1. Introductory

STUDIES IN T H E ELECTROCHEMISTRY O F T H E PROTEINS. VII. THE MODE OF FORMATION AND IONIZATION O F T H E COMPOUNDS OF PROTEINS WITH INORGANIC ACIDS AND BASES T. BRAILSFORD ROBERTSON

(From the Rudolph Spreckels Physiological Laboratory of the

University of Califorrzia) 1 . Introductory The investigations of Emil Fischer and of Kossel have shown us that the proteins are built up from amino-acids linked up in a catenary manner, and have demonstrated t h a t the mode of linkage is expressed by the formula:-COHN--.

By methods which are too well-known to require description here, Fischer has succeeded in building up chains of amino acids linked together in this manner which he terms "Polypeptids, " many of which exhibit characteristic properties of of the peptones, such as precipitability by ammonium sulphate, digestibility by trypsin, etc., and some of which have been found to occur in incomplete protein digests as intermedyate products of hydrolysis. These polypeptids and, presumably, the proteins, are as essentially amino-acids as the amino-acids out of which they are built up. Thus glycylglycin, NH,.CH,.CO.NH.CH,COOH is as typically an amino-acid as glycocoll itself (NH,.CH,.COOH), since it possesses an NH, group as well as a COOH group, and hence is capable of forming compounds both with acids and with bases. On undergoing electrolytic dissociation it may be supposed to yield either hydrogen (H+) ions, or hydroxyl ions, owing to the occurrence of a reaction with water of the type: R/", \COOH

+ H.OH = R

/",OH

\COOH

T . Brailsford Robertson

522

just as ammonia, in solution, partially reacts with water to form “,OH. It is usually conceded that these elements in the structure of the proteins afford an explanation of the power which they possess of neutralizing both acids and bases; the “ a m photeric” character of the proteins. To this opinion I have also formerly inclined, but an accumulation of data irreconcilable with this view have induced me to abandon it. The weight of evidence appears irresistible that some elements in the protein molecule other than terminal NH, or COOH groups are responsible for the acid- and base-neutralizing power which is possessed in such a marked degree by many proteins. In the first place the investigations of Levitesl have shown that only a very small proportion of the nitrogen in proteins is present within their molecules in the form of NH, groups, and even his estimates of the NH, nitrogen are probably in excess of the true values.’ Now Edestin, as Osborne has shown,s is insoluble, when in the free condition, in water. It forms an insoluble hydrochloride containing 14 X IO-^ equivalents of HC1 per gram and, on further addition of acid, passes into solution. Its combining capacity for acids does not remain constant, however, for at neutrality to Tropaeolin, which corresponds4 to a reaction of from 0.01t o 0.001N H f , i t neutralizes 127 X IO-^ equivalents of acid per gram. Hence, if the acid is neutralized by the -NH, groups of Edestin, the number of these groups must be at least E’ - ~ .

_____

~.

I4

=

9.5

S. Levites: Zeit. physiol. Chem., 43, 202 (1904); Biochem. Zeit., 20, 224 (1909). Emil Fischer : “ Untersuchungen ueber Aminosauren, Polypeptide und Proteine” Berlin, 1906, 5 2 . T. B. Osborne: Jour. Am. Chem. SOC.,21, 486 (1899); Zeit. physiol. Chem., 33, 240 (’90’). Ed. Salm: Zeit. phys. Chem., 57, 471 (1907). Osborne believes that a n insoluble “monochlorhydrate” is also formed, containing 7 x IO-^ equivalents of acid per gram, which would raise this number t o 18, but would, a t the same time, double the estimate of the molecular weight.

Studies in the Electrochemistry of the Proteins

523

From the former determination it would appear that the molecular weight of Edestin is 7000, and this corresponds with the molecular weight indicated by its tyrosin and glutamic acid content’ ( = I mol. tyrosin 3 mols. glutamic acid . . .). Nine -NH, groups in this molecule would correspond to over ten percent of the total nitrogen.’ From the investigations of Erb,s although the exact interpretation which is to be placed upon his results is not perfectly clear, it would appear that the combining-weight of egg-albumin for acids may be as low as 1 5 2 , while its molecular weight is, according to Hofmeister, 5400 or some multiple of this. Hence, upon the assumption that terminal -NH, groups bind the acids, ,~ there must be at least 35 of them in e g g - a l b ~ r n i n which would correspond to no less than 69 percent of the total nitrogen in this protein. The number of terminal -COOH groups cannot be much in excess of the number of terminal --NH, groups, since the protein would otherwise be overwhelmingly acid in characterb and the majority of the proteins possess a distinct capacity for neutralizing acids, even when they are thzmselves predoxinantly acid. Now free caseine is insoluble in water, but when combined with acids or with bases it is soluble. To carry one gram of casein into solution 11.4 X IO-^ equivalents of base just suffice, indicating a niolecular weight, for the casein, of about 8800 or a multiple of this. The tyrosin, glutamic

+

-+

A. Kossell and A . J. Patten: Zeit. physiol. Chem., 38, 39 ( 1 9 0 3 ) . Or almost exactly t o the -NH, content of the Arginin, calculated as the / m e diamino acid, which the Edestin molecule contains. Kossel : (Zeit. physiol. Chem., 2 5 , 165 (1898) attributes the basic properties of proteins to their content of hexone bases, and to this view electrochemical data lend very decided support. Edestin contains I 1.7 percent of arginin, but only insignificant amounts ( 1.0 percent and I . I percent respectively) of lysin and histidin. As a t least one of the -NH, groups of the arginin is almost certainly present in the protein molecule in the imino form, it is highly improbable that the acid is neutralized by terminal amino groups supplied by the arginin. W. Erb: Zeit. Biol., 41, 309 (1901). ‘ Gustav Mann: “Chemistry of the Proteids” London, 1906,147. F. Hofmeister: Ergeb. d. Physiol. I Abt., I (1902). T.Brailsford Robertson: Jour. Phys. Chem., 13, 469 (1909); 14, 528 (1910).


524

acid and sulphur contents of casein indicate a minimal molecular weight of from 4000 to 4400. In the presence of excess of base, however, casein attains a maximal combining-capacity (measured by the gas-chain) of 180 X IO-' equivalents per gram, so that it behaves like a 16-basic acid, and if -COOH groups bind the base there must be 16 of .them in the molecule, corresponding to 25 percent of the total oxygen or, almost exactly, to the number of -COOH groufis contained in the glutamic acid radicals in the casein molecwle, calculated for the fTee acid. In order to provide so many free carboxyls the form of the casein molecule would have t o be that of a branched chain, or the radiating spokes of a wheel, at the center of which must exist unions of the type:

and the regular decomposition of this protein into its constituent amino-acids, upon hydrolysis, would be unintelligible. Moreover, in the synthetical polypeptids, which closely reserrtble the natural peptones in their behavior, the linkage of the amino-acids is not radial but catenary in character2 and the peptids which have been isolated from the mixed products of partial protein hydrolysis are likewise catenary in structure. To account for the high acid- and base-containing capacity of the proteins we must therefore look to some other point in the molecule than terminal --NH, or -COOH groups. ~-

I

The point of union -C-N-C-

-

-~

.

1

l

l

immediately suggests itself . 3

I

Hofmeister: LOC.cit. Even when dicarboxylic acids enter into the compound. Cf. Emil Fischer and Ernst Koenigs : Ber. d. chem. Ges. Berlin, 37,4585 (1904), Emil Fischer and Julius Schmidlin: Liebig's Ann., 340, 123 (rgoj). It has been pointed out by Vernon (Jour. of Physiol., 31, 346, 1904) t h a t although the power of the sum of the decomposition-products of a protein to neutralize bases is somewhat, yet i t is only very slightly greater than that of the groups unhydrolyzed protein. Now in t h e process of hydrolysis the -COH.Nof the protein are split into -NH, and -COOH groups; yet this results in no


525

The type of this union which occurs in the polypeptids and presumably, in the proteins is either : -CO.NH-

or:

-C(OH) = N-

between which, the keto- and the enol-forms, synthetical data do not suffice to decide.’ According to Werner’s theory of valences the nitrogen in either of these unions contains two latent valences, positive and negative, which, whiln, the nitrogen is trivalent, neutralize one another internally, but which, when the nitrogen becomes pentavalent, are capable, respectively, of neutralizing a negative or a positive radical. The second of the above types of union (the enol-form) carries with i t the possibility of the following types of reactions : H I -COH = N-- + Na+ + OH’ = -CONa++ + W - .

I

OH

and :

H -COH

= N-

+ H+ + C1’

= -COH++

+ ” NI . . I

,

..

,

..

c1

yielding, in each case, only protein ions. I have already incidentally dwelt upon the fact that there is reason t o suspect that diamino- and dicarboxylic radicals in the protein molecule play a predominant part in accomplishing the neutralization of acids and bases, and electrocheniical data, as we shall see, lend most decided support to this view. Accordingly, the above formulae, which represent the reaction when only a single -COH.N-bond is involved, pronounced gain of combining-capacity for bases. The obvious conclusion is that the -COH.Ngroups within the protein molecule must be nearly as efficient in accomplishing the neutralization of bases as t h e -COOH groups of the constituent amino-acids out of which the protein is built up. Cf. Aders Plimmer “The Chemical Constitution of the Proteins” London, 1908, 2 .

T.Brailsford Robertson

526

should, in reality, be doubIed, and the following possibilities exist : In combination with bases :

H OH

+

+ KOH + H , O = R

$/ NNN-

' ' ' ' '

(3)

/\

H OH

&c

H

+ 2ROH

COK++ = R/ \COK+

OH

$- #N+

'

'

,

.

,

(4)

/\

H

OH

H

C1

In combination with acids:

\/ -COH. N

\R -COH .N/

+ HC1 + H,O =

-COH++

"N

-COH++

,,N

/\

H OH H

C1

/\

H

C1

It is obvious that in reactions 3 and 5 the molecule of water may or may not participate in the reaction; also that the anionic groups in reactions 3 and 4 may or may not be united to form a single quadrivalent anion (de.ived from a diamino-group) and that the cationic groups in reactions 5 and 6 similarly niay or may not be united t o form a single quadrivalent cation (derived from a dicarboxylic acid group). As we shall see, no evidence has been found (at least among

Studies in the ELectrochemistiy of the Proteins

527

the compounds of casein or of serum globulin with bases) for the occurrence of reactions of the type represented by equation 3. Equation 4 faithfully represents, so far as the electrochemical data are concerned, the mode of combination of bases with these proteins, and the anionic groups are united to form one quadrivalent anion. The union of serum globulin and ovomucoid with acids follows equation 5 (with or without the molecule of water) when the concentration of acid is low; btit at higher acidities ovomucoid, at least, combines with HCl in the manner indicated by equation 6. In the following pages I will endeavor to interpret some of the more important electrochemical data published in previous comniunications of this series, and also certain data not previously published, in the light of the above hypotheses. 2. The Non-ionic Character of the Inorganic Radical in

Compounds of Proteins with Inorganic Acids and Bases In previous communicationsZ I have pointed out that the inorganic radical, in the compounds of proteins with inorganic acids and bases, is not electrolytically dissociated as such; basing this conclusion, in the main, upon indirect evidence, ‘uiz., that the degree of dissociation of potassium caseinate is not affected by the presence within its solution of KC1, and that the sum of the equivalent ionic migrationvelocities of the ions into which the caseinates dissociate is in some instances less than the equivalent migration-velocity of the inorganic radical itself. Direct proof of this fact has, however, been obtained by Bugarszky and Liebermann. These observers employed the potentiometric method, using Unless the fact that the “apparent” molecular conductivity of potassium caseinate (calculated for the molecular concentration of KOH bound by the casein) rises somewhat as the proportion of base t o protein declines (Cf. T. Brailsford Robertson: Jour. Phys. Chem., 14, 528 (1910)) constitutes such evidence. As yet, however, I consider it inconclusive. T.Brailsford Robertson: Jour. Phys. Chem., 11, 542 (1907);14,528, 601 (1910).

Stefan Burgarszky and Leo Liebermann: Arch. f. d. ges. Physiol., 72, 51

(‘898).

T . 'Brailsjord Robertson

528

two different concentration-chains. nary gas-chain : P t saturated with H,

The first was the ordi-

Acid ( H C I )

Base ( NaOH)

2

s

P t saturated with . H,

4

the potential being measured, first with pure acid in 2 , and then with acid plus protein; the difference between the two reading?' yielding, by computation from the Nernst formula, the nunlber of hydrogen ions bound by the protein. The second concentration-chain was built up as follows : HgCl (solid) H C1

NaCl

NaBr, HgBr (solid)

2

3

4

I

5

and enabled them t o estimate, in a similar way, the number of C1' ions bound by the protein. Now the number of Cl' ions bound by a giTen mass of protein dissolaed in dilute HCl was found to he exactly equal to the number of H+ ions which it binds. The following data are compiled from those obtained by Bugarszky and Liebermann. -

Grams protein in I00 cc 0

0.4 0.8 I

Egg-albumin in 0.05 iV HC1 - - - . __ _ _ -

-

-

.6

3.2 6.4

~~

1 I

Percent of H + bound by the protein

I

0

9.0 18.9 33.3 60.2 96.6

Percent of C1/ bound by the protein 0

10.7

I

20.2

38 .o

64.0 76.0

I

The slight irregularities are, save in the last observation, no greater than might have arisen out of the uncertain magnitude of the correction for the potential between the elements 2 and 3 in the gas-chain. I

Less a correction expressing the potential between

2

and 3.

Studies in the Electrochemistry of the Proteiizs

529

Confirmatory evidence is not lacking. Loevenhart' and others have shown that rennet will not coagulate calcium caseinate unless a small amount of a dissociable salt of calcium is present. The calcium bound by the casein itself is not available for this purpose, but if a small quantity of acid be added then a proportion of the calcium is freed from its combination with casein and, if it forms a dissociable salt with the added acid, it is able to bring about coagulation. W. A. Osborne' has shown that if cacium caseinate be placed inside a dialyzing tube which is then immersed in a very dilute solution of mercuric chloride the mercury diffuses into the tube and is there held in an undissociated form, since the concentration of mercury zedhin the tube s found, after some time, to considerably exceed that of mercury in the outside fluid. Rohmann and Hirschstein have shown that solutions of silver caseinate contain no silver ions, since they fail to yield a precipitate on adding sodium chloride. The most obvious conclusions to be drawn 'from these results are that the protein-base or protein-acid compounds are not dissociated at all, or else that they dissociate the positive and negative ions of the inorganic constituent in equivalent proportions, i. e., undergo hydrolytic dissociation. That these assumptions are incorrect, however, is shown by a large number of experimental data, many of which will be found in previous communicat'ons of this series, which demonstrate that the protein compounds with inorganic bases and acids are true electrolytes, independent of any A . S. Loevenhart: Zeit. physiol. Chem., 4, 176 (1904). The statement of van Dam: Ibid., 58, 295 (1908) and 61, 146 (I909), that the extent of coagulation depends upon the quantity of Ca bound by the casein is not irreconcilable with Loevenhart's results. As is well known, the calcium bound in casein as calcium caseinate does not suffice to bring about coagulation. A dissociable salt of calcium must also be present. That this salt may combine with the calcium paracaseinate to form a double salt is not a t all unlikely. (Cf. T.Brailsford Robertson: Jour. Biol. Chem. W. A. Osborne: Jour. Physiol., 34, 84 (1906). F. Rohmann and L. Hirschstein: Beitr. chem. Physiol. und Path., 3, 288 (1902).

530


hydrolytic dissociation which they may undergo in solution. I will confine myself to two illustrations, Sjoqvistl has shown that if egg-albumin be dissolved in dilute hydrochloric acid, as the concentration of albumin is increased, keeping that of the HCI-solution constant, the molecular conductivity (calculated for 0.025 N HC1) diminishes until it reaches a constant minimum value, which is attained when about four grams are dissolved in IOO cc of 0.025 N HC1. Now the above quoted results of Bugarszky and Lieberniann show that in this solution at least 97 percent of the hydrochloric acid is bound by the egg albumin. The observed “ molecular ” conductivity ( = 67 X IO-^) is at least 7 times greater than could be accounted or by the maximum possible residual of unneutralized hydrochloric acid and must therefore be due to the protein-acid con: pound. Solutions of the caseinates of the alkalies and alkaline earths can be obtained which are neutral or even acid to litmus, these solutions therefore contain no free base; nevertheless they are excellent conductors of electricity’ a 2 percent solution of potassium caseinate which is neutral to litmus possessing a conductivity of between 80 and go X IO-’ reciprocal ohms per equivalent of base neutralized a t 30’ C.3 That this conductivity is not attributable to associated impurities, inorganic or other, is shown by the following facts : (I) It bears a definite relation to the amount of base neutralized by the protein. (2) The conduction of electricity is accompanied by migration of the casein to the anode, and the amount of casein transported to the anode is directly proportional to the quantity of electricity which is transported through the solution. Similarly, solutions of the serum-globulinates of the alkalies and alkaline earths may be obtained which are neutral to litmus and which nevertheless conduct electricity, the J. Sjoqvist: Skand. Arch. E. Physiol., 5 , 277 (1895). 0. Sackur: Zeit. phys. Chem., 41,672 (1902);T. Brailsford Robertson: Jour. Phys. Chem., 11, 542 (1907);14,528, 601 (1910). 3T.Brailsford Robertson: Jour. Phys. Chem., 14,538 (1910). Ibid., 15, 179(1911). 2


53 I

passage of a direct current through the solutions being accompanied by transport of the protein to the anode.' An experiment which demonstrates in a very striking manner the fact that protein salts do not undergo an appreciable amount of hydrolytic dissociation, in solution in water, nor split off the inorganic radical as an ion, is the following: It will be recollected that casein, deprved of its combined base or acid, is insoluble, and that if, to a solution of a caseinate of a base, exactly enough free acid be added (e. g., HCI) t o completely neutralize the combined base the free casein is entirely precipitated. Now one gram of ovomucoidZ combines with 45 X IO-^ equivalents of HC1 to form a compound such that less than I percent of the acid remains uncombined (as estimated by gas-chain measurements). One gram of casein combines with 90 X IO-^ equivalents of KOH t o form a compound such that less than I,/, percent of the KOH remains uncombined. If, now, these salts were appreciably subject t o hydrolytic dissociation, or if they yielded C1' and K+ ions respectively, then on mixing two volumes of a solution of the ovomucoid salt with one volume of a solution of the casein salt (each of the same percentage concentration) the K+ provided by the caseinate would be exactly neutralized by the C1' provided by the ovomucoid salt and it might be anticipated t h a t free, uncombined casein, would be precipitated. Nothing of the sort occurs, however. If to 25 cc of a 2 percent solution of the casein salt be added 50 cc of a 2 percent solution of the ovomucoid salt the mixture is no more opalescent than its constituent parts and the conductivity of the mixture i s the s u m of the separate conductivities of the two protein ~ a l t s . ~If the mixture be allowed to stand in the pres-

' W.

B. Hardy: Jour. Physiol., 33, 251 (1905).

T. Brailsford Robertson: Jour. Phys. Chem., 14,709 (1910). Ibid., 14, 528 (1910). Conductivity of a 0.5 percent solution of potassium casein to containing 90 x IO-^ equivalents of KOH per gram, a t SO0, = 38.0 X IO-^ reciprocal ohms. Conductivity of a I .o percent solution of ovomucoid chloride containing 45 x 10-6 equivalents of HC1 per gram, a t 30°, = 80.1 X o+ reciprocal ohms. Sum of these two conductivities = 118.1 X IO-' reciprocal ohms. Conductivity of a mixture containing the two salts in the above concentrations, at 30°, = 108.5 x IO-^ reciprocal ohms. 3

T . Brailsfovd Robertson

532

ence of toluol for a considerable period at 36', however, after 24 to 48 hours a marked increase in its opalescence is observed; after two or three days traces of casein begin t o be deposited, and after three'to four days all of the casein is found to have been precipitated. The precipitation of the casein is accompanied by a marked increase in the conductivity of the mixture, attributable to the setting free of KC1. It is therefore evident that at the beginning the mixture must contain only minute traces of K+ and C1' ions and that the protein salts only yield up these ions with extreme slowness. Only one conclusion is left open to us, therefore, namely, that the salts which proteins form with inorganic acids and bases do not dissociate at the point of union of the inorganic radical with the protein, but elsewhere, within the protein molecule its I f , yielding, not an inorganic and a protein ion, but two or more protein ions, in one or more of which the inorganic radical is bound up in a non-dissociable form. 3. The Electrolysis of Protein Salts

It was shown by Hardy' in 1899 that if a trace of acid be added t o a solution of dialyzed white of egg, modified by dilution and boiling, on passing a direct current through the solution the whole of the protein finally moves over to the cathode, where it is precipitated. If a trace of alkali is added, however, instead of acid, the whole of the protein finally migrates, under the influenc of the current, to the anode. He later showed that serum globulin, in solution, behaves similarly.' I have shown that if a direct currmt of about I milliampere be led through a solution of potassium caseinate, casein is deposited upon the anode and the amount of casein so deposited is proportional to the quantity of electricity which has traversed the solution.3 Under such circumstances, therefore the protein behaves I

W. B. Hardy: Jour. Physiol., 24, 288 (1899). W. B. Hardy: LOC.cit. LOC.cit.


533

like the anion or cation of a salt, like the cation when combined with an acid, like the anion where combined with a base. Here an apparent, but not a real difficulty confronts us in the application of t h ? hypothesis outlined above to the electrolysis of protein solutions. On the basis of this hypothesis the protein should migrate in both directions, the cation when the protein is combined with a base, and the anion, when the protein is combined with an acid, carrying the inorganic constituent with it. At first sight it might appear' as if the proteins should be deposited at both electrodes, but not when we look more closely into the matter. In both of the cases just cited the free, unconibined protein is insoluble, while the combined protein is soluble. Consider the electrolysis of a compound of such a protein with a base. The anion' H

I

R.N" will migrate to the anode, and after neutralizing any I OH excess of base which may be present (since the ion obviousl}., unless the neutralizing capacity of the protein is already fully satisfied, i. e., unless the solution is very alkaline, must contain -COH,N-groups and therefore, like the entire molecule, is amphoteric) must eventually, when the film in irnmediate contact with the electrode has become saturated with protein, be precipitated as the uncombined and therefore insoluble protein. The case is very different with the cation R.CONa++, for this, on arriving a t the cathode, will bring with i t an excess of base and the cathode region must become alkaline and, therefore, free protein cannot be precipitated there. Similarly, in the electrolysis of a salt of such a protein with an acid the uncombined protein must be precipitated a t the cathode, but not a t the anode. If the protein is of such a type that the free protein is soluble, for example ovomucoid, I have observed that no deposition of protein I n all thaL follows, for the sake of simplicity, unless actual error is involved bonds which are by doing so, I will consider only one of the two -COH.Na.ctually split in the electrolytic dissociation of protein salts.

,


534

occurs a t either electrode, but only the evolution of gas, presumably hydrogen at the cathode and oxygen at the anode. I have shown’ that the electrochemical equivalent of casein, precipitated from a potassium caseinate solution (neutral to litmus or phenolphthalein) by electrolysis is 0.0242 f 0.0019 gram per coulomb. Multiplying this by the Paraday constant, 96530, we obtain the weight of casein which transports one atomic charge. This is 2336 j, 183. Immediately preceding the precipitation of casein at the anode the proportion of casein to base in the film of solution which is in immediate contact with the anode must be that which obtains a t “saturation” of the base by casein. Now at “saturation” of a base by casein the proportion of base to casein is 11.4 X IO-‘ equivalents per gram.’ corresponding if, a t this reaction, casein combines with only one molecule of base, with the moleculer weight of 8772. If we assume that at saturation ” of the base with casein two, three, or four, etc., molecules of base are bound up in one molecule of caseinate the molecular weight of the casein would be two, three, or four, etc., times 8772. Either of two assumptions may now be made : ( I ) The potassium caseinate dissociates into potassiumand casein ions. If this be the case then the weight of the casein anion must be that of the molecule of casein, i. e., a multiple of 8772, and the valency of the casein ions must be a ‘ I

multiple of

__8772 - i. e., of 3.8 ;t 0.3 or, in round numbers, 4. 2336 f 183

The potassium caseinate dissociates into two pro(2) tein ions of approximately equal weight. If this be the case then the weight of the casein anion must be half that of the molecule of casein, i. e., a multiple of 4386, and the valency of the casein ions must be a multiple of

4.386 2336 f 183

2.

e., of

1.9 h 0.15, or in round numbers, 2 . Since, as we have seen above, the former of these two ~I

T.Brailsford Robertson: Jour. Phys. Chem., 15, 179 ( I 9 1 I ) .

* I b i d , 13,469 (1909);14,538 (1910).


535

assumptions is inadmissible, we may conclude that the valency of the casein ions in solution of a base “saturated ”with casein i s a multiple o j 2. This obviously corresponds with the view that the caseinate dissociates into two protein ions according to either of the schemes :

H

I

++

R.N”+ K0C.R

I

OH

or :

As we shall see, conductivity and freezing-point data point to the latter of these schemes. We may infer that, in all probability, the true molecular weight of the combined caseiii is about 17600 and that one molecule of the casein is combined with two molecules of a (monacid) base. 4. The Relative Masses of Protein Anions and Cations From the above experimental results it appears that the masses of the protein cation and anion must be nearly equal, for otherwise the weight of one ion would not be 1/2 but some other fraction of the entire molecule and the valency, deduced from the above experimental data, would not be a whole number but some fraction. Since the valency of an ion is necessarily a whole number and not a fraction, the equivalent weights of the cations and anions which the protein molecule yields must be nearly equal. The above data do not enable us, however, to decide whether or not the weights of the cations and anions are exactly equal, since the precision attained in these experiments was not sufficient to reveal with certainty a di erence of less than I O percent between the weights of the two ions.

536


It has been shown by Bredig' that the equivalent migration-velocities' of very heavy ions under unit potentialgradient at constant temperature tend to approach a minimal constant value of about 2 0 X IO+ cm per sec. a t 18' C. The conception developed above, therefore, of the mode of dissociation of the salts of a protein, leads to the conclusion that the equivalent velocities of migration of both the cation and the anion of a protein salt must be equal. Equal numbers of protein ions must therefore migrate to the anode and t o the cathode respectively. Now since only one of these ions is precipitated it would appear possible to determine whether or not they are of equal weight by measuring the change in concentration a t the anodal and cathodal regions. Employing Hittorf 's method of representation, in the accompanying diagram let the region to the right represent the cathodal and that to the left the anodal region. Let the black dots represent anions and the white cations. The initial state of the solution is represented by the first double row of spots, and its final condition, after the decomposition of six molecules by the current, in the second double row of spots.

............ ............ 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Fig. I

Since the velocities of the ions are presumed to be equal the number of undecomposed molecules of the original caseinate must, after the passage of the current, be the same on each side. The ions which have separated to the left (anions) have been precipitated, while those which have migrated to the right have remained in solution. It is clear from the above -

G. Bredig: Zeit. phys. Chem., 13, 191 (1894). That is, the migration-velocity under unit force. For a divalent ion under unit potential gradient the force exerted is twice as great as that which is exerted on a univalent ion; the absolzcte velocity is therefore twice as great as that of a univalent ion, but the equivalent velocity is the same.

StzGdies in the Electrochemistry of the Pyoteins

537

diagram that on the right three anions have been lost and three cations have been gained. If they were equal in weight, therefore, it would appear as if the concentration of casein in the right (cathodal) half should remain unaltered by the passage of electricity. A moment's consideration, however, will suffice to show that this is not correct. In attaining this conclusion the assumption has been made that the casein cations, after reaching the cathods, no longer participate in the carrying of current. Unquestionably this is not the case. The combining-capacity of casein increases markedly with increasing alkalinity of its solution, a t first in direct proportion,' each fresh equivalent of combined alkali splitting another -N.HOCunion. Therefore the potassium or other univalent metal which is transported along with the casein to the cathode, on being transformed there into the hydrate, will split the ion with which it has traveled into two, thus:

++

H,N.R.COH.N.R.COK -k zH,O = H,N.R.COH.N.R.COOH

+ KOH + H,; H

H,N.R.COH.N.R.COOH

I + KOH = H,N.R.COK + "N.R.COOH. ++

I

OH The resultant cation may be presumed to be retained in the cathodal region while the anion migrates to the anodal region. Each cation which the cathodal region gains, therefore, yields up, provided the masses of the cations and anions are in every case equal, one half its mass to the anodal region again.' Referring t o the diagram again, therefore, the true gain by the cathodal region will be only half of that represented, equal in weight to only three of the six ions which have been transported to the cathode, while its loss will have been three IT. Brailsford Robertson: Jour. Phys. Chem., 14, 528

(1910). This will be rigorously true, of course, only as long as the proportion of base to protein a t the boundary of the cathodal region is below that at which t h e combining-capacity of the protein ceases t o increase in direct proportion to the concentration of base, for casein about 100X IO+ equivalents per gram.

5 38


molecules, together equal to the total weight of casein lost from the anodal region.' Provided, therefore, the anions and cations in a solution of potassium caseinate are equal in weight, no matter what their absolute weight, then it is clear that the loss of casein from the cathodal region must be exactly half the loss jrom the anodal region.2 I have endeavored t o test this theoretical conclusiori by determining the loss of caseirl from the anodal and cathodal limbs of the U-tube employed in the experiments described in my communication upon the electrochemical equivalent of casein." The following are some experimental results. The measurements of the amounts of casein were made by means of the refractometer, as described in the communication to which I have referred. I. Solution of 3.75 percent in 0.03 N KOHneutral to phenolphthalein. Electrolysis for z hours a t 30'; current approx. I milliampere. Grams of casein lost from.the anodal arm 0.062 f 0.007 Grams of casein lost from the cathodal arm 0.036 f 0.009 Ratio 1.9 f 0.7 Solution of 4 percent casein in 0 . 0 2 N KOH; neutral to lit2. mus. Electrolysis for z hours a t 30'; current approx. I milliampere. Grams of casein lost from the anodal arm 0.093 f 0 . 0 0 8 Grams of casein lost from the cathodal arm 0,045 f 0 , 0 0 7 Ratio 2.15 f 0.55 3. Solution of 3.75 percent in casein0.03 N KOH; neutral to phenolphthalein. Electrolysis for 2 hours a t 30'; current approx. I milliampere. Grams of casein lost from the anodal arm 0.070 f 0.008 Grams of casein lost from the cathodal arm 0 . oog 0.045 Ratio 1.65 f 0.55

The anions retzwned to the anode, or their equivalent, are of course deposited on the anode and do not constitute a gain on the part of the anodal region. The same conclusion may be reached by another process of reasoning. It is evident that the end-result of the return of half of each cation to the anode must be the same as if the cation possessed one half the absolute migrationvelocity of the anion. Under such circumstances, the anion being deposited upon the anode and the cation retained in the cathodal fluid, the loss from the anodal region, as a reference to the Hittorf diagram shows, must be half the loss from the cathodal region. T. Brailsford Robertson: Jour. Phys. Chem., 15, 1 7 9 ( 1 9 1 1 ) .


539

Within the experimental error, therefore, the ratio of the anodal to the cathodal loss is z as demanded by theory. The experimental error in determining the ratio is large, however, as the above figures reveal, and although these results may be taken as confimatory of the general correctness of the above outline of the mechanism of electrolysis in these solutions, yet they do not suffice to enable us to determine whether or not the protein anions and cations are absolutely equal in mass. I’urther elaboration and refinement in the technique of these measurements will doubtless in the future enable us to measure the relative masses of the protein anions and cations with considerable precision. In passing it may be pointed out, however, that these results afford a striking confirmation of the view which I have developed above that the protein salts in solution in water do not yield protein and inorganic ions but only protein ions. Referring to the Hittorf diagram again it will be evident that if the cations were potassium ions the loss of casein from the anodal region should be a t least four times that from the cathodal region, since the equivalent velocity of potassium ions is at least four times that of heavy organic ions. The experimental fact that the loss from the anodal region is only about twice as great as that from the cathodal region can only be interpreted by assuming that protein material is transported into the cathodal region by the current, in other words, that the curred i s transported in both directions by protein ions. 5. The Migration-Velocity of Protein Ions W. B. Hardy endeavored to measure the migration-velocities of serum-globulin ions directly1by placing solutions of seruniglobulin, combined with acids or bases at the bottom of a U-tube and placing above the solutions, in both arms of the U-tube, a solution of the acid or base employed to dissolve the protein. A potential gradient was placed across the U-tube and it was observed that in acid solutions both boundaries of the W. B.Hardy: Jour. Physiol., 33, 286 (1905).

540


protein solution migrated towards the cathode, while in alkaline solutions both boundaries of the protein solution migrated towards the anode. The rate of migration TTaried between 7 . to I O X IO-^ cm per sec for unit potential gradient when strong acids or bases (HC1 and NaOH) were employed and about 2 0 X IO-^ cm per sec when a weak acid (acetic acid) was was employed. Following the view which I have developed of the mode of dissociation of protein salts it would appear as if the boundaries should move in opposite directions a t approximately equal velocities. A moment’s consideration of the method of measurement employed by Hardy shows, however, t h a t under the conditions of his experiments this would not occur. At the boundary of the protein solution (nearly neutral) and the HC1 solution (for example) a difference of potential of considerable magnitude would exist owing to the much more rapid diffusion of H+ into the protein solution than of C1’. This difference of potential would lower the potential gradient a t the cathodal boundary and raise it a t the anodal boundary. Bearing in mind the fact that the two protein ions are possesssed of the same equivalent migration velocities under unit potential fall, the effect of these inequalities in potential gradient must have been, at the anodal boundary, to urge the protein anions toward the anode more rapidly than the cations were repelled from the boundary, and at the cathodal boundary t o repel the protein anions from the boundary into the protein solution more rapidly than the cations crossed the boundary. The net result of these processes would be, obviously, a migration of the protein, as a whole, towards the anode. The contact-differences of potential at the boundaries would be equal in magnitude but opposite in sense and hence both boundaries would migrate a t the same velocity, but in the same direction. The fact that the highest velocities were obtained whqn weak acids (acetic) were employed, in which the difference between the equivalent velocities of the ions (H+ and acetanion) is greatest, strongly supports this interpretation of Hardy’s results. Alkali-globulin, in contact with alkaline solutions would, of course, move as a whole, but


541

somewhat more slowly (since the difference between the velocity of OH’ and that of Na+ is not so great as that between H + and Cl’), towards the cathode. This accords with the experimental observations of Hardy. The velocities measured by Hardy, therefore, do not afford a measure of the migrationvelocities of protein ions under a uniform potential gradient. 6. The Non-dependence of the Composition of the Com-

pounds of Protein with Acids and Bases upon the Dilution of their Solutions The view would appear to be very generally held’ that the salts which the proteins form with acids and bases are subject in a very high measure to hydrolytic dissociation. This view is nevertheless erroneous. When a base unites with an organic acid to form a salt by the neutralization of a -COOH group the reaction may be expressed as follows: R.COOH

+ KOH If

RCOOK

+ H.OH.

If the acid is a very strong one then the reaction will proceed completely from left to right and the salt will not be appreciably decomposed by dilution. If, however, the acid is tolerably weak (e. g., acetic acid) so that the water itself, acting as an acid, is able, if present in large proportion, to partially displace it from its combination with the base, then the reaction will not proceed completely from left to right but will pause, in accordance with the mass-law, in a condition of balance, which is shifted to the right by the addition of excess of base and towards the left by dilution. On adding excess of base to a dilute solution of an acid of this type, therefore, more of the base will be observed to be neutralized, while the addition of water, i. e., dilution, will result in partial decomposition of the salt. Similar considerations hold good, of course, for the salts of weak bases. When one examines the evidence which has been brought forward by different observers in support of the thesis that protein salts are subject to hydrolytic dissociation, one finds that it is all of the first kind, that is, con~~

~~

.~

Cf. for example H. Ley: Zeit. phys. Chem., 30,193 (1899); W. E r b : Zeit. Biol., 41,309 ( 1 9 0 1 ) ; L. von Rhorer: Arch. ges. Physiol., go, 368 ( 1 9 0 2 ) .


542

sists in the fact upon the addition of excess of acid or base to an acid or alkaline solution of protein a greater proportion of the acid or base is bound by the protein. From this the somewhat illogical, but natural assumption has been made t h a t the protein salts must exhibit the other characteristic property of salts of weak acids and bases, namely, decomposability by water. The fact that protein will bind more of an acid or a base, the greater, within certain limits, the excess of these reagents, admits of a very different explanation, however, groups by innamely the splitting of successive -N.HOCcreasing acidity or alkalinity of the solution, while numerous data contained in previous communications of this series and elsewhere enable us to conclude that water is not able to appreciably decompose the salts of proteins, at any rate the salts which casein forms with bases. For example, it has been shown by a number of observers' by direct titration that the quantity of a base which is bound by one gram of casein a t neutrality to litmus (i. e., absolute neutrality) or t o phenolphthalein is always the same whatever the total dilution of the system. I have been able to confirm this result with considerable precision with the aid of the gas-chain. Also, a survey of the gas-chain measurements enumerated in the communication just cited reveals the fact that the combining-capacity of casein for bases, when it attains its maximum in alkaline solutions, is independent of the dilution. That this should be so in alkaline solution is not surprising, since the excess of alkali might be expected to drive the reaction: R.COOH

+ KOH JJ R.COOK + H.OH

over towards the right; but that it is so in absolutely neutral solution, when the concentration of free KOH is evanescently small, is an extremely striking fact. But, not only is the composition of the caseinate of potassium independent of its dilution in neutral solution; it is -~ ~_____ ~

Cf. Van Slyke and Hart: Am. Chem. Jour., 33, 461 (1905).

2T.Brailsford Robertson: Jour. Phys. Chem., 14, 528 (1910).


543

also independent of its dilution in solutions which are pronouncedly acid in reaction. I have shown that for percentages of casein lying between 0.5 and 3.0 the quantity of base which is bound by the casein at “saturation” of the casein with base is o.ooo114 C, where C is the percentage concentration. In other words the proportion of base to casein is the same at all these dilutions. Now in these solutions the acidity is about I O - ~ H + . A glance at the formula: R.COOH

+ KOH

R.COOK

+ H.OH.

suffices to show that in acid solutions, when the KOH concentration is excessively small and the H+ concentration large, the tendency to hydrolysis of a salt of the type R.COOK must be exceptionally great, yet potassium caseinate does not reveal this tendency, and the conclusion is forced upon us that potassium caseinate is not formed according t o an equation of the above type. Reverting, now, to the hypothesis which I have advanced regarding the mode of formation of protein salts we see that in the equation : H -N.HOC-

++ + KOH I_ -NNI + KOC-

I

OH

n o water is involved, and consequently, the composition of the salt must be dependent on,ly u p o n the relative concentrations of protein and base and not u p o n the total dilution. Similarly in the formation of salts with acids: H

-N.HOC-

+ HCI J_ - N fIf + ++ HOCI

c1 no water necessarily enters into the reaction. Hence both the dependence of the composition of the protein salts upon the excess of acid or base and its non-dependence upon dilution receive a simple interpretation in the light of this hypothesis.

T . Brailsf ord Robertson

544

It will, I think, be clear from the foregoing discussion that the non-dependence of the composition of protein salts upon their dilution is one of the most emphatic proofs which we possess of the fact that terminal -COOH and -NH, groups are not responsible for their formation. 7. Data Derived from Conductivity and Freezing-point

Determinations In previous communications I have shown' that the caseinates and serum-globulinates of the alka ies and alkaline earths obey the Ostwald dilution-law? when expressed in the form :

where x is the conductivity in reciprocal ohms, m the equiva.lent concentration of neutralized base and p the number of equivalents of protein salt t o which one equivalent of neutralized base gives rise and u t v the sum of the equivalent velocities of the ions of the protein salt. For the caseinates of the alkaline earths (Ca, Ba, Sr) the average value of p ( u + v) is 38 X cm per sec at 30'; for the serum globulinates its value is slightly lower. Now Bredig has shown that the equivalent velocity of heavy ions at 15' is about 15 X IO-5 cm per sec. At 30' this would be some 30 percm per sec. Provided that one cent higher or 2 0 X equivalent of base yields one equivalent of protein salt and that the salt dissociates into two protein ions the value of p(zc t v) should therefore be about 40 X IO-^ cm per sec, which is very close to the value actually observed. For the caseinates and serum globulinates of the monoacid bases, however, p ( u t v) is almost exactly twice as great, and hence IO-j

IO-j

T. Brailsford Robertson: Jour. Phys. Chem., 11, 542 (1907); 12, 473 (1908); 14, 5 2 8 , 601 (1910). This does not necessarily imply that these salts dissociate only into two ions. The general form of the law for electrolytes which dissociate into n ions is: m = Ax Bx" and this relation may be satisfactorily represented by several different values

+

Of 41..

Studies in the Electrochemist,vy of the Proteins

545

we must conclude, as I have done in the communications referred to, that one equivalent of monacid base yields two equivalents of protein salt. Now the freezing-point depression produced by a solution of a caseinate of a monacid base is almost exactly that of a solution of the same molecular concentration as the neutralized base.l Since the caseinate of the monacid bases are very completely dissociated a t the dilutions employed (for their conductivities increase but slightly on further dilution) we must conclude that one molecule of neutralized monacid base yields one i o n of caseinate. The same is readily seen to be true for the caseinates of the diacid bases, when allowance is made for their relatively slight degree of dissociation. For the monacid bases, therefore the mode of reaction with casein must be that represented by equation 4 in the introduction. Hence dicarboxylic acid radicals play the leading part in the neutralization of bases by proteins. In this connection it is of interest to observe that the ratio of the maximum ( = 180 x IO-^ equivalents per gram) to the minimum ( = 11.4 X IO-^ equivalents per gram) combining capacity of casein for basesZis almost exactly equal to the number of glutamic acid radicals in the casein molecule, calculated on the basis of a molecular of weight 17600. Applying the dilution-formula t o the determinations made by Hardy on solutions of serum-globulin combined with 9.318 X IO-' equivalents of HC1 per gram we obtain, by the method of least squares :

+

whence p ( u v) = 140 X IO-^ cm per sec, or four times its value for the globulinates of the diacid bases. I find that solutions of ovomucoid combined with acids (45 X IO-^ equivalents HCI or H,SO, per gram) yield even larger v), namely 204 x IO-^ for HC1- ovomucoid values of (u

+

~

' T.

Brailsford Robertson and Theo. C. Burnett: Jour. Biol. Chem., 6 , 105

(1909).

T.Brailsford Robertson: Jour. Phys. Chem., 14,528 (1910).

546


and 189 X IO-' for H2S04- ovomucoid. Evidently each equivalent of HCl which combines with serum globulin yields four equivalents of protein salt and the same is probably true for the salts which ovomucoid forms with acids. Hence the reaction of acids with these proteins is represented by equation 5 in the introduction and the salts themselves (for the proportions of acid to protein named) are represented by either the formula H

C1

/\

H OH

or the formula: H R/Y ': \COH

C1

\/

++

+

"N

>R N

"

/\

H OH

If formula (I) represents the structure of the chloride of ovomucoid which contains 45 X IO-^ equivalents of HC1 per gram, then, since this salt is highly dissociated (for the conductivity of its solution increases but slightly with dilution) the freezing-point depression of its solution should be three times that of a solution of the molecular concentration of the neutralized acid. If formula ( 2 ) represents the structure of the salt then the freezing point depression of its solution should be two times that of a solution of the molecular concentrations of the neutralized acid. The experimental fact is that the freezing-point depression of a 0.018 m solution of -HC1 containing 4 percent ovomucoid ( I g per 45 x IO-^ equivalents) is 0.09' 5 o.o05O, corresponding to a 0.0055 m solution, or almost exactly three times the molecular concentration of the neutralized acid. We m a y conchde that formula ( I ) i s that of this chloride of


547

ovomucoid, that in the formation of salts of ovomucoid and serum globulin with acids diamino radicals Play the leading part, and that, in ovomucoid at least, the diamino radicals are not directly united to both the -COOH groups of dicarboxylic acid radicals. Continuing the addition of acid (HC1) to the above solution of ovomucoid a remarkable phenomenon is observed, namely, that doubling the amount of acid in the solution does not appreciably alter its freezing point' and, consequently does not alter the total number of ions per cc of the solution. Evidently, upon further addition of acid the remaining Nof the diamino radical becomes neutralized and the ion:

H C1

/\

H

C1

is formed. The form of the dilution-law which we should apply to these solutions is therefore :

The following are the experimental data. Ovomucoid was prepared from the whites of eggs in the manner described by me in previous articles.' This ovomucoid was dissolved in solutions of HCI and of H,SO, in such proportion that one gram of ovomucoid was introduced into the solution for every 45 X IO-^ equivalents of acid. Such solutions are very nearly neutral (H+ = about 3 X IO-^), over 99 percent of the acid being bound by the o v o r n u ~ o i d . ~These solutions, (containing 4 percent of the protein) were then diluted t o the various concentrations employed and the conductivities of the The freezing-point depression due to free ovomucoid is only one-third of t h a t of the above solution, namely: 0.03' & 0 . 0 0 5 ~for a 4 percent solution. T. Brailsford Robertson: Jour. Biol. Chem., 14,709 (1910); Jour. Phys. Chem., 7, 359 (1910). a T. Brailsford Robertson: Jour. Phys. Chem., 14, 709 (1910).

T . Brailsford Robertsolz

548

solutions measured at each dilution. The measurements were carried out at 30' in a conductivity vessel of resistancecapacity 0.1949. The following were the results obtained, the conductivity of the distilled water ( = 4 X IO-^) having been subtracted from each of the observed conductivities. TABLEI Ovomucoid Chloride ___

__

~ ~

Equivalent-molecular concentration of HC1 neutralized by ovomucoid

0.0180 o.oog0

I

276 X IO-^ 1 5 1 x IO-^ 80 X IO-^ 42 x IO-^ 2 2 x IO-^

I

I

i

0.0023 0.0011

__

= conductivity i n reciprocal ohms per cc at soo

I

0.0045

_

TABLEI1 Ovomucoid Sulphate -

- ~-

_ _

Equivaleut.molecular concentration of H,S04 neutralized by ovomucoid -

~_

I

-I--

= conductivity in reciprocal ohms per cc. a t 3oD -

I

o 0180 0 0090 0 0045 o 0023

1 I

1

0 0011

~~

---

X IO-^ 131 X IO-^ 73 x IO-^ 39 x IO-^ 2 1 x IO-^

232

Applying the above equation to the results enumei-ated in Table I we obtain : m =

5.63%

+ 0.117 X

1oBx3.

In the accompanying table the experimental and calculated values of m are compared. In the third column is given the degree of dissociation of the salt, estimated as the ratio of the ~ the calculated value of m. calculated value of 5 . 6 3 to


549

TABLEI11 Ovomucoid chloride (45 X IO-^ Equivalents HCl per gram) m x io4 (experimental)

45 23

,

x

(calculated)

Degree of dissociation Percent

46 24

IO0

m

104

98

550

T . B railsf ord Robertson

ting of -N.HOCgroups within the protein molecule in accordance with equations such as : H

-N,HOC-

+ KOH = -N”I + ++ KOCI OH H

-N.HOC-

+ HCI

I

= -N”

I

++ + HOC-

c1 and not byithe neutralization of terminal -NH, or --COOH groups. (2) That in the neutralization of acids and bases by proteins diamino and dicarboxylic radicals are chiefly involved.

1. Introductory

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