A Study of Some Properties of Thixotropic Gels Containing Egg

THIXOTROPIC GELS CONTAINING EGG ALBU&lIX

1113

to Professor J. M-ynian. Jr., Drs. 5. H. -Armstrong. Jr., J. D. Ferry and J . W. Mehl, and Mr. >I. Melin for making certain of the measurements recorded here; and to I'rof~ssor E. .J. C,ohn for his help throughout these inrestigations. REFEREKCES BUDO,FISCHER, A N D ,\IrY.iaIoTo: Physik. z. 40,337 (1939). COHP;:Ann. Rev. Biochem. 4,93 (1935). COHN:Chem. Rev. 24, 203 (1939). DEBYE:Polur M o l e c d e s . The Chemical Catalog Company, Inc., ?;en Tork (1929). (5) Dielectric Symposia: Trans. Faraday SOC.,Volume 30 (1934). (6) FERRY AND OXCLEY: J. Am. Chem. SOC.60,1123 (1938). (7) FERRY AND ONCLEY: J. Am. Chem. SOC.,in press. (8) MCMEEKIN:J. Am. Chem. SOC.61, 2884 (1939). ASD SIMHA: Science 92, 132 (1940). (9) MEHL,ONCLEY, (10) ONCLEY:J . Am. Chem. SOC. 60, 1115 (1938). (11) ONCLEY:rlnn. X . T.Acad. Sci., in press. (12) OSCLEY,FERRY, ASD SHACK: Cold Spring Harbor Symposia Quant. Biol. 6, 21 (1938). (13) ONCLEY, FERRY, A N D SHACK: Ann. X. Y. Acad. Sci., in press. (14) PERRIN: J. phys. radium 6, 497 (1934). (15) PERRIN:J. phys. radium 7, 1 (1936). J. Phys. Chem. 44, 25 (1940). (16) SIMHA: (17) SVEDBERGAh?) PEDERSIK:The ~%?'acenlri/uge. Oxford University Press, London (1940). (18) WYAIAN:Chem. Rev. 19, q13 (193s). (1) (2) (3) (4)

h STUDY OF SOME PROPERTIES OF THIXOTROPIC GELS CONTAINISG EGG ALBUMIX AS T H E DISPERSE PHASE' WILLIAM G. MYERS

AND

WESLEY G. FRANCE

Department of Chenustry, The Ohio Stale University, Cbluinbus, Ohm Receieed J u l y 3, 1940

Jirgensons (19) investigated thixotropic gels containing 2 to 5 per cent of egg albumin in which the percentage by volume of normal propyl alcohol was varied between 40 and 60 and the concentrations of various salts were found to be optimal between 0.2 and 0.5 mole per liter. In attempting to check his results it was found that acetic acid, when added to diluted egg white, produced thixotropic gels. This result is in marked contrast to that reported by Loughlin and Lewis (25), Loeb ( 2 4 , and others, who found Presented a t the Seventeenth Colloid Symposium, held at .4nn Arbor, Michigan, June 6-8, 1940.

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WILLIAM 0. MYERS AND WESLEY G . FRANCE

that the viscosity of such protein solutions is independent of the hydrogenion concentration when strong mineral acids are used. Preliminary experiments showed that the thixotropic properties of the gels were greatly altered by the addition of small quantities of various salts. In view of the little work that had been done on thixotropic protein gels and in order to clear up, if possible, the discrepancy between our findings and those of Loughlin and Lewis, it was deemed desirable to investigate carefully the factors which were responsible in producing the thixotropic gels. Only a few other thixotropic systems containing proteins have been studied. Freundlich and Abramson (1 1) found that solutions containing between 1 and 8 per cent of gelatin were thixotropic. Muralt and Edsall (27) were able to extract the globulin, muscle myosin, which constitutes the main portion of the interior of muscle fibers, and found solutions of it to be thixotropic. Both Chambers (4)and Faur6-Fr6miet (8) have described the thixotropic properties of protoplasm. Anson and Mirsky (1) studied the effects of acids, salts, and heat on the viscosity of denatured egg albumin and hemoglobin solutions. It is probable from their description that they were dealing with thixotropic gels in some of their work. Kopacaewski (22) studied thixotropic gels composed of impure serum proteins, casein, and mucoid in the presence of lactic acid and salts, and merely mentioned the fact that gels were produced when hydrochloric acid, pyridine, and sodium hydroxide were substituted for the lactic acid. Many theories of protein structure have been formulated in attempts to depict the complex interrelationships among the thousands of atoms constituting each huge protein molecule and to correlate these with the changes in properties and thermodynamic quantities which result when these massive entities are acted upon by external agencies. The modern picture is far from a complete one, as the controversy around several points in the current literature will attest (31,41,42),but the main framework has been established to the satisfaction of most of the investigators in the field. I n 1906 Emil Fischer (9) introduced the concept of the peptide bond, and his subsequent brilliant researches established that this bond is the essential one in protein structure. From this arose the idea that a native protein molecule is composed of amino acid residues linked together by peptide bonds into a single continuous polypeptide chain. As the result of many experiments of great diversity, it is now generally agreed that this chain is not an extended one, but that it is twisted and folded upon itself to form a compact globule having either a spherical or a more or less elongated shape (29). Svedberg (38,39) and his collaborators have shown that the particle weights of these globules vary for different protein species from about fifteen thousand to several millions. In 1931 Wu (43) first expressed the idea that the pattern of folding of the polypeptide chain im-

THIXOTROPIC GELS CONTAINING EGG ALBUMIN

1115

parts to the molecule of each protein species the unique configuration which gives the molecule its specific characteristic properties. Since the extended chain is a more random condition than the globular form and therefore is the more probable one, it follows that some kind or kinds of bonds must exist among different points on the chain to hold it in a rolled-up state. The nature of these non-peptide bonds is the principal cause of argument among workers in the field of proteins (2,31,37,41,42) a t the present time. It is not our purpose to enter the controversy here; rather, we shall adopt the concepts which seem best fitted to explain the phenomena under study. Those most useful to us have appeared in the works of many authors, but they are summarized principally in the recent theoretical discussions of protein structure and protein behavior by Mirsky and Pauling (26), Anson (36, Chapter IX), and Eyring and Stearn (7). According to these workers there are essentially two different types of cross-linkages, one of which, the cystine link (included here, also, are any possible inner ester links), plays a minor part in holding the chain wound up because of its paucity in most proteins. The other type of cross-link is an internal salt-bridge, arising from the interaction of excess basic and acidic groups along the chain as well as from a basic and a carboxyl group not involved in a peptide bond at each end of the chain. The excess acidic groups along the chain are due to the monoaminodicarboxylic acids, aspartic, glutamic, and hydroxyglutamic, whereas the excess basic groups are due to the diaminomonocarboxylic acids, arginine, histidine, and lysine. The nature of the interaction of these basic and acidic groups is unsettled. We shall adopt the concept of Mirsky and Pauling (26): “Side-chain bonds in proteins we consider to involve usually an amino and a carboxyl group, the nitrogen atom forming a hydrogen bond with each of two oxygen atoms and holding also one unshared hydrogen atom. I n acid solutions hydrogen bonds may be formed between two carboxyl groups, as in the double molecules of formic acid (30). . . .The bond is essentially electrostatic in nature.’’ A scheme of such a bond between a lysine and a glutamic acid residue located a t points along the polypeptide chain separated by . . .n. . . interposed amino acid residues appears in figure la. Figure l b shows how the electrons are arranged in the salt-bridge according to the above description by Mirsky and Pauling. The small light-inked symbols between the heavy ones represent electrons the source of which is self-indicated. The dotted lines Salt-bridge COO-Ht NCHsCHi CHn C Hz CHn CHz . . OCCHNH. .n--0CCHNH.. FIQ.Is

I

FIQ.Ib

1116

WILLIAM 0. MYERS AND WESLEY 0. FRANCE

show where the hydrogen bonds are located. Rodebush (33) “regards the hydrogen bond as a case of coordination of the simplest cation, the proton.” A fuller description of the nature of the hydrogen bond and of its applications appears in the reviews by Huggins (18) and Lassettre (23). Now there is an excess of twenty-eight each of basic and acidic groups in egg albumin (5), among which there are presumably salt-bridges in the native protein molecule to hold it rolled up in its globular form. Evidence for this is that the native protein molecule loses many of its characteristic properties in a process known as denaturation, when it is acted upon by various agents which break the links holding the parts of the molecule in their specific relationships. “The denatured protein molecule is characterized by the absence of a uniquely defined configuration” (26). Many different explanations have been offered by various authors in attempting to account for thixotropy. The most widely accepted theory is that thixotropy is due to the formation of solvated hulls around each of the particles of the dispersed phase (12, 13, 14, 15, 16, 17, 20, 32). These thick envelopes or lyospheres of oriented solvent molecules are supposed to be disrupted by shaking and to re-form again when the system is left undisturbed; the rigidity of the gel state arises from the fact that these lyospheres become so large as to interlock throughout the system. Several lines of evidence may be adduced in favor of this hypothesis. Chief among these are the observations of the sol-gel transformation made under the ultramicroscope (10, 13, 16, 34, 35). Evidence which may make the solvated hull theory seem untenable is the excessive thickness of these hulls that would be necessary in order for them to touch. Werner (40) calculated that the thickness would have to be up 3 p for zt gel containing kaolin, and Freundlich (10) found that it would have to be about 100 mp for a bentonite gel. A popular alteiiiative hypothesis, which has been expressed variously (12, 13, 14, 34), attributes thixotropy to the formation of a network composed of chains, filaments, or sheets of the dispersed phase that pervades the entire gel and cntraps the dispersion medium. These structures are readily broken down by shaking and form anew when the system is undisturbed. Factors affecting thixotropic gel formation are discussed fully in the reviews of Freundlich (13), Pryce-Jones (32), and Myers (28). EXPERIMENTAL

Solutions of thrice-crystallized egg albumin were prepared by the method of Kekwick and Cannan (21). They were electrodialyzed free of salt and analyzed for protein content by evaporating a weighed amount of the solution to dryness a t 11O-14O0C. The pH of the solutions was measured with a Coleman glass-electrode assemblage and found to be around 4.85. This is in agreement with the


1117

results of Errera and Hirshberg (6), who found that prolonged electrodialysis always brought the p H to the isoelectric point of the protein. An Ostwald-type viscosimeter immersed in a bath constant to &O.O2"C. was used. Time intervals were measured in fifths of seconds with a stop watch. Whenever protein was a reagent being used in conjunction with a weak organic acid or base, the emptying periods of the viscosimeter were used only as bases for comparisons of the actions of reagents or of different durations of time upon the protein. This qualification is necessary, since chemical interactions within the systems caused their viscosities to increase continuously even while the emptying periods were being determined. Also it is doubtful that the emptying periods of the very viscous systems could be used validly for the determination of their viscosities, even though they had remained constant during the times of measurement, because such highly viscoud systems probably do not obey Poiseuille's law relating viscosity with pressure for flow through a capillary. Greenberg (36, page 452) designates the viscosities of highly viscous systems, which vary with the rate of shear, as structural viscosities or plastic flow. The method of mixing the reagents when 1 per cent egg albumin solution was used along with acetic acid was as follows: 2 cc. of a 5 per cent protein solution was allowed to flow into a test tube from a buret. Then an appropriate concentration of a salt solution in a volume calculated to give the desired final concentration when diluted to 10 cc. was added. Next, water was added to bring the total volume up to 7 cc. Finally 3 cc. of glacial acetic acid was added as quickly as possible. A cork was inserted in the test tube, and the contents were mixed by inverting the tube five times. Then the contents were quickly poured into the viscosimeter in the water bath and forced by air pressure to a point above the second file mark of the capillary of the viscosimeter. In order to obtain as comparable results as possible, 1 min. was allowed to elapse from the time that the addition of acetic acid was begun until the beginning of the emptying period of the viscosimeter was clocked. After the emptying periods had been determined over the desired intervals of time, the contents of the viscosimeter were poured back intp the test tube. The systems were then stored in test-tube racks for further observations of the effect of lapse of time upon them. Thixotropic gelation, flocculation, or syneresis were noted in particular. i n figure 2 the emptying periods of a viscosimeter containing a 1 per cent protein solution in the presence of 30 per cent of glacial acetic acid by volume a t 25°C. are plotted against the time intervals after the mixing of the components. The curve is very smooth up to a point where the emptying period exceeds 260 sec., when it is thought that plastic flow or "structural" viscosity becomes dominant.

1118


The emptying periods of a viscosimeter containing a 1 per cent solution of egg albumin in the presence of 30 per cent of glacial acetic acid by volume a t 27OC., when increasing concentrations of sodium chloride are added, are given in table 1. Emptying periods are given in seconds and time intervals after mixing the components are given in minutes. The data are plotted in figure 3.

Time a f t e r mixing in hours FIG.2. Plot of the emptying periods of a viscosimeter against time intervals after mixing components. The viscosimeter contains 1 per cent protein solution in the presence of 30 per cent of glacial acetic acid by volume; no salt is present. Temperature, 25°C. The curve indicates gradual unwinding of the egg albumin molecule with time, owing to thermal agitation with simultaneous solvation by acetic acid via hydrogen bonds.

The systems which were 0.006-0.020 N with respect to sodium chloride showed increasing gel structure. They were thixotropic. Similar data were obtained when sodium sulfate and sodium ferrocyanide were used. Here the emptying periods increased much more rapidly for a given concentration of salt than was the case with sodium chloride, as figure 4 illustrates. Some qualitative observations concerning the effects produced by


TABLE

1119

1

Emptying period8 of viscosimeter containing egg albumin, acetic acid, and sodium chloride CONCENTRATION O?

N&l

I

EYPTYINO PERIODS IN BECONDB

Timeintervals ............

0.002 N . . . . _ . . . . . . . . 0.004N . . . ., . . . . . . . , 0.006 N . . . . . . . . . . . . _

0.008N . . . . . . . . . . . . . 0.010N . . . . . . . . . . . . . 0,020 N . . . . . , . . . . . . . 0.040 N * . . . . . . . . . . . . 0.060 N t . . . . . . . . . , . .

* Set to gel immediately. t Formed suspended flocs immediately and was white and opaque. Syneresis soon occurred.

Time after mixing in

minutes

FIQ. 3. Plot of the emptying periods of a viscosimeter against time intervals after mixing components. The viscosimeter contains a 1 per cent solution of egg albumin in the presence of 30 per cent of glacial acetic acid by volume and added sodium chloride. Temperature, 27'C.

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WILLIAM G. MYERS AND WESLET G. .FRANCE

weak organic acids and bases, including pyridine, were made. Propionic acid and lactic acid appeared to have the same effect as pyridine. Nicotine also produced thixotropic gels, but they were very viscous and showed very prolonged setting times. Piperidine produced very basic solutions when added to egg albumin, but these showed no tendency to develop a gel structure.

ob

1

Time

i 2;

t

40 5L after mixing in minutes 30.

a!

FIG.4. Plot of the emptying periods of a viscosimeter against time intervals after mixing components. The viscosimeter contains a 1 per cent solution of protein in the presence of 30 per cent of glacial acetic acid by volume and added sodium ferrocyanide, sodium sulfate, or sodium chloride. Temperature, 27°C.

Figure 2 shows how the emptying period of a viscosimeter containing 1 per cent of egg albumin mixed with 30 per cent by volume of glacial acetic acid increases with the interval of time after the mixing of the components. This result is entirely different from that reported by Loughlin and Lewis (25) and by Loeb (24), who found that the viscosity of solutions of egg albumin was independent of the hydrogen-ion concentra-* tion when strong mineral acids were added. We also found that the viscosity of a 3 per cent solution of egg albumin was constant when sulfuric acid was

1121


used to produce the same hydrogen-ion concentration as that obtained by the addition of 30 per cent of glacial acetic acid. Wow this difference between the effect of a weak organic acid and that of a strong mineral acid cannot be accounted for on the basis of a difference in the number of salt-bridges neutralized, because the hydrogen-ion concentration in each case was great enough t o insure the complete neutralization of all salt-bridges. We believe that, in addition to the formation of albumin acetate in a process identical with the formation of a protein salt by strong mineral acids, acetic acid present in sufficient con-

(b)

(C)

(Solvated by five acetic acid (Solvated by three acetic molecules through five acid molecules through hydrogen bonds) three hydrogen bonds) FIG.5. A possible solvation mechanism

centration to give a pH of about 2 also solvates (through hydrogen bonds) the loose ends of the salt-bridges after neutralization has permitted them to separate. This is in harmony with the view of hlirsky and Pauling (26). Huggins also believes that such solvation can occur readily. Carboxyl groups seem to be especially well adapted to bridge formation. The carboxyl hydrogen is held loosely enough so that it can be shared readily with a suitable electronegative atom elsewhere. The carboxyl oxygen atoms likewise act as receivers for hydrogen atoms held loosely enough elsewhere (18). -4depiction of a possible solvation mechanism appears in figure 5, where

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WILLIAM 0. MYERB AND WESLEY 0. FRANCE

the source of the electrons is indicated by the small symbola and the hydrogen bonds are indicated by the broken lines. The two hydrogen bonds between the nitrogen atom and the two oxygen atoms in figure 5 a, -postulated by Mirsky and Pauling,-are broken a t the pH (about pH = 2.0) under consideration, with a subsequent solvation of the free carboxyl to the maximum extent of five molecules of acetic acid (figure 5 b) and of the free H:Nto the extent of three molecules of acetic acid (figure 5 c) through hydrogen bonds. It is unlikely that the carboxyl group could form the maximum of five hydrogen bonds with acetic acid if correct bond angles and bond distances were observed. An even greater solvation is probable if one considers the possibilities of hydrogen-bond formation a t each peptide link, as shown in figure 6. There are three lone pairs of electrons and a hydrogen attached to the nitrogen which are available for solvation by four acetic acid molecules through hydrogen bonds. Since there are 288 amino acid residues in the egg albumin molecule (3), the total maximum solvation by acetic acid (28 X 8) = 1372 molecules. through hydrogen bonds is (287 X 4) Multiplying by the molecular weight of acetic acid adds 82,320 to the par-

+

FIQ.6. Possibilities of hydrogen-bond formation at each peptide link

ticle weight, owing to solvation. As pointed out before, the maximum solvation is probably prevented when the bond angles and distances are considered. However, the above calculation is concerned only with the maximum solvation directly through hydrogen bonds. If one examines figure 5 it is apparent that each directly solvated molecule presents a great many opportunities for further indirect solvation by acetic acid. It is known that acetic acid readily forms hydrogen bonds with itself and with water as well, so that one immediately realizes that he is here dealing with a logical mechanism for lyosphere formation without having to resort to the postulation of any vague and unknown forces (10, 13, 15,20,40)acting over comparatively enormous Eitanc.es. The picture thus far then is that weak organic acids and bases, which form hydrogen bonds readily, neutralize the salt-bridges in the protein molecule and then solvate onto the loosened ends as well as onto the atoms attached to and involved in the peptide bonds. These solvate molecules are in turn solvated by others like themselves as well as by water, so that a huge lyosphere is built up with a more or less extended polypeptide chain as its central strut. The interlocking of such lyospheres in moving


1123

past each other is thought t o impart an increase in viscosity to the system which is a function of the size and shape of the lyospheres as well as of the fraction of the space occupied by them and the amount of interlocking. We believe that figure 2 indicates that when the salt-bridges are neutralized the polypeptide chain does not unwind a t once to form its extended and most random configuration but that unwinding occurs slowly from rotation on bonds produced by thermal agitation. The unwinding successively makes more of the polypeptide chain available for solvation. The gradual increase in the duration of the emptying periods with the lapse of time is interpreted to mean that the increase in the elongation and solvation processes is responsible. The maximum in the duration of the emptying periods, which was reached 284 hr. after the acetic acid and the solution of egg albumin had been mixed, is interpreted to mean that the configuration of the polypeptide chain has reached its highest state of randomness and that the solvation has become a maximum. I t is quite probable that the polypeptide chain also unwinds after the salt-bridges have been neutralized by sulfuric acid, but, since there is very little of the sulfuric acid present, solvation of the chain does not have an appreciable effect. We believe that the lyospheres, which are formed according to the mechanism described above, may penetrate each other somewhat, but that in general they repel each other, owing to like charges on the polypeptide chains. In the case of denaturation by acetic acid each lyosphere should carry twenty-eight positive charges, which are balanced by a like number of acetate contraions. On this basis it is believed that, by selective adsorption, the anions of added salts neutralize the positive charges on the protein cation. After electrical neutralization there are no repelling forces between the elongated lyospheres, so that collisions between them can result in cohesion and flocculation. However, the addition of variable quantities of salts, which are sufficient to effect only a partial electrical neutralization, results in variable degrees of cohesion of the lyospheres when they collide, owing to the interpenetration of the overlapping portions of the lyospheres with hydrogen-bond formation among the solvating molecules. In this manner chains of lyospheres are built up which cross each other in entirely random directions. At the points of crossing of these chains there will be cohesion by the same mechanism that is operative when lyospheres link together to form chains. That a multivalent anion can also act a t a point of crossing of these chains to hold them in association by electrically neutralizing a positive charge on each one of as many lyospheres as the valency of the anion is indicated by figures 3 and 4. Such a mechanism accounts for the greater effectiveness of salts having multivalent anions over those having univalent anions in increasing the viscosity.

1124


Any of the liquid medium which is not immobilized in the formation of the lyospheres has a high viscosity, owing to hydrogen-bond formation among acetic acid and water molecules. It is then thought to become entrapped in the three-dimensional meshes of the cohering lyosphere chains. The rigidity of the resulting gel depends upon: (1) The protein concentration. (8) The completeness of unwinding of the denatured egg albumin molecules, and hence the time interval which has elapsed since the mixing of the components. Figures 2, 3, and 4 all illustrate this point. (3) The amount of solvation of the polypeptide chain, and hence the concentration of the solvating molecules as well as their relative ability to form many and strong hydrogen bonds. (4) The concentration of salt and the valency of the effective ion. This is well illustrated by figures 3 and 4. Figure 4 shows the greater effectiveness of higher concentrations of a tetravalent anion over corresponding concentrations of a divalent anion and, in turn, of the divalent anion over a univalent anion. Varying these factors in all possible ways gives systems which have any degree of viscosity or which have gel structures of any rigidity. There result sols of variable viscosity, labile gels, thixotropic gels, rigid gels, gels in which the disperse phase shows variable flocculation, and coagulated systems having low viscosities. We believe that the solation of the thixotropic gels by mechanical shear is due to the breaking of the relatively weak hydrogen bonds which hold the lyospheres together. After the shearing stress is removed, there occurs a re-formation of these hydrogen bonds either at the same or (much more likely) at different points than was the case previous to solation. The amount of time required for the gel to re-form depends upon the degree of disruption of the three-dimensional meshwork of lyospheres and hence upon the vigor with which and the duration of time for which the system is shaken. RE BUM^ The explanation given above to account for the gel structures and the thixotropic breakdown and re-formation of the systems studied is a combination of the common theories of gel structure. We have given a logical explanation of the elongation and solvation of the disperse phase to form anisometric lyospheres without postulating any unknown forces acting over large distances in order to account for the formation of solvated hulls. In this way the greatest weakness of the solvated hull theory has been avoided. Also, we do not follow this theory to the extent that the interlocking of the lyospheres is alone thought to be responsible for the establishment of a gel structure. Instead we describe a mechanism according to which the elongated lyospheres are able to link together toform chains and three-dimensional meshworks which entrap any of the disper-


1125

sion medium not involved in the formation of the solvated hulls. I n this way we have modified the network theory of gel structure by postulating that the filaments making up the network are highly solvated previous to their linking together, instead of simply being the disperse phase alone. Also, we have modified the repulsion-attraction equilibrium theory by postulating that added salts decrease the repulsion between the likecharged particles of the disperse phase which have already become highly solvated before the salt is added. The salt is then thought to decrease the repulsion between the elongated lyospheres to the extent that they can cohere enough so as to permit them to link together by the formation of hydrogen bonds among the molecules of the interpenetrating solvated hulls. REFERENCES (1) ANSON,M. L.,

MIESKY,A. E.: J. Gen. Physiol. 16, 341 (1931). (2) ASTBWRY,W. T.: Trans. Faraday SOC.29, 193 (1933). (3) BERGMANN, M.,AND NIEMANN,C.: Science 86, 187 (1937). (4) CHAMBERS, R.: Proc. Soc. Exptl. Biol. Med. 19, 87 (1921). (5) COHN,E. J.: Cold Spring Harbor Symposia Quant. Biol. 6,8 (1938). (6) ERRERA,J., A N D HIRSHBERG, Y.: Biochem. J. a7, 764 (1933). (7) EYRING,H., A N D STEARN,A. E.: Chem. Rev. 24, 253 (1939). (8)FAURB-FRBMIET, E.: Trans. Faraday Soc. 1, 779 (1930). (9) FISCHER, E. : Untersuchungen ziber Aminosdiuren, Polypeptide und Protein. J. Springer, Berlin (1906). (IO) FREUNDLICH, H.: Kolloid-Z. 48, 289 (1928). (11) FREUNDLICH, H., AND ABRAMSON, H. A.: Z. physik. Chem. 131, 278 (1928). (12) FREUNDLICH, H. : Kapillarchemie, 4th edition, Vol. 11. Akademische Verlagsgesellschaft m.b.H., Leipzig (1932). (13) FREUNDLICH, H.: Thizotropy. Hermann et Cie., Paris (1935). (14) FREUNDLICH, H.: Proc. Roy. Inst. Gt. Brit., March 20, 1936. (15) HAMAKER, H. C.: Rec. trav. chim. 58, 727 (1937). (16) HAUSER,E. A.: Kolloid-Z. 48, 57 (1929). (17) HAWSER, E . A.: J. Rheol. 2, 5 (1931). (18) HWGGINS, M.I,.: J. Org. Chem. 1, 407 (1936). (19) JIRGEXSONS, B.: Kolloid-Z. 74, 300 (1936). (20)KALLMANN, H., AND WILLST~TTER, M.: Naturwissensohaften 20, 952 (1932). (21) KEKWICK,R. &4.,A N D CANNAN, R. K.: Biochem. J. So, 22? (1936). (22) KOPACZEKSKI, W.:Protoplaama 29, 180 (1937). (23) LASSETTRE, E. N.:Chem. Rev. 20, 259 (1937). (24) LOEB,J.: Proteins and the Theory of Colloidal Behavior. McGraw-Hill Book Company, Inc., Yew York (1924). (25) LOUGHLIN, W.J., A X D LEWIS,W. C. M.: Biochem. J. !&,476 (1932). (26) MIRSKY,A E., AND PAULING, L.: Proc. Natl. Acad. Sei. U. S. P,439 (1936). (27)MURALT,A. L., AND EDSALL,J. T.: J. Biol. Chem. 89,315 (1930). (28) MYERS,W.G.: Master's Thesis, The Ohio State University, 1937. (29) NEURATH, H.: J. Bm. Chem. Soc. 61, 1841 (1939). (30) PAULING, L.,AND BBOCKWAY, L. 0.: Proc. Natl. Acad. Sci. U. S. 20,336 (1934). (31) PAWLING, L., AND NIEMANN,C.: J. Am. Chem. SOC.61, 1860 (1939). (32) PRYCEJONES,J.: J. Oil Colour Chem. Assoc. 17, 305 (1934). AND

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A. M. BUBWELL, K . F. KAEBB AND W. H. RODEBUBH

(33) RODEBUSH, W. H.: Chem. Rev. 19, 59 (1936). (34) RUSRELL, J. L., AND RIDEAL,E. K. : Proc. Roy. soc. (London) MW,540 (1936). (35) SCHALEK, E., AND SZEGVARI, A.: Kolloid-Z. Sa, 318 (1923);33, 326 (1923). (36) SCEMIDT, C. L. A. (Editor): The Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois (1938). (37) SPONSLER, 0.L., AND DORE,W. H.: Ann. Rev. Biochem. 6, 63 (1936). (38) SVEDBERG, T.:Chem. Rev. 10, 81 (1937). (39) SVEDBERG, T.: Ind. Eng. Chem., Anal. Ed. 10, 113 (1938). (40) WERNER,H.:Ber. 62B, 1525 (1929). (41) WRINCH,D.M.:R’ature 137, 411 (1936);198, 241 (1936);139, 972 (1937). (42) WRINCH,D.M.: Phil. hlag. 26, 313 (1938). (43) Wn, H.: Chinese J. Physiol. 5, 321 (1931).

THE INFRARED ABSORPTION OF PROTEINS IN THE 3 fi REGION. XII‘ A. M. BUSWELL, KARL F. KREBS, AND W. H. RODEBUSH Department of Chemistry, University of Illinois, Urbana, Illinois Received J u l y 3, 1940 INTRODUCTION

.

The study of bound water in biological tissues has been handicapped from its inception by the weaknesses inherent in the methods used for the determination of bound water. None of the various methods gives any clue as to the mechanism of bonding, and all are subject to criticism on the basis of the fundamental assumptions involved (12). A possible mechanism for the bonding of water was suggested by Buswell in 1928 (3). His suggestion that water mas bonded to gelatin by means of the hydrogen bond followed from a correlation of the paper by Latimer and Rodebush (17), who suggested the hydrogen-bond hypothesis, and the classic work in spectroscopy of Coblentz (9), who had noticed peculiarities in the infrared absorption of water a t 1.5 p when present as “water of constitution” in certain minerals and in a gelatin film. The work of Wulf and Liddel (21, 13), who showed that the first harmonic of the hydroxyl group disappeared when opportunities for hydrogen bonding were present, indicated that the infrared absorption technique offered a method of testing this proposed mechanism, and also gave promise of being adaptable to quantitative determination of bound water in biological hydrogels. 1 Presented at the Seventeenth Colloid Symposium, held at Ann Arbor, Michigan, June 6-8, 1940.

A Study of Some Properties of Thixotropic Gels Containing Egg

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