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STUDIES ON DENATURATION OF PROTEINS XIII.

A THEORY OF DENATURATION*t
By HSIEN WU
The Department of Blochemlatry, Pelplng Union Medical College, Peiping

Received for publication June 17, 193 I

INTRODUCTION
It is a matter of common experience that natural, soluble proteins easily become insoluble under a variety of conditions. Even dialysis against distilled water frequently results in some change of the protein. That the immune bodies and the enzymes are easily inactivated is well known. Recent work on urease (41, 42) and on pepsin (32) and trypsin (33) indicate that the enzymes are proteins. While the immune bodies have not been isolated and identified, it is highly probable that they are also proteins, since the conditions of inactivation of these bodies and those of denaturation of proteins are generally the same. Denaturation of the protein is therefore a phenomenon of wide biological significance.
T h e ternf denaturation (denaturization or denaturalization) has been used loosely to include all changes in the solubility of proteins brought about by a variety of reagents. The terms coagulation and denaturation are often used indiscriminately, and coagulation, in turn, is sometimes confused with precipitation and flocculation (or agglutination). We will define these terms to avoid confusion. Precipitation is the separation of the protein from a solution without any change in the nature of the protein. It may be brought about in three ways. (a) By means of reagents which form insoluble compounds with the protein. Examples of such reagents are salts of heavy metals, complex acids and dyes. (b) By changing the nature of the solvent, e.g., by adding alcohol, acetone, etc., which are miscible with water. (c) By salting out with salts of alkali metals, etc.

* A preliminary report was read before the Xlllth International Congress of Physiology at Boston, Aug. 19-24, 1929 and published in the Am. J . Physiol. for Oct. 1929. t Reprinted from the Chinese Journal of Physiology, 1931, Vol. V, No. 4, pp. 321-344.
ADVANCES IN PROTEIN CHEMISTRY, Vol. 46

THEORY OF PROTEIN DENATURATION

Precipitation by any of the above methods is a reversible process, as it involves no chemical change of the protein molecule. The precipitate redissolves as soon as the precipitating agent is removed or diluted. However, proteins precipitated by the first and second methods mentioned above, if allowed to remain with the precipitating agent for some time, often undergo a further change which is, for most proteins at least, irreversible. The protein then will not redissolve even after the removal of the precipitating agent. Denaturation is a change in the natural protein molecule whereby it becomes insoluble in solvents in which it was previously soluble. Denaturation can occur at any reaction. If denaturation occurs at reactions far removed from the isoelectric point, the denatured protein remains in solution. That a change has occurred is shown by the fact that when the denaturing agent is removed and the reaction of the solution brought to the isoelectric point, the denatured protein flocks out. If denaturation occurs at or near the isoelectric point, the denatured protein should flock out directly. This is what apparently happens when a protein is heated at the isoelectric point and the view of Chick and Martin (9) that heat coagulation consists of two stages-denaturation and flocculation-is generally accepted. However, the protein which is coagulated in one operation is not identical with the protein which has been denatured and flocculated in two separate operations. Thus, the latter (metaprotein) is readily soluble in slight excess of acid and alkali, while the former (coagulated protein) cannot be so dissolved. Similarly, protein denatured by alcohol in an acid or alkaline solution and subsequently flocculated (after dilution with H 2 0 in order to avoid coagulation by alcohol) by bringing the reaction to the isoelectric point is not identical with the protein coagulated by adding alcohol to an isoelectric solution of protein. The metaprotein is also more soluble in strong urea solution than the coagulated protein. In physical appearance, the metaprotein is flocculent and translucent while the coagulated protein is compact and opaque. Only after a second heating at the isoelectric point does the denatured and flocculated protein assume the properties of the coagulated protein (48). Whatever may be the cause of the difference between the properties of the coagulated protein and the metaprotein, these differences should not be ignored, and coagulation is not the exact equivalent of denaturation plus flocculation. Without any reference to theory we may define coagulation as a change in the natural protein whereby it separates oul directly from the solution or a change in the already denatured protein whereby it becomes insoluble in solvents in which it was origtnally soluble. When a dilute solution of protein is heated at the isoelectric point but in the absence of electrolytes, the coagulated protein remains finely dispersed in solution. There is nothing except a slight opalescence to indicate that coagulation has occurred. The statement sometimes found in the literature that an electrolyte free solution of protein is not coagulated by heating is erroneous, since by adding electrolyte to such a solution after cooling the coagulated protein flocks out. Similarly, when an acid or alkaline solution of denatured protein is brought to the isoelectric point the solution becomes turbid, but the precipitate is very finely divided. Addition of electrolyte will cause flocculation. Flocculation is the clumping together of fine particles o f denatured or coagulated protein in the presence o f electrolyte and generally in the isoelectric region. Flocculation of coagulated or denatured protein is apparently the same phenomenon as the flocculation of ferric hydroxide or arsenous sulphide solution. The relation of the different processes discussed above is shown in the following diagram. It should be noted that there is no sharp pH line which divides coagulation and denaturation. Both processes can occur at the same reaction, but the relative rates differ greatly. The maximum rate of coagulation occurs at the isoelectric point while the minimum rate of denaturation occurs at the point of neutrality (24, 55).

HSIEN WU

alkaline

5 c3

E $ r d

2 .5

E:

-&Z 2

E:8 .o 0
$ 2

gj

Denatured protein in filterable form

$-

Coagulation by heat

Coagulated protein * in filterable form

Denaturation (in the broad sense, that is not differentiated from coagulation) has been variously supposed to be depolymerization (2), anhydride formation (35, 40) or hydrolysis (22, 25, 26, 56). It appears that the different theories do not refer to exactly the same phenomenon, but they can be reconciled if the difference between denaturation and coagulation is recognized. However, there is something in common between denaturation and coagulation (as we shall see in the concluding section of this paper) and for the sake of brevity, we shall use the term denaturation in the broad sense unless otherwise indicated. It should be pointed out that all the previous theories of denaturation are based mainly upon observations on heat denaturation and to some extent on denaturation by alcohol and by acids and alkalies. Other modes of denaturation were not considered. It is evident that such theories, though possibly containing some element of truth, are not comprehensive enough to correlate all the facts known about denaturation. We, ourselves, were at one time of the opinion that denaturation was a hydrolysis of some non-peptide linkage, basing our view on studies of denaturation by acids and alkalies. But acquaintance with other modes of denaturation has compelled us to formulate a more general theory which will be presented in this paper. Some colloid chemists prefer to regard protein as a colloid and the

THEORY OF PROTEIN DENATURATION

denaturation of protein as a colloidal phenomena, therefore not necessarily involving any chemical change (4). In the language of colloid chemistry, denaturation is a change of the protein from the lyophile to the lyophobe state. But what is the cause of this change? The change in state is the effect and not the cause of some fundamental change in the protein molecule. The line between physical change and chemical change being difficult to draw, especially for molecules as large as the protein molecule, it seems best not to press the point whether denaturation is physical or chemical. We will simply ask ourselves the more fundamental question what happens to the protein molecule when it is denatured. There can be no doubt that the liability to denaturation is associated with some peculiar structure of the protein molecule, because no other class of substance possesses the same property. Let us see whether our knowledge of the structure of the protein molecule can offer any explanation of denaturation.

Configuration of Individual Molecules The configuration of a molecule containing only two atoms is constant. Excepting vibrations along the linejoining the atoms, they bear a constant spatial relation with respect to each other. Likewise, the configuration of a molecule containing only one C atom is constant. The configuration of a molecule containing several atoms are in general not completely defined by its structural formula on account of the possibility of free rotation. Thus in the acetic acid molecule, although the CH, group bears a constant relation to the COOH group each considered as a whole, no particular H in the CH, bears a constant relation to any atom in the COOH group or vice versa. Taking the CH, and COOH groups as two tetrahedra joined together by a common axis, they can rotate freely around this axis. T h e acetic acid molecule, therefore, does not have a constant configuration, unless it is held in a constant field of force as in the crystal. In a mass of acetic acid molecules in the gaseous or liquid state, all possible configuration are present. An individual molecule may have one configuration in one moment and another configuration in the next moment. The properties of acetic acid is the liquid or gaseous state are not those of the individual molecules but the statistical average of a very large number of them, just as the temperature of a body does not represent the kinetic energy of the individual molecules but the average of a large number of them. In the case of carbon compounds the possibilities of configuration due to free rotation are greatly increased as the C-chain is lengthened. But even more important than free rotation is a new factor which comes into play when the chain contains more than two atoms. The chain being

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flexible, it need not be straight or rather zig zag in accordance with stereochemical theory, but may be curved, bent to form a polygon or twisted into a spiral. It is an established fact of organic chemistry that carbon compounds tend to form rings, especially rings with 5 atoms. As the atoms forming part of the chain cannot rotate, the configuration of a ring without side chains is completely defined by its structural formula. In other words the atoms in a molecule of ring compound bear constant spatial relation to one another as in a crystal. Rings with which we are familiar are formed by primary valence bonds, such as

But a ring may conceivably be formed also by secondary valence bonds, such as
H,C

CH,
HO-C=O

and

H,C

RI

H,C

HO-C=O

H,C H,C

O=C-OH

H-N-H

CH,

H,C

R 2

in which the secondary valence is denoted by the sign%. Unlike the primary valence rings which have constant configuration, the rings formed by secondary valence represent only one of the many possible configurations. These rings, therefore, have only temporary existence. A ring may be formed in one moment only to be opened again in the next moment by collision with another molecule. There is a dynamic equilibrium between the ring form and many possibilities of open form. T h e life of the ring depends on the strength of the secondary valence bonds and on the kinetic energy of the average molecule. Although the individual secondary valence bonds are not strong

THEORY OF PROTEIN DENATURATION

11

enough to withstand the force of collision, more or less stable configuration can be formed if a large number of secondary valence bonds are arranged in parallel. This is evidently possible with large molecules containing many secondary valence linkages. We have examples of such molecules in proteins. The NH, and COOH groups of the proteins are strongly polar, that is, they form relatively strong linkages. X-ray studies of silk fibrin have shown that these consist of many chains arranged with their axis in the direction of the fiber (29). The forces which hold these chains together are those of the secondary valence bonds between groups in adjacent chains. The attraction between polar groups is of course not confined to groups in different molecules but must hold also for different groups in the same molecule. If a chain is very long, it should be able by intramolecular attraction to fold upon itself repeatedly. The structure o f the protein molecule. The structure of the protein molecule has not yet been worked out. Nearly 30 years ago Fischer propounded the theory of peptide linkage, the NH, groups of one molecule of aminoacid uniting with the COOH group of another molecule, the NH, groups of the latter molecule combining with the COOH group of still another molecule and so on. Doubt has been thrown on this theory by certain facts for which the theory offers no explanation and a number of hypotheses have been advanced that the protein molecule is composed of rings (diketopiperazine, pyrrol, etc.). None of these hypotheses, however, rests on sufficient experimental evidence and the peptide linkage remains the best supported theory regarding the constitution of the protein (21, 45). Whatever may be the constitution of the protein molecule, its configuration is not completely defined by its structural formula even if this be known. The configuration is completely defined only if the protein molecule is made up entirely of primary valence rings so that there is no possibility of free rotation or bending-a possibility which seems remote. As long as there are open chains, and different parts of the molecule can move with respect to each other, many configurations are possible to a single protein molecule.
Some Important Facts about Denaturation

I. Diversity of conditions. The most remarkable fact about denaturation is the diversity of conditions which can bring about this change. Acids and alkalies, salts of heavy metals (44), alcohol, ether and other organic solvents (47),concentrated urea and related compounds (19), heat, ultraviolet light (6, 11, 57), high pressure (7, 8), shaking (51), supersonic

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waves (36, 52) and even drying:* all these can induce denaturation of the protein. Proteins denatured by different methods are probably not identical, but it is remarkable that they are all insoluble which indicates a fundamental similarity in the underlying processes. 2. Ease o f denaturation. The ease with which proteins undergo denaturation is remarkable. Oxyhemoglobin is instantaneously denatured at a reaction of pH 3. Dialysis of pseudo-globulin of the serum always results in some of it becoming insoluble in distilled water. It is impossible to filter an isoelectric solution of egg albumin without some coagulation occurring at the tip of the funnel. 3. No change o f molecular weight. The ease with which proteins undergo denaturation by the action of mild reagents and purely mechanical forces seems to exclude the possibility of decomposition in the ordinary sense of the word, that is, the breaking of primary valence linkages. Huang and Wu (20) have shown, in fact, that egg albumin denatured by alcohol and methemoglobin denatured by urea have the same molecular weight, within the limits of error, as the respective natural proteins. Although changes in molecular weight do occur in denaturation by acids and alkalies these can be explained as due to secondary reaction. Denaturation per se involves no change of molecular weight. 4. Order of reaction. Heat denaturation has been shown to be a monomolecular reaction by several investigators, (9, 12, 24, 27) although other modes of denaturation have not been studied from this stand point. 5 . Effect o f hydrogen ion concentration on rate of denaturation. Lewis (24) showed that the rate of heat denaturation of egg albumin and oxyhemoglobin is a minimum at the neutral point of water and not at the isoelectric point. Wu and Yang ( 5 5 ) found that in denaturation of egg albumin by urea, the point of minimum denaturation is also at neutrality. 6. Absence o f characteristic chemical change. Certain chemical changes have been attributed to denaturation, for example, the liberation of ammonia and other non-protein nitrogenous compounds, H,S and compounds which react with the phenol reagent of Folin and Denis (56). On closer examination it appears that these substances are produced by secondary changes and not by denaturation itself, because the liberation of these substances depends on the method of denaturation employed. Denaturation by alcohol and by mechanical methods is not accompanied by these changes. Harris ( 16) found that denatured albumin gives the nitroprusside reaction whereas the natural albumin does not, and he believed that this

* Drying of oxyhemoglobin over sulphuric acid causes some denaturation. This, however, has not been carefully studied.

THEORY OF PROTEIN DENATURATION

13

is a characteristic of denaturation. Hopkins (18, 19)has also studied this reaction and found it to be in all cases associated with denaturation, although in certain cases the reaction is given only after treatment with cyanide. There is, however, no parallelism between the rate of the reaction responsible for the nitroprusside reaction and the rate of denaturation, as judged by the change of solubility. Solutions of serum albumin or paraglobulin when mixed with high concentrations of urea will give at once after treatment with cyanide an intense nitroprusside reaction, but no precipitate on dilution or dialysis even after several hours standing. It seems therefore that the change responsible for the nitroprusside reaction is only incidental to denaturation. 7. Change o f acid and base binding power. Wu and Chen (49)found that the acid and base binding power of egg albumin is increased in denaturation in the strict sense by acids or alkalies. Coagulation, however, causes a decrease in acid and base binding power. Hendrix and Wilson (17)obtained similar results. Booth (5)found no change of acid and base binding power of egg albumin denatured (and in fact partly coagulated) by heating in neutral solution. It is probable that the same protein denatured under different circumstances will show different amounts of change in acid and base binding power, but his experiment has been repeated in our laboratory with different results (53).We concluded from the shape of the titration curves that the ionization constant of some acidic and basic groups are changed in denaturation but the total number of such groups probably remains unchanged. 8. Increase in tryptic digestibility. T h e rate of digestion of denatured egg albumin is much greater than that of the natural egg albumin. This is true even in denaturation by alcohol and by shaking where the probability of secondary changes seems to be excluded (23). 9. Change o f antigenic properties. T h e antigenic power of egg albumin is much decreased by denaturation (54).In anaphylactic tests, animals sensitized to the natural albumin gave reactions in dilutions as high as 1 : 1,000,000, whereas animals sensitized to the denatured albumin rarely responded to a dilution higher than 1 : 1,000. The antigenic character is changed by denaturation. Animals with denatured albumin respond specifically to denatured albumin, although sometimes a slight cross reaction with natural albumin is observed. 10. Viscosity. Schorr (37) observed an increase in viscosity of an alkaline solution of serum albumin on standing. This was interpreted by Wu and Yen (56) as due to denaturation of the albumin. In fact the increase in viscosity of the protein when denatured by acid or alkali is so obvious that it can scarcely escape notice by all who have studied the phenomenon of denaturation. Mirsky and Anson (30) have reported that the increase

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in viscosity of protein-urea solution runs parallel with the course of denaturation.


Configuration of the Protein Molecule

The molecular weight of certain proteins is known with a considerable degree of certainty. For example, the values for egg albumin, serum albumin, hemoglobin, and serum globulin are 34,000, 68,000, 68,000, 103,400, respectively. Svedberg (43) found that the molecular weight of 12 proteins studied by means of his ultracentrifugal method are all multiples of 34,000. The significance of this finding is not yet clear, but a molecular weight of 34,000 may be regarded as small for natural proteins. Taking 100 as the average molecular weight of amino-acids minus that of H,O, then each molecule of egg albumin is made up of 340 molecules of amino-acid. From X-ray studies the length of each peptide linkage CO-NH is known to be 3.5 A.U. (28). If all the aminoacids were linked end to end in the form of a polypeptide without branches, the molecule of egg albumin would have a length of mm or 0 . 1 2 ~ (which should be visible under 1200 A.U. or 1.2. x the microscope) but a thickness of only 6-7 A.U. This cannot be the structure of the natural and soluble protein molecule for several reasons. 1 . Crystallization. In a crystal the atoms are arranged regularly, and the process of crystallization consists in the laying-on of molecules in regular orientation. For small molecules containing few atoms, the number of possible orientations of molecules, one with respect to another, is small. Hence, the probability of the regular orientation is high. For large molecules, the number of possible orientations is large and the probability of the regular orientation is low. Hence for compounds of the same nature small molecules crystallize more easily than large molecules. Taking 10 as the average number of atoms in each amino-acid residue in the protein molecule, the total number of atoms in a single molecule of egg albumin is 3,400. Each of these atoms must be orientated correctly in order to form the crystal. The probability of such occurrence must be so small as to be practically negligible. I t is probably true that when a certain number of atoms are correctly orientated the rest will automatically follow. Such atoms are of course those of the polar groups NH and CO. There are two of these in each mono-amino monocarboxylic acid and three in the dibasic or dicarboxylic acids. Each molecule of egg albumin contains between 200 and 300 polar groups. Even assuming that when such a group as a whole is correctly orientated the rest of the amino-acid residue will automatically fall into the right position, the probability of all the 200 or so groups simultaneously assuming the

THEORY OF PROTEIN DENATURATION

15

correct orientation is still very small. Proteins certainly crystallize with difficulty, but it is remarkable that they crystallize at all. T o explain the crystallizability of natural proteins we have to assume that these protein molecules are more or less rigid, that is, the atoms in each molecule occupy fixed positions with respect to one another. If this is the case, it will be necessary only to orientate the molecule as a whole or only several parts of the molecule in order to form the crystal. The crystallizability of the natural and soluble proteins thus indicates that their molecule is not an open and flexible chain but a more or less rigid and compact structure. 2. Biologzcal specificity. The specificity of immunological reactions has been shown to be due to specific groups in the protein molecules. In some artificially modified proteins the nature of the specific group is known. In natural proteins the specific groups are not yet known, but they are believed to reside in those amino-acids containing aromatic rings (46). However, these groups do not have in themselves the antigenic power which must be ascribed to some peculiar feature of the protein molecule. What is this feature? Certainly it cannot be the size of the protein molecule. The size of the polysaccharide molecule is comparable with that of the protein. The molecular weight of type I11 specific polysaccharide of pneumococcus is 118,000 (3), but it is not antigenic. The antigenic power of protein is weakened or destroyed by denaturation which does not necessarily involve a change in the molecular weight of the protein. T h e antigenic power must be due to a certain structure which is unstable and which is not found in any other class of substance. What kind of structure can there be in the natural protein molecule and not in the denatured protein or in the polypeptide? All the aminoacid radicals in the natural protein molecule are in the molecule of the denatured protein or polypeptide. If there is any structure present in the former but absent in the latter, it must be a structure formed by intramolecular union. The only kind of intramolecular union which we are sure of is that formed by means of secondary linkages. Such a union must give rise to a more or less rigid structure, and it is to certain parts on the surface of this structure that the antigenic power of the protein must be ascribed. Furthermore, it should be pointed out that if the natural protein is an open chain of amino-acids it should be able to assume practically an infinite number of configurations. There is a grave philosophical difficulty in this conception. Biological specificity in general certainly lies in the individuality of proteins of different origin. If a protein molecule can assume practically an infinite number of configurations, it is difficult

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to see how there can be any specificity, because there can be nothing but mixtures of all possible configurations. On the other hand, if the different parts of the same protein molecule are united to form a rigid structure, it can have only one or at any rate a small number of configurations and biological specificity can be easily explained. 3. Organization in living matter. The fundamental characteristic of living matter is organization. Organization means a definite, and more or less permanent relation between parts of the whole. There are definite relations between organs in the same organism, between parts of the same organ, and between parts of the same cell. There is no reason to think that the organization stops abruptly at a point which happens to be the limit of microscopic vision. It must extend down to the ultramicroscopic and molecular dimensions. The size of the smallest living things, the virus, has been estimated at 20-30 pp in diameter (34).T h e size of most protein molecules calculated from the molecular weight lies between 4-8 pp in diameter, although hemocyanin has a diameter of 24.4 pp (43). T h e protein molecule is therefore not much smaller than the virus. If there is organization in the virus, which we have no reason to doubt, that organization must extend to parts of the protein molecule. It is highly probable that the molecule of the substance which forms the fundamental machinery of life is in the natural state organized within itself, and not a mere statistical average of many possible configurations. The hypothesis of the compact structure of the natural, soluble protein molecule is supported b~ some direct evidence. N N 1 . Spreading ofprotein on water. On acid (- HC 1) or alkaline (- NaOH) 10 10 water the maximum spreading of proteins (for example hemoglobin, plasma proteins) is reached almost instantaneously. In neutral solutions (pH 6-8) the maximum spreading is not reached until 4 hour after the protein has been placed on the surface (15). The time required to reach the maximum spreading decreases as the reaction becomes more acid N or alkaline. T h e thickness of the film on -NaOH is the minimum, 10 namely 7-8 A.U., which corresponds to the average length of the aminoacid molecule. T h e thickness at pH 6-8 is a little more than the maximum (12 A.U.) while at intermediate reactions, it is several times that of the minimum. The final thickness of the film depends not only on the pH but also on the salt concentration and the reason is not clear. But the difference in the rate of spreading is significant and easily interpreted. N N In - HCI or - NaOH solution the protein is rapidly denatured, and 10 10

THEORY OF PROTEIN DENATURATION

17

the thin film formed is not that of natural protein but denatured protein. In the isoelectric region the rate of denaturation is slower and it spreads to a film of minimum thickness only after denaturation. 2. Surface tension. Du Nouy found that the surface tension-concentration curve of egg albumin showed three maxima. He believed that at these maxima the surface is covered by protein molecules in the same orientation. Knowing the surface area and the concentration of the solution, he calculated the thickness of the protein film at the maxima. These thicknesses are of course the dimensions of the protein molecule. T h e values obtained by Du Nouy, are 41.7, 30.8 and 30.8 A.U.(13). A molecule of this size will have a molecular weight of 30,800 which is not far from the value obtained in other ways. The values of these dimensions are of course only approximate at best, but it is certain that the three dimensions are not very different. 3. Frictional constant. Svedberg (43) in his study of the molecular weight of proteins by means of the ultracentrifuge has calculated a quantity which he called the molal frictional constant. He also calculated the theoretical frictional constant of a sphere by a formula derived from Einsteins formula for diffusion. T h e actual frictional constant for egg albumin is identical with the theoretical for a sphere with a radius of 21.7 A.U. The values of the ratio of the actual to the theoretical frictional constant for 10 natural proteins studied by Svedberg are all about 1. If the three dimensions of the protein molecule are not equal, they are at least of the same order of magnitude. 4 . Double refraction offlow. Muralt and Edsall (31) in two recent papers reported that muscle globulin solution shows double refraction of flow. The authors point out that the double refraction may be produced in two ways: (a) the elastic deformation of the globulin particle, which may be isotropic itself, due to the shearing stresses which occur in flow; or (b) the orientation of anisotropic particles. They believe, however, that the double refraction of the globulin is primarily due to the orientation of anisotropic particles. In support of this conclusion we may mention the fact that muscle itself shows double refraction and that denatured muscle globulin shows no double refraction of flow. If the globulin particles are themselves anisotropic, they must have crystalline structure, that is, the atoms in the globulin must be arranged regularly. It is not certain whether a muscle globulin particle represents a single molecule or an aggregate of a number of molecules. Muralt and Edsall have reason to believe that muscle globulin particles are of uniform shape and size which seem to indicate that they are probably individual molecules. At any rate, if an aggregate of several molecules has a crystal-

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line structure, the atoms in the individual molecules must have a fixed configuration. Muscle globulin is the only protein which has been shown to produce the double refraction of flow, but it is the only one which has been studied. If this is shown to be a general phenomenon for all natural, soluble proteins it will be a highly significant fact. In the crystalline state all the atoms occupy fixed positions with respect to one another. If the protein molecule is a mere chain of amino-acids it will have no fixed configuration, that is, cannot be crystalline. A single molecule can be in the crystalline state only when the atoms in the molecule occupy fixed positions, that is, if the molecule is rigid. The foregoing considerations lead to the conclusion that the individual molecules of a natural protein have a compact structure formed by intramolecular attraction. There is no difficulty, nor, indeed, novelty in this conception. The force which holds together different parts of the single protein molecule is exactly the same as that which holds different molecules together in a large crystal. In fact, a crystal has been regarded as a single molecule. In a crystal of NaCl for instance, the Na ion is surrounded in an identical way by six C1 ions, and each C1 ion is surrounded by six Na ions. There is no reason to assign a Na ion to any particular C1 ion or vice versa. When NaCl is dissolved in water the Na and C1 ions are pulled apart by H,O molecules. If we imagine that Na and C1 ions are alternately linked together by hypothetical bonds to form a chain, then in the crystal, the chain will fold upon itself in a regular way. This is essentially the picture of the natural soluble protein molecule according to our hypothesis. The only difference between the mono-molecular crystal of protein and an ordinary crystal is that a primary valence chain linking all atoms in the crystal exists in the former but not in the latter. A Theory of Denaturation The compact and crystalline structure of the natural protein molecule, being formed by virtue of secondary valences, is easily destroyed by physical as well as chemical forces. Denaturation is disorganization of the natural protein molecule, the change from the regular arrangement of a rigid structure to the irregular, diffuse arrangement of the flexible open chain. Let us see how the facts of denaturation may be explained and correlated by this theory. I . Denaturation under different conditions. (a) Denaturation by drying. Water is usually an integral part of a crystal, and loss of water of crystallization is accompanied by loss of crystalline structure. The molecule of the natural protein contains water molecules

THEORY OF PROTEIN DENATURATION

19

which probably fill the gaps between chains and stabilize the structure. Just as removal of H,O of crystallization from a crystal of copper sulphate causes the crystal to crumble, so removal of water from the natural protein molecule causes denaturation. (b)Denaturation by heat. This may be explained as the result of molecular collision. When the kinetic energy of the protein molecule reaches a certain critical value corresponding to the coagulation temperature, the force of impact is sufficient to break the secondary valence linkage and the regular arrangement within the molecule is destroyed. T h e high temperature coefficient (9)of heat denaturation suggests such a mechanical process. (c) Denaturation by high pressure. When a protein solution is subjected to high pressure, molecules of water are crushed into the protein molecule and cause denaturation. Different protein molecules also crush into each. (d) Denaturation by ultraviolet light. Radiant energy is absorbed and converted into kinetic energy by certain parts of the protein molecule. When this kinetic energy reaches a certain value, the secondary valence bonds are loosened. (e) Denaturation by shaking. It has been shown that denaturation by shaking occurs on the surface of the solution. Shaking does not cause, but only accelerates, denaturation by removing the film of denatured protein formed on the surface. In the interior of the solution the individual protein molecules are much further apart than on the surface where they are concentrated by adsorption. Hence the collision of protein molecules, one with another, is more frequent on the surface than in the interior. Furthermore, in the interior of the solution the collision is buffered by a large number of water molecules, whereas on the surface there is little or no such buffer. Hence protein molecules are more likely to collide into each other on the surface than in the interior. (f) Denaturation by high frequency sound waves. This has been shown by Wu and Liu (52) to be due to vibrating gas bubbles, and the mechanism is therefore the same as shaking. (g) Denaturation by acids and alkalies. Two explanations may be offered for denaturation of protein by acids and alkalies:i. The direct action of H + and O H - . The catalytic action of these ions in hydrolysis of all kinds is well known. If primary valence linkages are readily broken under the influence of these ions, secondary valence linkages should be loosened all the more easily. T h e exact mechanism, however, is a matter of conjecture in both cases. ii. Ionization of NH, and COOH groups. The intramolecular attraction in the protein molecule is due largely to free NH,, COOH and CO-NH

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groups. In the isoelectric state, the NH, and COOH of an amino-acid molecule are not ionized or, according to the modern view, combined in a hermaphrodite ion such as

c=o
But in acid solution,

NH;
0-

R
COOH
is formed. The

NH :

R-NH; COOH
ions carrying charges of the same sign repel each other. Similarly in alkaline solution

R-NH,

I cooions repel each other. The NH, and COOH groups in the protein molecule form similar ions. This electrostatic repulsion between different parts of the same protein molecule causes disorganization. The CO-NH groups probably can also form ions, although to a much smaller extent than the COOH and NH, groups. In the isoelectric condition the CO-NH groups attract each other, but in acid solutions

ions should be formed. Similarly in alkaline solution the ions should

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be formed. These ions must also repel each other. (h) Denaturation by concentrated urea and related compounds. Any molecule which can work its way into the space between the primary valence chains in the protein molecules should cause denaturation by disruption. Such molecules must possess groups similar to the polar groups in the protein molecule and must have a certain shape and size suitable for penetration. Hopkins (19) found methyl-ethyl-urea, butyl-urea, unsymmetrical dimethyl- and diethyl-urea, thiourea, acetamide, forrnamide and urethane could all induce denaturation of egg albumin, while symmetrical diethylurea, acetyl and methyl-acetyl-urea, biuret, allatoin, semicarbazide, benzamide, etc., were not effective. It would seem that the latter group of substances does not have the right shape and size of molecule for penetration into the protein molecule. Hopkins found that the denaturation of egg albumin by urea had a negative temperature coefficient. This unusual phenomenon is readily explained by our hypothesis. T h e cause of denaturation by urea is the attraction between the urea molecule and the polar groups which hold the protein molecule together. Now the force of molecular attraction is counteracted by thermal agitation, hence the lower the temperature, the more rapid is the denaturation. (i) Denaturation by alcohol. Alcohol removes the water from the interior as well as from the surface of the protein molecule. The loss of water from the surface facilitates collision between the protein molecules. T h e removal of water from the interior causes the molecule to crumble. It appears that the dehydrating action of salts which can precipitate natural proteins is not as powerful as that of alcohol. (j) Denaturation by salts of heavy metal. It appears probable that the denaturation of protein by heavy metals is in reality due to the acid or alkali formed by hydrolysis of the salt or purposely added to produce precipitation. However, if salts of heavy metals do by themselves cause denaturation, it can be explained as due to the impact of the dense molecule of metallic ions or salts upon the protein molecule. We have seen that the theory is able to explain the diverse ways of denaturation. The other facts about denaturation can also be readily explained. The loosening up of the compact structure does not necessarily involve a splitting of the molecule, that is, change of molecular weight, although this may happen. Disorganization of the individual molecule

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is obviously a monomolecular reaction. There is no destruction or formation of new groups, hence there is no characteristic chemical change, although certain groups hidden in the interior of the natural protein molecule may be exposed by denaturation. T h e increase in tryptic digestibility is due to the increase of exposed surface. The change in antigenic properties is due to the change of the surface structure. The increase in viscosity can be explained as due to the increase in the molecular surface when the compact structure is replaced by the open structure. There remains one fundamental fact which has to be explained, that is, the change in solubility. Why should the protein with compact structure be soluble and why should the protein with open structure be insoluble? T h e solubility of the amino-acid in H,O is due to the NH, and COOH groups. The solubility of the protein is due to these groups as well as the CO-NH groups. Now these groups are present in the denatured as well as in the natural protein, the peptone and peptides which are all soluble. Hence the insolubility of the denatured protein must be due to a peculiar arrangement of these groups. It is conceivable that in the natural protein molecule the majority of polar groups are directed outward, while in the denatured protein molecule, the polar groups are surrounded by the non-polar groups which are more voluminous. T h e solubility in water is regained only when the size of the molecule is reduced by hydrolysis. Reuersibility o j denaturation. If denaturation is disorganization and not decomposition, it should be possible for a denatured protein to revert to the natural form. For egg albumin which has been most studied, reversion of the denatured to the natural form has never been observed. However, for hemoglobin and serum albumin reversibility of denaturation seems to be an established fact. Anson and Mirsky (1) were the first to observe that some hemoglobin is formed by neutralizing an alkaline solution of hemochromogen and they suggested that denaturation might be reversible. Wu and Lin (50) were able to show that methemoglobin and reduced hemoglobin denatured by dilute HC1 could revert almost completely to the natural form under certain conditions, namely in a slightly alkaline solution. Using a concentrated carbon monoxide solution Mirsky and Anson (30) have recently succeeded in obtaining crystals of natural carbon monoxide hemoglobin from the denatured form. SpiegelAdolph (39) found that coagulated serum albumin could also revert to the natural form in slightly alkaline solution. According to our hypothesis reversion of denatured protein to the natural form is essentially similar to crystallization. In both processes the

THEORY OF PROTEIN DENATURATION

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atoms are laid on regularly. In reversion only one molecule is involved, whereas in crystallization many molecules are involved. The conditions required for reversion of denatured protein are also similar to the conditions required for crystallization of natural protein. Ample time must be allowed for both processes. If the natural protein is quickly precipitated from the solution by salting out, the precipitate is always amorphous. If the denatured protein solution is rapidly brought to the isoelectric point, it is precipitated, and little or no reversion occurs. T o obtain crystallization of the natural protein o r reversion of the denatured protein it should be brought almost but not quite to the precipitating or isoelectric point. There is only a narrow zone of reaction where crystallization or reversion is possible. If the denatured protein is so insoluble that it can be dissolved only in strong acids or alkalies, then reversion is practically impossible, because such reagents in bringing the denatured protein into solution cause secondary changes which are irreversible. This seems to be the reason why reversion of denatured egg albumin has not been accomplished. An important corollary of our theory is the non-unity of denatured protein. If denaturation is disorganization, the same protein denatured in different ways cannot be identical, because of different manners of disorganization. Even when a pure protein is denatured by a single agent, the product is probably not a single chemical individual but a mixture of proteins with varying degrees of disorganization. In the introductory part of this paper we have pointed out the difference between denatured protein and coagulated protein. In the preceding sections we have for the sake of brevity used the term denaturation to include coagulation. We will now explain the difference between the two phenomena in terms of our theory. Denaturation in the broad sense is the disorganization of the individual protein molecules. In acid or alkaline solution the molecules carry charges of the same sign, so they remain apart. When the charge is removed they attract each other by virtue of the secondary valences. This is flocculation. Coagulation is the interpenetration of many protein molecules. The deeper the penetration, the more compact is the resulting coagulum. Deep penetration is possible only when the individual molecules carry no charge and collide with a large amount of kinetic energy. This is why the most compact coagulum is formed by heating at the isoelectric point. Interpenetration of protein molecules necessarily causes disorganization, but disorganization may not be followed by interpenetration. In flocculation the disorganized protein molecules come together, but only the secondary valences on the surface are engaged.

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The validity of mechanical concepts to molecular dimensions. The above theory of the mechanism of denaturation and ccagulation may be criticized on the ground that it is too mechanical and that we know of no chemical phenomenon which can be explained in such a mechanical way. Blood corpuscles may be broken by mechanical force. Yeast cells can be ground. Bacteria can be injured and proteolytic enzymes destroyed by shaking (38). Virus can be inactivated by pressure (14). If there is a limit of dimension below which mechanical forces cease to have any effect, the dimension of the protein molecule must be on the border of this limit. If the protein molecule is large enough to be centrifuged, why can it not be torn, crushed or entangled? In conclusion it should be emphasized that the theory proposed in this paper does not presuppose the absence in the protein molecule of linkages other than those at present known. Unless the protein molecule is a rigid system of rings formed entirely by primary valence linkages, the theory will not be invalidated by any new knowledge about the constitution of the protein molecule which organic chemistry may bring in the future. SUMMARY Evidence is adduced in support of the hypothesis that the molecule of natural, soluble protein is not a flexible open chain of polypeptide but has a compact structure. The force of attraction between the polar groups in a single molecule of protein holds them together in an orderly way, just as the force of attraction between different molecules holds many molecules together in a crystal. In denaturation or coagulation the compact and orderly structure is disorganized. If denaturation occurs in acid or alkali or in urea solution, the individual molecules are disrupted but they remain separate. In coagulation they interpenetrate and are entangled. T h e facts known about denaturation and coagulation in diverse ways are explained and correlated by the theory.

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