ovalbumin denatured by heat and urea. In the heat denaturation studies, the dialyzed protein solutions were adjusted to pH 8.0 and heated for 7 minutes a t 100" C. In the studies on urea, 1 gm. urea per cubic centimeter protein solution was added and the solution allowed to stand for 1 hour. In each case, in order to suppress electroviscous effects, phosphate buffer was

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added to 0.02 M concentration during the dilution necessary to make viscosity runs. The viscosity increments, taking the volume fraction of protein as 0.746 X (g. protein per c.c.), were 5.5 for native, 9.3 for heat-denatured, and 12.3 for urea-denatured protein. The Polson equation gave axial ratios of 3.9/1, 7.4/1, 9.2/1, respectively, ratios much less than the 100/1 ratio Bull suggests for the fully extended polypeptide chain. Clark (277) interprets the negative temperature coefficient of denaturation in 40 % and 50 % urea in terms of two opposed reactions with positive temperature coefficients; one of these is denaturation (insolubility) and the second is called splitting, although it is not clear that this is meant in the physical sense. The following interesting observation was made. A 0.6% isoelectric ovalbumin solution (pH 5.4) is made 40% to 50% in urea by adding solid urea, and the mixture is allowed to stand for several days at 13 to 25" C., which produces a maximum of "splitting." This is indicated by a marked difference in opalescence, after dialysis, between this sample and a similar sample showing maximum opalescence (after ca. 2 hours urea treatment). If, now, the dialyzed material is filtered, the clear filtrate will not precipitate on boiling. The changes produced are such that the usual loss in solubility can no longer take place. It is of course possible that on long standing in urea the groups responsible for intermolecular linkage are rehidden in the internal volume. Foster and Samsa (278) have extended their studies of denaturation described above to include the effect of heat in the presence of urea. As before, determinations of particle size are dependent upon measurements of streaming double refraction. In their experimental technique the samples before adding urea contained 0.6% albumin in veronal buffer a t pH 8.0 and ionic strength 0.1. Heating was carried out at 100" C. for 5.5 minutes. There is a pronounced decrease in average particle length with increasing urea concentration until at 4.0 M urea a homogeneous system of particles having lengths of 700 A. is obtained. Comparison of this value with those values obtained by heat denaturing at pH 2.5 or 9.1, a t both of which aggregation is a minimum (see above), suggests that it represents the length of the single unfolded molecule. The same authors found that heat denaturation in the pH ranges just named, but in the presence of 7.5 M urea, led to heterogeneity (aggregation) if heating were prolonged, but for short heating times homogeneous systems having lengths of 500 to 700 A. appeared. Protein concentration effects were found to be small at pH 2.5. According to Foster and Samsa the fully extended ovalbumin molecule has a length of 1400 A. Frensdorff, Watson, and Kauzmann (279) have examined the viscosity and gelation characteristics of urea-ovalbumin solutions. Gelation occurred in solutions containing 3 % ovalbumin, 10 M urea, and 0.1 M sodium

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chloride at least over the pH range 4.9 to 10.5. Beyond these pH limits no gels formed. Huggins et al. (207) found that a t pH 8.0, 0.1 M phosphate, 37" C., and 8.0 M urea, at least 3.2 % ovalbumin was required. Electrolytes accelerate gelation (279)-a circumstance which the authors suggest indicates that salt linkages are not important. The authors, on the basis of the effects of agents such as p-chloromercuribenzoate, which binds SH groups, and cysteine, which would be expected to reduce interprotein SS links, conclude that a mercaptan exchange is involved. This exchange was postulated previously by Huggins et al. (207) to account for the gelation characteristics of serum albumin. As Firensdorff and coworkers note, certain of the viscosity changes observetd on dilution are difficult to explain on this basis. It should be noted that Halwer (280) has come to the same conclusion, links are involved in intermolecular interaction. namely, that S - S Ovalbumin solutions were denatured by heat a t pH 8.0, in solutions containing 0.02 M phosphate buffer and 0.25 % protein. The heat treatment was 1 to 5 minutes a t 100" C. After cooling they were diluted and simultaneously brought to 6 M urea concentration. When reagents that break SS bonds were now added (thioglycollate, sulfite, cysteine, mercaptoethanol; all 0.02 M) there was a decrease in light scattering of about 35% to 50% as compared to control suspensions. The light scattering of native albumin solutions treated in the same way increased, however, and other evidence, such as the failure of SS splitting agents to act in the absence of urea, requires careful consideration. The p roblems generally encountered will be considered below. The increases in viscosity and rates of gelation are much slower than the changes in optical activity observed under the same experimental conditions. Quite possibly, as with /3-lactoglobulin, the changes in optical activity also precede the "unfolding" of the protein which is very generally thought to be a necessary condition for aggregation. Simpson and Kauzmann (187) have recently considered the complicated series of changes which take place in the optical activity of ovalbumin in urea. We can only note here that the temperature coefficient was negative below 20" C. and positive above, that complexes between ovalbumin and urea must be assumed, and that first-order kinetics are not followed. The authors suggest that the values of the constants in their derived equations are not reasonable if urea acts only by breaking hydrogen bonds within the protein. 15. Fibrinogen

Like many other proteins fibrinogen may be denatured and rendered insoluble by heat, acid, urea, alkali, etc. Indeed its susceptibility to

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denaturation suggests that it has less structural stability than most of the proteins examined here. It has been selected because its most interesting aggregation reactions follow an enzymatic activation (normally by the enzyme thrombin). To all intents in the following discussion of the aggregation of “activated” fibrinogen, the activation process may be considered irreversible. Laskowski et al. (281) indicate the difficulties attending reversal. The most recent determination of the molecular weight of fibrinogen by Shulman (282) yields a particle of mol. wt. = 340,000 with, provisionally, a length of 520 A. Thrombin has been demonstrated to split an acidic peptide or peptides from this particle (Bettelheim and Bailey, 283; Lorand and Middlebrook, 284)-a circumstance whichmust make activated fibrinogen differ from fibrinogen in net charge. It is, of course, possible that the splitting off of peptide material is incidental to the process of activation and that some more subtle change is involved. However, a change in charge configuration suggests that linkage may be attributed primarily to the interaction of charged groups-a possibility which has been considered by Ferry (285) and Ferry et al. (286). One can hardly doubt that the clotting process proceeds by two sets of reactions, the initial set which can lead to the production of activated fibrinogen and the subsequent set of reactions by which the activated fibrinogen polymerizes to form strands which then cross link. The kinetics of activation have been examined by Waugh and associates (287, 288) over a range of fibrinogen concentration (0.036 to 0.36 mg. clottable nitrogen per ml.), thrombin concentration (0.009 to 0.45 NIH unit per ml.), and ionic strength (0.05 < p < 0.6). At a pH of 6.85 and p = 0.15 the activation reaction was found to be given by

where $0 and Tho are the total initial concentrations of fibrinogen and thrombin. The term on the right is obtained on the basis that thrombin adsorbs equally to fibrinogen and fibrin. Equations similar to 27 were obtained in studies on the effect of ionic strength (288). The activation rate and the adsorption of thrombin to fibrinogen and fibrin were found to have maxima in the vicinity of p = 0.15 and to fall off rapidly on either side. The fibrinogen “molecule,” as determined from behavior in solutions, has a considerable asymmetry. In view of this it is of interest that the reaction kinetics suggest that a single critical contact between thrombin and fibrinogen is all that is required for activation. This observation will be considered again. In the process of transforming activated fibrinogen into the cross-linked network there must appear, by some sequence of

410

PROTEIN-PROTEIN INTERACTIONS

aggregation processes, strands of an asymmetry much greater than that of the fibrinogen molecule. Such strands are shown in the electron micrographs of Hall (289) and Porter and Hawn (290). The striking cross 220 A. periodicity have not been related as yet to the striations of mechanics of polymerization. The over-all rate of clot formation and the physical characteristics of the resulting clot have received considerable attention. Ferry and Morrison (291), Shulman and Ferry (292), and Edsall and Lever (293) describe the relationships between clot structure and factors such as pH, ionic strength, and concentrations of interacting substances. Clots vary from the coarse type, which has considerable mechanical strength, is opaque, and has a strong tenden’cy to synerize, to the fine type, which is translucent, has a low tensile strength, and little tendency to synerize. In the latter the fibrils are thin, and in the coarse clots they are thick (289, 290). In general, coarse clots are obtained under conditions of pH and ionic strength which lead to rapid and strong interactions between activated fibrinogen molecules, such a s pH values approaching the isoelectric point (e.g., pH < 6.8) and low ionic strengths. In addition, coarse clots are favored by an optimal rate of formation of activated fibrinogen. Too high a rate, i.e., at high thrombin concentrations, has a tendency to produce a h e clot. Quite possibly there exist in the clotting system complications in aggregation similar to those encountered in the nucleation and growth characteristics of insulin fiibrils (see Section IV, 4, Insulin); namely, that a rapid formation of activated fibrinogen leads to many nuclei, consequently, many fine fibrils. In addition there must be factors which are dependent upon the time allowed for the structural integration of an activated fibrinogen molecule or an intermediate polymer before subsequent aggregation “fixes” its position within the fibril. Edsall and Lever (293) examine, inaddition to pH and ionic strength, the effects of neutral molecules and certain specific ions. Anions such as iodide, thiocyanate, and acetyl tryptophanate a t pH 6.3 produce increasing fineness, friability, and a marked decrease in the tendency toward syneresis, effects which are similar to an increase in pH. Urea in the unusually low concentrations of 0.1 to 0.5 M exerts a marked effect in decreasing the clotting rate, the final fibrin yield, and clot turbidity. Guanidinium ion at concentrations below 0.075 M retards clotting, but in distinction to the substances given above, it increases clot turbidity. At pH 6.3, increasing ionic strength (0.15 to 0.45) retards clotting and decreases turbidity, the effect being more pronounced with calcium chloride than with sodium chloride. It is interesting that reagents such as p-choromercuribenzoate which react with SH groups have no effect on the clotting process, although they do effect the solution in urea of clots formed in the presence of calcium and serum factor. It is apparent from the extensive examination given

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411

DAVID F. WAUGH

(293) that the processes of activation, aggregation, lateral association, cross linking, etc., show differential susceptibilities to an environmental change. As will be detailed below, some believe a t the present time that the first step in polymerization is the formation of aggregates of relatively constant size and shape. The occurrence of such aggregates was observed first in systems where polymerization was partially inhibited, as will be discussed below. More recently intermediate polymers have been observed by using flow double refractions by Backus et al. (294) and Shulman et al. (295) in normal clotting systems just before the moment of gelation (clotting). Shulman and Ferry (296) about three years ago reported that a partially polymerized fibrin could be observed in the presence of 0.6 M hexamethylene glycol. Since then a number of studies of intermediate polymers have been made. These include flow double refraction studies of systems inhibited by hexamethylene glycol by Foster et al. (297) and Scheraga and Backus (298), sedimentation and viscosity studies on urea-inhibited systems by Ehrlich et al. (299), sedimentation and viscosity studies of the influence of pH and ionic strength on hexamethylene glycol-inhibited systems by Shulman et al. (295), similar studies on systems inhibited by neutral salts (Shulman, Katz, and Ferry, 300) , light-scattering measurements on systems inhibited by hexamethylene glycol (Katz et al., 301), and sedimentation studies of systems inhibited by high pH (Tinoco and Ferry, 302). Kaesberg and Shulman (303) have taken electron micrographs of systems inhibited by hexamethylene glycol. Such studies bring forth an impressive array of evidence which shows that under the various conditions studied activated fibrinogen first aggregates to form polymers of relatively constant size and shape having sedimentation constants in the range 18 to 23, but closer to the latter value on extrapolation to infinite dilution. These polymers are calculated to be twice the width of fibrinogen and to consist of an average of 15 monomeric units of mol. wt. 340,000. Lengths, depending upon particular circumstances, would be variable but approximately 4000 A. It has been noted above that similar polymers have been observed in noninhibited systems (294, 295). Ferry et al. (286) propose a polymerization mechanism based largely on the properties of intermediate polymers observed in inhibited systems. The splitting out of an acidic peptide on thrombin activation is presumed to leave a site of positive charge, for example near the center of the elongated molecule, which can then interact with negative ends of other molecules. I n this way a polymer of twice the monomer width can be elaborated. These authors suggest that charge interactions alone cannot account for polymerization and that short-range attractions also contribute. They are inclined to implicate histidine residues, directly or indirectly. Sizer and Wagley (304) have oxidized the tyrosine groups by tyrosinase and have prevented

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PROTEIN-PROTEIN INTERACTIONS

clotting. Laskowski et al. (305, quoted in Ferry et al., 286) suggest hydrogen bonds between histidine and tyrosine side chains. Hydrogen bonds between CO and N H groups are also proposed by Seelich et al. (306). The factors which limit the growth of micelles in inhibited systems remain obscure (286). I n examining the kinetics of polymerization a t ionic strength 0.3, Waugh and Patch (288) observed the temporary accumulation of some intermediate fibrin polymers under conditions where most of the activated fibrinogen went rapidly into a compactible clot structure. It was suggested that imperfections in monomer packing during aggregation eventually give rise to micelle ends whose molecules cannot accommodate an additional molecule in stable linkage. A similar limiting mechanism might well operate in other inhibited systems. As will be seen shortly, considerable revision in our views of activation and polymerization may be necessary as a consequence of investigations on the fibrinogen LLmonomer.’l That the rate of polymerization of activated fibrinogen depends also on the level of residual fibrinogen has been shown in an analysis of clotting time and the rate of appearance of activated fibrinogen (Waugh and Livingstone, 307). I n clotting under near normal physiological conditions, although there is an immediate extensive build-up of intermediates, these intermediates disappear rapidly and thereafter the activated fibrinogen is removed as rapidly as it is formed (287). When fibrin is prepared from purified fibrinogen, the resulting clot can be dissolved in a variety of agents such as concentrated urea (Laki and Lorand, 308) and lithium bromide and other salts (Shulman et al., 300). The fragments thus produced have the same shape as fibrinogen, suggesting that there is no extensive unfolding on activation (Steiner and Laki, 309; Katz et al., 310). In the presence of calcium ion and a serum factor (Robbins, 311 ; Laki and Lorand, 308) clots become stable and can no longer be dissolved in urea or acid. Serum factor has many of the characteristics of a labile protein, as has been more recently detailed by Shulman (312). Evidently the serum factor increases considerably the average interaction energy of activated fibrinogen molecules, or fibrin strands, possibly by making infrequent but extraordinarily strong cross 1ink.ages between the latter. Apparently serum factor does not increase the size of intermediate polymers observed in the hexamethylene glycol-inhibited systems described above but reduces their tendency t o dissociate on being diluted with the inhibiting concentration of hexamethylene glycol (301). Almost all of the examinations of the activation and aggregations of fibrinogen have assumed that units of the size (mol. wt. = 340,000) and shape (- 60 X 540 A.) indicated by hydrodynamic properties were involved (see ref. 29). The electron micrographs of Hall (289‘) and Porter and

413

DAVID F. WAUGH

Hawn (290) indicated that the molecules were nodose and of variable length, indeed, that they consisted of strings of spheres. I n cases large single spheres were seen (290). Siegel et at. (313) observe nodose filaments, but Mitchell (314) observes predominantly single spheres of 50 A. diameter and suggests the possibility of a dissociation. All of these authors used either concentrated solutions, in which strings of spheres predominate, or used a spray drying technique which does not avoid the unknown association-dissociation changes which can take place in drying liquid droplets, these being due to changes in protein concentration, ionic strength, surface spreading, etc. Some time ago it was felt that the possibility that fibrinogen was a reversible association system of monomeric units of mol. wt. 80,000 was of considerable importance in formulating the correct mechanism of activation and aggregation. Fitzgerald (315) has obtained electron micrographs of specimens prepared by a spray-freeze drying technique designed to eliminate many of the difficulties mentioned above. The technique is a modification of that of Williams (316). Fibrinogen solutions of IV 0.76 y per milliliter a t a low ionic strength and pH 9.5 (ammonium hydroxide) are sprayed on a prepared grid held at the temperature of liquid nitrogen. They are dried from the frozen state and shadowed with chromium. At the concentration given, predominantly single globular units of diameter less than 100 A. are seen. At high concentrations nodose filaments are observed. The undiluted fibrinogen solution was checked for clottability. By using an internal standard consisting of polyvinyltoluene spheres, it was apparent that the number of subunits observed was several times the calculated number of fibrinogen units of mol. wt. 340,000. These, coupled with other observations, suggest that fibrino80,000. On this gen is capable of dissociating into subunits of mol. wt. basis both the process of activation and aggregation must receive further attention. For example, it is quite possible that the process of activation involves the subunits of mol. wt. 80,000 and that there is a transfer of subunits from the larger fibrinogen unit of mol. wt. 340,000 to fibrin. It is interesting that Siegel et at. (313) observe, in the normal clotting sys80 A. diameter. It is also possible that tem, linear arrays of subunits of the subunits are not structurally alike and that thrombin affects one or more of the group. On this basis the varied responses observed by Edsall and Lever (293) would be more easily understood. The mechanisms proposed by Ferry et at. (286) and Waugh et al. (288, 307) might be generally correct but require considerable revision in detail. An additional observation of Fitzgerald (315) suggests that the action of charged groups may be indirect. It was found that fibrinogen dissolved a t pH 6.85 and brought rapidly to pH 1.6 will remain in solution but that such a solution will gradually gel. There is evidence that intermediate polymers, of a size similar to those

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414

PROTEIN-PROTEIN INTERACTIONS

described above, form under these conditions. Of course, it must be realized that the mechanism of coagulation at this extreme in pH may be quite different from that a t pH 7. The occurrence of bonding groups in the surface is expect,ed to be a function of structure with the latter dependent upon pH, ionic strength, etc.

V. DISCUSSION 1. Structure The present view of the protein molecule proposes a structure in which the polypeptide a-helix (the secondary structure of IJnderstrgm-Lang, 317) is integrated with the neighboring helices to produce an internal volume of closely packed side chains (the tertiary structure, 317). The packing is not completely space filling, there being indications for about 10 % unoccupied space. Such space must be distributed within the internal volume so that i t is either considerably smaller than the water molecule or is inaccessible t o the surrounding water. The properties of native proteins require that most of the nonpolar residues plus compensating small residues be placed in the internal volume, leaving the polar residues, certainly the residues charged over a range of pH near neutrality, to occupy the surface preferentially. To a considerable extent the internal volume also contains masked groups. The view of the native protein presented so far has not specifically pointed out the fact that the approach to a protein molecule (of the assumed structure) will be quite different from directions perpendicular (side) and parallel (end) to the directions of the helices. In the former case the distribution and characteristics of the side chain spikes will be important. For end approach, in addition to the interactions of side chains, we find the possibility that the “fit” of the helices may be such as to bring into play the same type of CO-HN hydrogen bond as operakes in giving stability t o the continuous helix. For endwise association, the joiiiing of helices by proline and other residues will have a major effect in limiting “fit.” Ultimately, of course, we cannot expect to be deding with perfect helices. Lateral bonding through SS linkages, the interactions of side chains, and the shifts in OH and SH groups previously recognized may produce significant structural deviations. As is evident from an examination of the previous section dealing with specific proteins and as has been pointed out recently by Lumry and Eyring (318), changes in structure of sufficient magnitude to produce a protein having an altered biological activity and solubility (trypsin, soybean trypsin inhibitor, chymotrypsinogen, and pepsinogen) are attended by relatively small changes in free energy. The heat changes and entropy changes are large but opposed, the energy required to break bonds being largely offset

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415

by a positive entropy change (318). In the instances given the changes are reversible. One would expect that the energy trough in which the native protein exists has a rippled base, suggesting a family of structures easily convertible into each other through the heat effects of ordinary temperatures. These would be the transconformations of Eyring (see ref. 318). There can be no doubt that the native protein exists in states of relatively low free energy, the energy barriers between the family of native structures and irreversibly altered states being sufficiently high that a slow or negligible rate of transformation of native protein into these other states is ordinarily observed. The tertiary structures of most of the representatives of the family of native proteins are such that they are not attacked by endopeptidases. LinderstrGm-Lang (88, 317, 319) has examined the possible pathways through which degradation may occur. The pathway to be pointed out here is that which may involve an equilibrium, within the family of structures, of native and denatured protein, the latter being attacked by enzyme. Thus, in all considerations of interaction, the probability that only the frankly irreversible changes may be placed outside of normal behavior must be kept in mind. This will become apparent in the discussion which follows, although in no instance have structural relationships between the family of structures of the native state been elucidated. For interaction of the type considered here to be made evident there must be an appropriate change in free energy on interaction. In those cases where a reversible association-dissociation is evident, the standard free energy change has been obtained through the application of the equation

AF"

=

-RT In K

where K is an equilibrium constant descriptive of the association involved. If the variation in K with respect to T is determined the heat of reaction may be obtained from

The heat of reaction may be determined independently. From these values the entropy change is obtained as usual from AF' = AH" - TAS".

(30)

Where equilibrium constants have been determined, the changes in standard free energy, as would be expected from Eq. 27, have been between -5 and - 10 kcal. per mole (for antigen-antibody combinationandpolymerization of insulin). A change in free energy much more energetic than

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PROTEIN-PROTEIN INTERACTIONS

-10 kcal. per mole would displace the equilibrium so that, with all but the most sensitive techniques, only the associated form \vould be observed. I n contrast to reversible denaturation reactions, few measurements have been made on association reactions. One would expect that the free energy change is, as in the case of reversible denaturation, the result of a delicate balance between heat and entropy changes, these thermodynamic quantities being different from one protein to another. Once having tabulated values of AH", AF", AS", etc., the problem of physical interpretation appears. As has been noted in specific instances, physical interpretations are not only difficult to make, but where made must be Laken as tentative. The balance in AH" and AS" leading to a free energy change may be made possible by a number of physical pathways. On dimerization, for example, there are the heat and entropy changes attending, among other things, the changes in bound water, the approximation of charged groups, the approximation of nonpolar residues, the redistributions of protons among ionizing groups, and the rearrangements of hydrogen bonds.

2. ClassiJication The cataloguing of protein-protein interactions should have sufficient flexibility t o include, eventually, as yet ill-defined types of interprotein interactions and interactions between proteins and other substances. A number of schemes have been examined with no obvious choice to be made. Recognition of the central position of the native protein in any scheme, however, suggests that a promising lead is t o catalog primarily with respect to the progressively more drastic changes in surface or internal volumes and secondarily, with respect to the specific manifestation of interaction (association, dissociation, aggregation, disaggregation). The cataloguing would then follow lines such as are suggested in the following outline: I. Interactions with minimal structural changes. ,4. Single protein. Dissociation, association, aggregation, disaggregation. B. Different proteins. 1. Structural change limited to one of the interacting proteins. 2. etc. 11. Interactions with reversible structural changes. 111. Interactions with irreversible structural change. There must be superimposed on this classification a cognizance of the characteristics of the particular intermolecular linkage, a few of the specific factors being: the element of specificity, the distribution of the linkage among the various bond types, the mediation of coupling agents such as

DAVID F. WAUGH

417

mercury and formaldehyde, enzymatic activation, the size and shape of the product of association or aggregation. Finally, due consideration must be given to the velocity with which the product of interaction appears. This is particularly the case when reversible denaturations are examined. The very factors which make proteins of extraordinary interest are, at the same time, the factors which have prevented the accumulation of sufficient information to allow us to place proteins in such a classification. Thus, as is evident from an examination of almost any well-studied protein, the single protein engages in a wide variety of interactions only a few of which are as yet characterized. Another rather obvious complication is our lack of knowledge concerning the distribution of the types of bonds operating in intermolecular linkage. Only in the interactions of antigens and antibodies, insulin fibrils, and the mercaptan exchange have the intermolecular linkages been related to specific bond types. Additionally, an important part of any view of interaction is the structural changes which occur as a prelude to interaction. Again, a lack of information concerning structure prevents our assigning any specific designation with assurance. For example, it would be expected that the interaction of an antigen with an antibody, the dissociation of hernocyanin, the interaction of egg albumin and pepsin a t pH 3.1 to 4.2 (Yasnoff and Bull, 320), or the crystallization of insulin would involve minimum structural changes in the internal volume (if not the surface). Yet there is evidence which suggests that an appropriate configuration of surface groups is not only necessary but is established as a result of the approximation of surface volume regions. There are brought into play on surface contact forces which become significant only when the interacting groups are separated by small distances. Although individually weak, collectively such forces might well induce structural change, as, for example, the shifting of side chains from the internal volume into the surface or a distortion of the helix. For example, Pauling and Pressman (28) show that the antibody molecule can accommodate its cavity to haptens of a size and shape different from that of the immunizing hapten. Linderstr9m-Lang (88) has suggested that an interaction between enzyme and substrate, presumably nonhydrolytic, can alter the latter so that its peptide linkages may be attacked. The nucleation and growth characteristics of insulin fibrils (lei), even when aggregation takes place at low temperatures, require that a structural change take place on linking (see below). Configurational changes are felt to attend the interactions of serum albumin and small molecules (Karush, to be discussed below). Frequently it will be impossible to tell whether an unusual distortion has taken place or whether the spectrum of structures of the family has been channeled into a few of the normal but less probable configurations.

418

PROTEIN-PROTEIN INTERACTIONS

3. Associations Interactions leaaing to limited association-dissociation phenomena present features of special interest. The first group of interactions of this type would comprise those in which interaction is limited by a mechanism similar to that proposed by Debye for the limitation of micelle growth in soap solutions (see Section 11, Group Interactions). ‘The energy gained through short-range interactions would be balanced by the energy lost through opposing electrostatic repulsion. T o a first approximation no special geometry of the interacting centers need be assumed. Even in the case of insulin association, where a certain uniformity of lateral association might be assumed, the present evidence suggests that successive interactions are physically different. Thus, the dissociation of the assumed monomer of 12,000 into subunits of 6000 is not readily observed even at concentrations satisfactory for light-scattering experiments. The author and Mr. Yphantis, a t the somewhat lower concentraticlns usable with an ultracentrifuge separation cell, have obtained evidence for a normal dissociation of insulin in acid into units of mol. wt. = 6000. No reliance is placed on these data alone, but in conjunction with dat,a presented under Section IV, 4, Insulin, a 12,000 -+ 6000 dissociation a t very low concentrations is probable. If such be the case, the energetics of this particular reaction must be quite different from those attending the construction of other polymers from the 12,000 unit. Figure 3A refers 150 the 12,000 unit, one half of which may be taken as the 6000 unit. The complexion of side chains involved in joining two 6000 units would, on the basis of previous considerations, be expected to be largely nonpolar. The free energy change on interaction would be expected to be quite large, giving rise to the stable unit of 12,000 ordinarily observed. The further association of 12,000 units would have available considerably fewer nonpolar groups. If these again were distributed in an asymmetric fashion, the dimerization reaction (mol. wt. 24,000) would involve the approximation of the faces leading to the next most energetic interaction of such groups, and giving, on the basis of an insulin model, a structure represented in cross section by Figure 7 A . Here the approximation of two faces or one face per “monomer,” is apparent. The approximation of the next monomeric

-

A B C FIQ.7. Association of a dimer and monomer in insulin.

DAVID F. WAUGH

419

unit, Figure 7C, would necessarily involve the approximation of four faces. As found by Doty and Myers (139) and Steiner (141), the free energy changes on dimerization and trimerization are not much different, but a linear trimer (141) need not necessarily be assumed. Assume that the groups available on the free faces of the dimer are structurally different from, and less compatible with, the faces of the entering monomer than those already linked in the dimer. A similar free energy change might be expected on dimerization and trimerization. The above speculations are made to suggest the possibility that successive interactions in any system may be energetically different and yet not involve the group specificity which seems to be required by the hemocyanins and similar proteins. The reversible association-dissociation systems of hernocyanins, plant seed proteins, etc., seem to require an additional strong element of specificity, for such materials are often homogeneous over a wide range in pH, concentration, and ionic strength near their isoelectric points. The mechanism which limits association must have some of the elements involved in the limitation of aggregation in antigen-antibody systems where one component is present in excess or where, as suggested under antigens and antibodies, three different kinds of molecules are present leading to a system of the kind

Ax X - R Ag In these systems we must note at once that the larger the free energy change, -AF, involved in an association having surface specificity, the more will the equilibrium be displaced toward the associated state, without, however, any necessity of proceeding beyond that state of association which utilizes all linkages. The possibility of an antigen-antibody-like specificity in hernocyanin association, as has been discussed, was demonstrated by Tiselius and Horsfall (113) in experiments on hernocyanins from different genera and species. The reversible dissociation of a large molecule into as many as 16 subunits would seem to require more than the simple specificity embodied in Eq. 31, particularly since the association is not a simple linear one such as A . B . C . D - - - - - .K

It would seem that a sharp upper limit to association could be obtained most appropriately by a mechanism depicted in simple form by Fig. 8. Here the smallest stable subunits have a specificity X , which linkage we must assume from the pH dissociation curves to be that of the most favorable free energy change. On the association of two subunits there are brought into existence specificities S and 2 in these surfaces, respectively.

420

PROTEIN-PROTEIN INTERACTIONS

The specificities S and 2 could be localized at the point of juncture or could be removed from it, depending upon the structural changes attending the approximation of X faces. The stepwise construction of specificity through association would cease with the step proceeding the final association, Such a scheme would provide a simple explanation for the fact that association or dissociation follows frequently a sequence such as 1-2-4-8-16 or a partial sequence of the same type. Another mechanism by which specificity may be controlled is well illustrated in the reversible aggregations exhibited by sickle-cell hemoglobin. Here, as shown by Pauling, Itano, and associates and by Harris, combination with oxygen expands the protein structure in such a way that specific fit is destroyed. The observations just made prompt a few further rather obvious remarks about the internal volume and intermolecular linkage. Starting with the simplest reversibly dissociable subunits, it is seen a t once that the association of two such subunits will create an additional internal volume with respect to which the next association is to be examined. Each successive internal volume formed will have a pH stability less than that of its immediate predecessor. With respect to conditions of pH and ionic strength where the largest association product is observed, the most susceptible linkage should be that between halves. Brohult (110) observed that the immediate physical manifestation of ultraviolet radiation, and presumably other splitting agents, is an irreversible splitting into halves. The author has pointed out elsewhere (150), in connection with the relatively stable associations of insulin molecules in insulin fibrils, that the observation of reversibility of a linkage is dependent upon the stability of internal structure (internal volume) with respect to the stability of the intermolecular linkage (the newly formed internal volume). Reversibility would be difficult to observe under conditions where the environmental change necessary to produce dissociation produces a t the same time irre-

Fro. 8. The stepwise appearance of interaction areas having specificity.

DAVID F. WAUGH

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versible changes in the regenerated protein. Thus the smallest subunit reversibly dissociated with hemocyanin is not always the smallest observable subunit ; production of these smaller subunits is usually attended by irreversible changes leading to insoluble aggregates, As noted above, certain splitting agents can produce irreversible dissociation without producing insolubility. Presumably they do so by altering surface specificity without liberating the groups which lead to a general insolubility. Prolonged application of splitting treatments does not lead to further dissociation but leads to insolubility. Most reversible association-dissociation systems share with other proteins the characteristic appearance of insolubility on extensive denaturation.

4. Aggregation Reactions The behavior of insulin illustrates a possible complication which may well operate, in a less noticeable way, in the aggregation of proteins denatured by heat and other physical agents. Here we observe that the addition of preformed fibrils to an otherwise stable solution of insulin causes a rapid transformation of the soluble insulin to the insoluble fibrous form. The rate of removal of insulin is proportional to the surface area of the population of seeding fibrils. It has been proposed that a group of molecules in the fibril structure co-operate in producing a stable linkage. In aggregation reactions dependent upon the transformation of a protein into a reactive form it is generally presumed that the rate of production of the reactive form is the rate-limiting step. The particular kinetics of the aggregation steps are not important in analyzing the over-all reaction under conditions where aggregation takes place as rapidly as the reactive species appear. Where such conditions can be realized experimentally essentially first-order kinetics are generally observed. In some instances, however, aggregation does not follow first- or second-order kinetics (which is the case with insulin). In these cases effects similar to the co-operative effects described for insulin are to be suspected. The nucleation and growth reactions of insulin fibrils are instructive in still another respect. Granting that the co-operative effect accounts adequately for the kinetics of growth reaction at low temperatures, the rates of nucleation, for example, at 80 to 90" C. and 25 to 35" C. suggest that a different species of molecule must be present in the temperature ranges given. The growth of insulin fibrils at the low temperature then suggests that the co-operative effect is more than that associated with the simultaneous formation of three interinsulin linkages. It is expected that the co-operative effect also involves the induction of a structural change in the monomer as it enters the fibril structure, a change which is the near equivalent of that induced by increasing the temperature.

422

PROTEIN-PROTEIN INTERACTIONS

5 . Reversible Denaturation

Many of the reversible and irreversible denaturation phenomena lead to aggregation and therefore have a direct bearing on the nature of proteinprotein interactions and the stability characteristics of the internal volume; particularly in the latter to the appearance of a t least two different levels of clearly demonstrable structural alteration, thle first level being “reversible” (under a given series of treatments), and the second irreversible. I n all cases the native protein is subject to a denaturing treatment which renders it differentfrom the native protein. Such treatments include heat, urea, acid, and alkali. That the treated protein is different is shown as follows. The altered protein is transferred rapidly from the denaturing conditions (under which it is soluble and presumably has a molecular weight equivalent to that of the native protein) to a set of more “normal” conditions in which pH adjustment and salt addition suppress electrostatic repulsion. The protein is found to be insoluble and t o precipitate with such rapidity that no appreciable energy barrier or bond rearrangements are involved. Under these same conditions the native protein has lost its characteristic physical properties or biological activity, or cannot be activated in the customary way (proenzymes) . If the denaturing treatment be replaced by more normal conditions where electrostatic repulsion still prevents aggregation, such as a p H removed from the isoelectric point and the absence of salt, there may be a more or less rapid return of the protein to the native state, that is, a recovery of solubility, biological activity, crystallizability, etc. Frequently, a complete return in all measurable detail has not been demonstrated. Irreversible structural changes take place when the denaturing treatment is prolonged and/or is applied at its maximum intensity. Irreversibility is evidenced by the fact that reversibility as described above is absent and the protein remains insoluble. Not all proteins have been shown (or are expected to be able) to exhibit a reversible denaturation of this type. A number of reversible denaturations dependent upon pH changes exhibit a trigger mechanism; the transformation to the denatured form, or to a dissociated or some other form, involves a large change in properties which is dependent upon a relatively small change. The model system of trigger mechanisms is that found by Steinhardt and Zaiser (231, 232) with hemoglobin. Other proteins showing a similar behavior are edestin and arachin. Those proteins which have been studied exhibit a small free energy change on reversible denaturation. As indicated at the start of this discussion, such facts show that the reversibly denatured state is one of the

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normal states of these proteins. The large heat and entropy changes largely offset each other, the former indicating that group attractions must be overcome in yielding the increased freedom indicated by the latter. Lumry and Eyring (318) point out that in complicated molecules such as proteins a number of reaction patterns must occur between the set of states of the native and denatured proteins. The effects of a number of agents such as pH, pressure, heat, radiation, proteolytic enzymes, and various types of solutes have been reviewed extensively elsewhere (58, 59, 60). A few situations more pertinent to the problems of protein-protein interactions have been examined under discussions of specific proteins. Additional comments need be made only with respect to the effects of pressure. Moderate pressures, in a range up to a thousand or so atmospheres, will generally retard denaturation, as found for example by Johnson and coworkers (321, 322, 323). Eyring, Johnson, and Gender (322) point out that the volume increase which occurs during activation is not associated with the rupture of primary bonds but is probably due to the unfolding and rupture of secondary bonds. In the view taken here the volume increase is expected to be primarily associated with the separation of the side chains in the internal volume. Such a view can also account for the insolubility which appears when proteins are subjected to high pressures ( >5000 atm.). Any extraordinary compressibility of the protein is probably associated with a physical property such as the unoccupied space of the internal volume. On compression the side chains are rearranged and compacted-a circumstance which leads to the structural changes producing insolubility. The universal appearance of insolubility on the application of physical and chemical treatments which have a tendency to denature (i.e., to distort the structure of the helices and the internal volume) suggests that proteins be examined for some common basic property. Mirsky and Pauling (57) have suggested that hydrogen bonds might represent such a property, hydrogen bonds between the CO and NH groups of neighboring main chains or hydrogen bonds between side chain groups. There is also, of course, a possibility that a particular charge distribution might account for interaction, and finally there is the possibility that interaction could be ascribed t o a substantial extent to electronic and other van der Waals’ forces. Each of these possibilities merits consideration in turn. As has been pointed out here, there are substantial reasons for believing that the polar groups of proteins exist in surface regions in native proteins. Since the native protein can show its soluble characteristics under such conditions, one must assume that there is no initial complementariness of surface structure so far as polar groups are concerned. Since it is generally agreed that native refers to a unique set of structures and that alterations

424

PROTEIN-PROTEIN INTERACTIONS

in structure generally lead to the introduction of randoimness, it is difficult to conceive of the process of denaturation leading to the introduction of complementariness, that is, to configurations of positive and negative charges on the opposing surfaces which differ from randomness in such a way that a net attractive force arises. This is more SO the case since in most instances the changes leading to insolubility may be produced while the proteins are still essentially in solution. One should also remember that, once the insoluble precipitate has been allowed t o form, it may be insoluble over a surprisingly wide pH range and becomes soluble only when large excesses of negative or positive charges have been introduced. All of these observations suggest that the chief effects of the charged groups in proteins are: (1) to aid in establishing the structural alterations which lead t o interaction through electrostatic repulsion, changes in hydration, and similar effects, and (2) for similar reasons to introduce an energy barrier which prevents close approach and thus modifies the interaction energy which results from short-range attractive forces (or of course from covalent linkages depending upon SS bridges). The balance between these two effects will in general determine the p H stability characteristics of a given protein-for example, whether or not reversible or irreversible effects will be observed in the presence or absence of aggregation. As has been indicated under Section 11, Group Interactions, interactions can be expected where, in a single collision, a number of hydrogen bonds can be formed between appropriate groups. The same line of reasoning as was applied in the case of charged groups can be applied to those capable of forming hydrogen bonds. In this case also, since the hydrogen bond has a certain specificity with respect to distance and direction, one would not expect the process of randomization to lead so frequently to the correct spatial distribution of hydrogen bond forming groups. Mainly on the basis of X-ray diffraction evidence (see Astbury and I,omax, 324) it has been supposed that the structural alterations leading to insolubility are in the nature of an unfolding, to the extent that main chain hydrogen bond forming groups can be approximated. The conditions for achieving any number of such hydrogen bonds, however, require a particular geometry on the part of the main chain. One would expect that the ;size and prevalence of non-hydrogen bond forming side chains would introduce steric factors which would prevent main chain approximation. Indeed, it appears most probable that the strong interactions which lead to insolubility can appear under conditions where the native protein, although expanded (swollen), is not drastically unfolded. It would appear then that insolubility is the result of the random appearance of groups which can engage in interactions which do not require a particular orientation or surface distribution. Reference has previously

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been made to Table 111, which lists the distributions of selected groups in a variety of proteins. The relatively high frequency of occurrence of large nonpolar residues in all proteins has, in the author’s opinion, a direct bearing on the problem of the production of insolubility. We have seen in another place that a free energy change of -10 kcal. per mole will lead to the formation of a stable bond. An interaction energy of 1.18 kcal. per mole CH2 groups suggests that, neglecting other effects, the mutual interaction locally of as few as four average large nonpolar side chains on each protein surface would be sufficient to produce not only a bond stable with respect to thermal effects at ordinary temperatures but stable also with respect to pH. Hopkins (206) noted, as has been noted often since, that changes leading to insolubility involve a loss of lyophyllic (polar) properties with the appearance of lyophobic (nonpolar) characteristics. It should be re-emphasized that, although the possible extent of nonpolar interactions has been brought to attention, interactions dependent upon structural change will be expected to be varied in character, As pointed out previously, certain types of linkages have been ascribed predominantly to one bond type. Even in these cases important contributions may be expected from bonds of other types. Reversibility of association reactions involving minor structural changes has been discussed. Here again, in the cases of denaturation reversibility of the aggregation reaction will depend upon the stability of the internal sructure of the original monomer with respect to the stability of the intermolecular linkage.

6. Organic Molecules as Stabilizers or Denaturing Agents The extensive literature concerning the interactions of proteins with small organic and inorganic ions and with neutral molecules has been reveiwed several times in the past few years, for example, by Putnam (325) and by Klotz (326). Certain molecules induce an unusual instability or a stability, and since these lead to (or prevent) interaction they are of concern here. That bile salts and synthetic detergents are, on a molar basis, extremely effective denaturing agents was shown by Anson (327) some time ago using bovine methemoglobin. The strong denaturing action of anionic detergents in particular has since been verified many times. Detergents may denature and yet L‘protect”the denatured protein from precipitation, they may protect a protein from the interactions which usually follow heat denaturation, or they may solubilize the precipitate of heat denaturation (325). Usually these effects occur with relatively high detergent concentrations in a certain range, too high concentrations leading again to precipitation. One suspects, as, for example, has been pointed out by Gibbs

426

PROTEIN-PROTEIN INTERACTIONS

et al. (328), that the primary combination of protein. and detergent is followed by a series of interactions which are mainly dependent upon detergent-detergent interactions, the latter being related to the critical micelle concentrations. Considerable attention has been given t o the electrostatic component of the primary interaction between detergent and protein (see Klotz, 326, and Schellman, 329). One finds general agreement t'hat the maximum primary binding of an anionic detergent is related to the number of cationic groups on the protein. The nonpolar portions of the detergent also must be intimately involved, however, as shown by Boyer et al. (330), Teresi and Luck (331), Friend et al. (332), Laurence (333), and Klotz and Ayers (334). The increase in binding and -AF with nonpolar chain length (330, 331) in interactions with fatty acids, the effect of the nonpolar structure in the adsorption of positively and negatively charged dyes (333), the possibility that adsorption of an anionic detergent may introduce negative charges (332), and the interaction with neutral organic molecules (334) all show that nonpolar interactions must play an important role. The interaction of a negatively charged detergent with the native protein would involve a juxtapositioning of positively charged and nonpolar side chains. The liberation of nonpolar residues has been presumed to accompany denaturation. On this basis one would expect the denatured protein to exhibit an increased binding for molecules having nonpolar moieties. That such is the case has generally been observed. Thus Haurowitz et al. (335) find that urea-denatured serum albumin has an increased affinity for methyl orange, and Colvin (337) finds increased binding for a number of dyes by denatured lysozyme, between 0-lactoglobulin and methyl orange, and ovalbumin and methyl orange. Colvin found a decreased binding, however, between serum albumin and methyl orange. Interestingly, insoluble denatured pepsin did not show an increased binding, but insoluble denatured ovalbumin bound methyl orange to a greater extent than that observed with soluble denatured ovalbumin. Hanna and Foster (338), in studies of streaming double refraction and light scattering, observed that several cationic detergents in low detergentprotein ratios produced an unfolding of ovalbumin (presumably t o the rods 600 A. long observed in other systems; see Section IV, 14, Egg Albumin) followed by aggregation. These effects took place a t room temperature. Anionic agents required a temperature sufficient, to cause heat denaturation before aggregation was observed. It was quite apparent that the mechanism of detergent action might well involve an electrostatic interaction through polar groups followed by an interaction between the nonpolar portions of the adsorbed molecule, and where instability results, the nonpolar residues of the internal volume.

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A refolding of the protein results with the liberation of nonpolar side chains. At low detergent concentrations the intermolecular interactions of the latter are possible; a t higher detergent concentrations the nonpolar side chains released are masked through interaction with excess detergent. The shift in the absorption spectrum of the phenolic hydroxyl groups with pH has been mentioned (see refs. 91, 92, 93, and Section 111, 3, d). In line with the view developed here, it is noteworthy that Schauenstein and Gold (339) observe that unsaturated fatty acids produce a shift on combination with proteins, but not on combinations with polytyrosine compounds. Serum albumin, and to an extent p-lactoglobulin, present features of special interest. Luck and co-workers (340,341, 342, 343) have found that the combination of serum albumin with anions such as mandelate, caprylate, and dodecyl sulfate renders the protein unusually resistant to denaturation by heat, urea, and guanidine. Denaturation has been measured by determining changes in digestibility, solubility, viscosity, and crystallizability. The combination of eight molecules of dodecyl sulfate with each serum albumin molecule is optimal for this compound (343) in stabilizing against the denaturing action of urea. T o account for the fact that the first dodecyl sulfate molecules are not bound statistically Karush (344, 345) proposes that the structure of the serum albumin molecule can adapt in such a way that the binding is affected by side chains in the near vicinity of the charged group. Prior to binding the sites exist in a number of configurations of approximately equal energy. Combination is presumed to stabilize the most appropriate configuration. Lumry and Eyring (318) point out that ‘(configurationaladaptibility” may be a consequence rather than a cause of binding. The serum albumin molecules are so far unique in their capacity to interact with a variety of small molecules. As has been pointed out in a discussion of serum albumin, the protein is also one of those which undergoes most readily reversible denaturation (in the absence of aggregation). Protection against the structural changes producing insolubility as observed by Luck and associates would seem to require more than an interaction with surface residues and to require a stabilization of the internal volume. Electrostatic interactions with charged surface residues which place the nonpolar portions of the stabilizing molecules in the internal volume would seem most appropriate. The binding data of Teresi and Luck (331) suggest that a structural rearrangement occurs which allows unoccupied space to be filled without straining neighboring helices (or perhaps with a release of helix strain). The greater “internal” stability is reflected as a resistance to denaturation. A stabilization of the internal volume or a decrease in the agents produc-

428

PROTEIN-PROTEIN INTERACTIONS

ing instability may likewise be involved in stabilization against interaction as found when proteins are dried, dissolved in high concentrations of glycerol and other nonelectrolytes, or dissolved in moderate to high concentrations of salt (Anson, 60). Irreversible changes generally follow reversible changes closely and are usually connected with the long application of conditions which produce reversible effects or with an increase in the intensity of the denaturing agent itself. If N represents the native protein, D represents the reversibly denatured protein, I represents the irreversibly denatured protein, LA represents limited association, and UA unlimited aggregation, an equation describing the behavior of many proteins could be formulated as:

-s

N1 ct Nz

c-*

Ni

c-)

LA, UA

D1 cs D2 ts Di

s

4

It

e+

UA

Iz 4 1 2 etc.

-1

LA, UA

(32)

Certain species of N, D, and I may not interact or may give rise to LA or UA .

7. Directional Aggregation The process of aggregation may lead t o a dense pre,cipitate, to loose flocs, to a gel, or to some combination. Whenever a solution of low viscosity containing not more than a few per cent protein gels, it is apparent that a transformation has taken place in which there have been formed fibrils of an asymmetry much greater than that of the parent molecules. A t one time it was thought that such fibrils were the result of an extensive unfolding of globular protein molecules into polypeptide chains with their subsequent lateral alignment , We have seen in previous considerations of insulin, serum albumin, and fibrinogen that the process of fibril formation is reversible; on reversion of fibrils a product resembling closely the initial native protein may be obtained. It is most likely that the process of fibril formation involves molecules having structures simil.ar to those of the native protein or structures which represent no greater degrees of unfolding than those involved in reversible denaturation. One must conclude that the fibrils result from the linkage of protein “molecules.” That the transformation of proteins between globular and fibrous forms is partially reversible has been emphasized recently by Rarbu and Joly (346) for serum albumin and actin but not for ovalbumin or horse pseudo-y-globulin. The author and associates have examined the reversion of fibrous gels of insulin and bovine serum albumin in which all of the moiiomer was in the fibrous form. Complete reversibility is shown in the former case and about 70% reversibility is obtained in the latter. There can be little doubt that clots formed from purified fibrinogen may be disaggregated into subunits

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of the size and shape of the original fibrinogen (see Section IV, 15, Fibrinogen). The formation of asymmetric aggregates from precursors of low asymmetry, which retain their structural identity in the process, can take place by a number of pathways of which two will be sufficient for illustrative purposes. First, as Rees (347) has shown, when the attractive energy is sufficient to overcome electrostatic repulsion (and other effects) and produce linkage, the effect of electrostatic repulsion is to cause the aggregates to assume an elongated or fibrous form. This results from the fact that the electrostatic potential is a minimum a t the ends of any aggregate, including that resulting from the linking of the two particles. In the mechanism proposed by Rees it is not necessary that the groups responsible for linkage impose any geomerty of their own. When the groups responsible for linking occupy local surface regions, the geometry of the aggregate mill be the resultant of electrostatic effects and the positions of the links. Thus a linear aggregate will result when essentially two linkages are situated a t opposite ends of the molecule (147), whereas a helix will be more probable if the linking groups on combination cause the axes of the combining molecules to make an angle other than 180” (348). Pauling (348) considers the properties of helices resulting from this type of linkage in considerable detail. Barbu and Joly (346) consider some of the complications which arise when “limited” aggregates of asymmetric molecules are to be accounted for in terms of directional linkages, energy barriers, activation process, and interaction energy. Ferry, in a previous article in Advances in Protein Chemistry (349), has reviewed the relationships between structure and properties of protein gels.

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