ELECTROCHEMICAL PROPERTIES OF PROTEIN

PHYSICAL CHEMISTRY OF T H E PROTEINS IN NON-AQUEOUS AND MIXED SOLVENTS. I1

ELECTROCHEMICAL PROPERTIES OF PROTEIN SOLUTIONS IN CERTAIN GLACIALORGANIC ACIDS DAVID M. GREENBERG AND CLARENCE E. LARSON T h e Division of Biochemistry, University of California Medical School, Berkeley, California Received September 17, 1984 INTRODUCTION

In general the proteins are insoluble in the most commonly used organic solvents, but there are known a few anhydrous and a larger number of mixed solvents which will dissolve many of the proteins. These solvents consist largely of a number of alcohols and hydroxy compounds, some aliphatic carbon acids, and certain not very closely related nitrogen compounds. The separation of the prolamines by alcohol mixtures is the best example of the use of a mixed solvent in protein chemistry. In the first paper of this series a study was made of the state of aggregation of certain proteins in mixtures of urea and water and of glycerol and water (1). In this communication there will be presented the results of experiments on the electrochemical properties of protein solutions in the aliphatic carbon acids, formic, acetic, and lactic. GLACIAL ALIPHATIC CARBON ACIDS THAT ARE PROTEIN SOLVENTS

Formic, acetic, lactic, and pyruvic acids have been reported to be solvents for proteins. The earlier literature of the subject contains the information that gelatin dissolves in glacial acetic and lactic acids (see ref. 12), that Mathewson (13) prepared solutions of gliadin in glacial acetic acid, and that Robertson (16) found that casein would dissolve in anhydrous formic acid with ease. A more systematic survey of the subject has recently been published by Loiseleur (10). In the course of the present work, most of Loiseleur’s observations were confirmed, but in a few instances there was a disagreement with his statements. With Loiseleur, it was observed that anhydrous formic acid readily dissolves most proteins in the cold, including such diverse members as gelatin, casein, fibrin, edestin, gliadin, egg albumen, zein, etc. Apparently the only proteins not dissolved are certain of the keratins. 665

666

DAVID M. GREENBERG AND CLARENCE E. LARSON

Loiseleur states that acetic acid dissolves gelatin in the cold and edestin on heating, but does not dissolve the other mentioned proteins. The only dry protein we were able to dissolve in acetic acid was gelatin, and in the course of this there was noted the paradox that solutions could be prepared and were stable when the gelatin concentration was above a certain critical value, but that it could not be brought into solution in concentrations below this critical level (9). Attempts to dissolve edestin in acetic acid were unsuccessful even after prolonged heating on a water bath. According to Loiseleur, propionic acid dissolves none of the proteins, but the substituted propionic acids, lactic and pyruvic, readily dissolve many proteins. In our experience casein and edestin swell and slowly go into solution in lactic acid at room temperature. Heating markedly accelerates the rate of solution. Shreds of fibrin become hugely swollen in cold lactic acid, but only on being heated do they completely disperse. Loiseleur emphasizes the point that protein solutions in the organic acids do not have the usual properties characteristic of colloidal solutions in water. They are not turbid, do not foam, have a low viscosity, and are not coagulated by the protein precipitant acids, tannic, trichloroacetic, and picric, nor by salts of such heavy metals as iron, chromium, and lead. However, by merely diluting these organic acid protein solutions with water, the characteristic colloidal properties are at once restored, Because of these effects, Loiseleur takes the point of view that in the anhydrous solutions the proteins are actually in “true” solution. Just what is implied by this is somewhat ambiguous, since no further elaboration of the author’s conception of the distinction between true and colloidal solutions is given. We assume it is implied that these acid solvents cause a disaggregation of the proteins into units of low molecular weight, since it is generally accepted that colloidal properties are associated with a high molecular size. If this hypothesis is correct, and it were possible to demonstrate that the proteins disaggregate into small units in any group of solvents, it is readily seen that it would be a matter of great importance for chemical theories of the structure of the proteins. One class of substances, namely the soaps, is known to exist as aggregated colloidal micelles in aqueous solution and as simple molecules in alcoholic solution (11). If we interpret Loiseleur correctly, he views the change of properties from aqueous protein solutions to those in the glacial acids as a similar transformation. This conception is not completely novel. Similar suggestions have been proposed, for example, by Herzog and Kobe1 (7) and by Troensegaard and Schmidt (21). Experimental evidence for such a transformation was offered in what was supposed to be the low molecular weight, 200 to 600, obta#inedby freezing point measurements of proteins in phenol solution. However, Cohn and Conant (4))who reexamined the question, showed that the above measurements actually were in error owing

PROTEIN SOLUTIONS I N GLACIAL ORGANIC ACIDS

667

to the traces of moisture which were introduced with the protein samples. When this error is eliminated, no disaggregation of proteins in liquid phenol can be demonstrated. The claim that proteins dissolved in the aliphatic carbon acids lose all of their colloidal properties, moreover, is not completely correct. Concentrated solutions of casein in formic acid, dissolved by heating, set t o gels on cooling, and these solutions also show a distinct turbidity. One of the colloidal precipitants, phosphotungstic acid, is able to precipitate proteins even from formic acid. Moreover, the lack of the properties usually associated with colloidal solutions is not a sufficient criterion for invariably assuming a change in the state of aggregation. A similar lack of colloidal properties is found with protein solutions in concentrated urea, which also show no turbidity, do not foam, and are not coagulated by heat. Yet, as was shown by Burk and Greenberg (1) and by Burk (2), many of the proteins have the same molecular size in urea as they do in aqueous solution. The experiments reported here were undertaken because of their interest to the subject of the structure and the physicochemical state of the proteins, and also for their significance to theories of solubility. Since most of the solvent mixtures for proteins are weak acids or bases, it has been proposed that the solvent properties depend upon the formation of electrically charged protein ions by the interaction with the solvent acid or base. This is undoubtedly the case in many instances, but is by no means universally true. Burk and Greenberg have shown that urea does not act as a protein solvent because of basic properties, and in this communication it will be shown that the same is true of certain of the glacial acids. EXPERIMENTAL

The conductivities were measured by the Kohlrausch telephone method, using a General Radio Company oscillator as the alternating current source. The semi-micro cell of 2.5 ml. volume shown in figure 1 was employed in all the measurements. An interesting feature of the cell, aside from its size, is that the electrodes, indicated by “s” in the drawing, were formed by spiraling 22 gauge platinum wire. These electrodes were coated with platinum black electrolytically, and the cell constant was determined against standard solutions of potassium chloride in the usual manner. The solvent materials used for preparing the solutions were as follows: Kahlbaum’s 100 per cent formic acid, specific conductance about low4mhos; Baker or Merck’s 85 per cent glacial lactic acid1 with a conductance of around 5 X mhos; and Baker’s 99.5 per cent glacial acetic acid with a conductance of less than lo-’ mhos. The proteins used were purified preparations of low ash content. Casein a

Completely anhydrous lactic acid cannot be used for solvent purposes, as it is crystalline solid a t ordinary temperatures.

668


was prepared by the method of Van Slyke and Baker (ZZ),edestin by that of Osborne (14). The gelatin was an Eastman Kodak Company’s electrolytically deashed preparation, and deaminized gelatin was prepared from this as described by Hitchcock (8). All of the protein concentrations are recorded in the tables on the basis of grams of dry protein per liter of solvent. Wherever necessary for this purpose, a correction has been applied for the moisture content of the protein. Conductivity measurements

The conductivity data obtained in this investigation are recorded in tables 1 to 5 . The observations in glacial lactic and acetic acid, which

FIG. 1. SEMI-MICRO CONDUCTIVITY CELL

are given in table 1, may well be considered together, separately from t h e results in formic acid. The three proteins, casein, edestin, and gelatin, as noted in table 1, add practically nothing to the conductance of glacial lactic acid. In acetic acid, gelatin increases the conductivity to values more than a hundredfold that of the solvent, but even so, the magnitude of the conductance is comparatively small. The inference to be drawn from this is that the solvent power of these two acids for proteins is not dependent upon an ability on their part to react with the proteins to form protein ions. From the conductivity values of table 1, there can be no question regarding this view about lactic acid. However, to accept the same viewpoint for acetic acid, the increased conductance produced by gelatin, even though small, has to be explained.

669


That the increased conductivity in these solutions is probably not due to protein salt formation follows from the fact that the conductivity increases TABLE 1 Specific conductivity of certain proteins in glacial lactic and acetic acids CASEIN I N LACTIC ACID

EDESTIN I N LACTIC ACID

Concentration

Specific conductivity

8. per l i b of solvent

nhos X 10

0 2.80 5.65 8.45 11.25 14.05 16.90 19.70 22.50 25.30 28.15

5.14 5.06 4.95 4.93 4.90 4.88 4.87 4.86 4.96 4.97 5.04

x CONCENTRATION

SPECIFIC CONDUCTIVITY'

Concentration

S ecifio ionJuctivitj

per liter of solvent

mhos X 106

0 2.85 5.70 8.55 11.40 14.25 17.10 19.95 22.80

5.25 5.24 5.23 5.30 5.34 5.46 5.49 5.53 5.56

7.

1 -

QELATIN IN LACTIC ACID

ConcenS eoific tration cond'uotivity per liter of aolvent

g.

0 4.30 8.60 13.00 17.30 21.60 26.00 30.30 36.10

EQUIVALENT CONCENTRATION N 990 (MEAN OF DYE AND HEXONE BAED CONTENT)

1

.

TIT~ATION

per liter of solvent

g.

0 27.0 36.0 45.0 54.0 63.0 72.0

7.37 7.48 7.53 7.75 8.03 8.11 8.52 8.82 9.03

I

S ecifio oonluctivity

mhos x

0.01 (about) 1.22 1.97 2.75 3.64 4.71 5.79

x CONCENTRATION

0.0046 0.0094 0.0140 0.0187 0.0235 0.0254 0.0282 0.0328 0.0395 0.0470 0.0563

SPECIFIC CONDUCTIVITYt

EQUIVALENT CONCENTRATION N = 990 (MEAN OF DYB TITRATION AND HDXONE BASE CONTENT)

Casein I1 mhos

solvent

17.3 37.6 55.1 73.2 95.8 102.8 110.6 121.1 138.8 169.8 200.0

IO5

Concentration

TABLE 2 Conductivity. of . casein i n formic acid

Casein I

4.55 9.30 13.90 18.55 23.20 25.10 27.85 32.50 39.10 46.40 55.70

mhos X

QELATIN I N ACETIC ACID

37.7 40.0 39.3 39.1 41.7 41.5 39.2 36.7 35.2 36.2 35.5

* Specific conductivity of solvent

mhos

solvent

1.90 4.70 9.45 14.15 18.90 23.60 28.30 33.10 37.85 47.20 5G.65

7.2 17.3 39.9 56.6 74.6 91.6 109.1 126.7 140.4 170.5 198.4

0.0019 0.0047 0.0095 0.0143 0.0191 0.0239 0.0286 0.0334 0.0382 0.0477 0.0572

38.0 36.8 42.0 39.6 39.1 38.3 38.2 38.0 36.8 35.8 34.7

-

= 14.0 X lo-' mhos has been subtracted from

the total. f Specific conductivity of solvent = 10.15 X the total.

mhos has been subtracted from

670


much more than proportionally to the gelatin concentration. Doubling the gelatin content produces about a threefold increase in conductance. This increase may well be due to the effect of the moisture added with the gelatin. In qualitative tests it was found that small quantities of water markedly increased the conductance of glacial acetic acid. The gelatin employed had a 10 per cent moisture content. TABLE 3 Conductivity of edestin in formic acid EQUIVALENT !ONCENTRATION

N = 760

A

CONCENTRATION

A

(HEXONE BASE CONTENT)

liter of solvent

g. per

mhos X 105

4.75 9.50 14.25 19.00 23.80 28.50 33.30 38.10

29.7 55.9 85.4 115.3 144.7 165.1 195.7 216.8

mhos

0.0074 0.0148 0.0223 0.0297 0.0372 0.0445 0.0518 0.0595

mho8

40.1 37.8 38.3 38.9 38.9 37.1 37.8 36.5

0.0063 0.0127 0.0190 0.0254 0.0318 0.0380 0.0444 0.0508

47.2 44.0 45.0 45.4 45.5 43.5 44.0 42.7

* Specific conductivity of solvent = 10.15 X low6mhos has been subtracted from total. TABLE 4 Conductivity of gelatin in formic acid CONCENTRATION

g. per

liter of solvent

9 .o 18.0 27.0 36.0 45.0 54.0

X SPECIFIC CONDUCTIVITY’

EQUIVALENT CONCENTRATION N = gbo (MEAN OF D Y B TITRATION AND HEXONE BASE CONTENT)

* Specific conductivity of solvent = 10.8 X the total.

A

mhos

mhos X 106

37.5 77.0 116.1 151.9 185.4 217.1

i

0.0095 0.0190 0.0285 0.0379 0.0475 0.0568

39.5 40.5 40.8 40.0 39 .O 38.2


Even if it were granted that the conductivity increase is due to protein ionization, a rough calculation shows that the density of charge produced must be very small. Assuming the equivalent conductivity of gelatin acetate would be of the same order as of gelatin formate (table 4), it is calculable from the data of table 1that the gelatin would have one equiva-

671


lent of charge for each 75,000 to 100,000 grams. This figure can hardly be as much as one charge per protein molecule. From this evidence another explanation must be sought for the ability of lactic and acetic acids to dissolve proteins than through an interaction to form protein ions, The true explanation of the solvent power remains to be determined.

Conductivity in formic acid On the other hand, it can hardly be doubted that formic acid reacts with proteins to give ionized protein aalts. In tables 2 to 5 are recorded the Fonductivity values of solutions of casein, edestin, gelatin, and deaminized gelatin in 100 per cent glacial formic acid. These proteins all markedly increase the specific conductivity of the formic acid, the increase being nearly proportional to the amount of protein in solution. The casein TABLE 5 Conductivity of deaminized gelatin in formic acid EQUIVALENT :ONCENTRATION N = 1705 (DYE TITRATION VALUE)

liter of solvent

mhos X 106

10.0 20.0 30.0 40.0 50.0 60.0

25.7 53.4 77.1 90.8 126.0 147.4

g. per

.

* Specific conductivity of

EQUIVALENT CONCENTRATION

A

N = 1515

mhos

mhos

0.0059 0.0117 0.0176 0.0235 0.0294 0.0352

solvent = 10.8 X

A

(HESONE BABE CONTENT)

43.6 45.6 43.8 38.6 42.9 41.9

0.0066 0.0132 0.0198 0.0264 0,0331 0.0397

39.0 40.4 38.9 34.4 38.1 37.2


the total.

measurements were carried out on two separately prepared samples and in different lots of the solvent. The good agreement between the two series is evidence that the conductivity is an intrinsic property of the protein and not of any contamination therein. The magnitude of the conductivity values attained is quite considerable, being nearly of the same order as is found with solutions of the alkali and alkaline earth formates in formic acid. While the ionization reaction is probably no more essential to account for the solvent properties of formic acid than for the other organic acids, still it must be beneficial in this respect and it indicates why formic acid is a so much more powerful solvent than either lactic or acetic acid. To obtain a more quantitative picture of the electrochemical nature of the protein solutions, it is desirable to evaluate the values for the equivalent conductivity. But to obtain these physical quantities for the proteins in

672


formic acid is a problem offering formidable difficulties because of the uncertainty of the equivalent weights of the proteins.2 We were aware of no means by which it would be possible to determine the equivalent weights of proteins in these solutions directly. The extent of the interaction between the proteins and formic acid is unknown, and in aqueous solutions of acid or alkali the equivalent weight of a protein is not a constant but instead varies with the pH, so an estimate is not readily obtained in this way. The only possible method of approach appears to be through the application of plausible assumptions regarding the equivalent weight, derived from other sources. With such an approach, to obtain what seemed a reasonable estimate of the equivalent weights, recourse was had to the following: From the many studies on the acid combination of proteins in aqueous solution, it has become probable that the maximum combining capacity in this medium is determined by the content of the amino acids, arginine, histidine, and lysine, in a protein. Chapman, Greenberg, and Schmidt (3) have shown that the titration of proteins with acid dyes is in good agreement with this view. Because of the huge excess of formic acid it may be reasoned that the basic groups of the proteins are completely neutralized in formic acid solution. Accordingly, it seems not improbable that the equivalent weight of a protein in formic acid solution may be measured by the value given by the content of arginine, histidine, and lysine or, what usually closely agrees with it, the dye titration figure. On the basis of this assumption, the equivalent conductivities of casein, edestin, gelatin, and deaminized gelatin were calculated from the content of the hexone bases as determined by Van Slyke (see ref. 3) and,.where there waa a significant difference, also from the dye titration data of Chapman, Greenberg, and Schmidt. The results of these calculations are incorporated in tables 2 to 5 for each oi the proteins respectively under the columns designated by the symbol A. The equivalent weight chosen for casein in table 2 is 990, which is the average of the value from the dye titration of 1000 and the hexone base content of 980. The equivalent weight of 950, chosen for gelatin (table 4), also is the mean of the figures from dye titrations and the hexone base content. For the other two proteins separate calculations are given, using values of equivalent weight derived from both of the above sources. The equivalent conductivity of all four measured proteins has nearly the same calculated values, This correspondence upholds the choice of the equivalent weights, since because of their closely related properties, it may well be expected that the mobility of the proteins in formic acid would be

* This is an illustration of the great importance which the equivalent weights have for the solution of electrochemical problems and of how baffling the formulation of the electrochemical state is without these constants.

PROTEIN SOLUTIONS IN GLACIAL ORGANIC ACIDS

673

virtually the same. An erroneous basis for the selection of the equivalent weights would be expected to lead to widely varying calculated values of A. The close correspondence in equivalent conductivity, it is true, is mainly determined by the high value of the mobility of formate ion in comparison with the mobilities of other ions in formic acid. The work of Schlesinger and coworkers (19, 20) shows that a similar condition prevails among the alkali and alkaline earth formates. The magnitude of the equivalent conductances obtained with the proteins seems plausible when compared with the values for the alkali and alkaline earth formates. Most interesting are the results with gelatin and deaminized gelatin. The calculated values of the equivalent conductivities are about the same for each, and both agree well with the figures obtained for casein and edestin even though it is to be remembered that the equivalent weight of the deaminized gelatin has been deliberately altered by the removal of the e-amino group of lysine through interaction with nitrous acid. That the calculations based on this known change should give such harmonizing results is strongly in favor of the reasoning given here. The results obtained lead to the picture that the proteins in formic acid form ionizable salts rather analogous in electrochemical behavior to the alkali and alkaline earth formates in formic acid. The proteins behave in a manner that might be expected of formic acid salts of rather high valency.

Electrical transference Since the conductivity curves of the proteins are not satisfactory for the purpose of evaluating the limiting equivalent conductivities at infinite dilutions, a number of electrical transference experiments were carried out to get at this data. The Hittorf method was employed in the first instance. The cell, with some slight modifications, was the type introduced by Landsteiner and Pauli (15). Five electrode portions were obtainable for analysis, but experience soon showed that only in the outermost anode and cathode portions was there produced any concentration change. The electrodes were of platinum wire and no correction was applied for the hydrogen evolved at the cathode and carbon dioxide at the anode, which have been shown by Schlesinger and Bunting (17) to be the main products of the electrode reactions. The current passing through the cell during the course of an experiment was measured with an iodine coulometer, A motor generator developing a D. c. current at a voltage of 250 supplied the current for electrolysis. Naturally, the change in formate ion was not measurable, so only the change in protein content could be followed. This was determined by Kjeldahl analysis of the nitrogen content. The results obtained on each of the proteins are given in table 7. As the table shows, very closely agreeing figures were obtained from both cathode and anode portions in

674


repeated experiments. From the transference numbers the ho values of the proteins shown in table 6 were calculated, using the value of 51.6 for the equivalent conductivity of formate ion as given by Schlesinger and Bunting (19). Compared to the figures in aqueous solution, the proteinTABLE 6 Electrical transference of proteins i n formic acid Temperature, 25°C. a. Casein CASEIN CONCEN-

EX* 'ICR1-

COULOMETER CURRENT

9,"."

TRATION

1 1

liter

30.0

30.0

2

1

0 . per

NITROGEN TITRATION CIiANQE OF COMPARTMENT

E :",

COMPART-

PARTMEN'I

milliequivalents

milliequivalents

:::::

Anode Cathode Anode Cathode

{ {

0.443 0.430 0.995 1.025

17.5 17.0 19.6 17.5

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

Average

CASEIN CZANGE I N COMPARTMENT

miEliequivalents

-42.4 +41.6 -95.5 $97.5

-0.043 $0.042 -0,097 $0.099

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

Ao+cssein........................................

0.095 0.092 0.090 0.093 0.093 5.5

b. Edestin

-EXPERIMENl NO.

EDEBTIN CONCENTRATION

COULOMETER CURRENT

0 . p

milfiequzpvalent8

COMPARTMENT

-lzter

3

27.0

4

28.5(

VOL- NITROGEN EDEBUME OF TITRATION COMCAANQE PARTOF COM- IN 'OMM E N T PARTMENT

EDEBTIN CEANQE

milliequivalents

milliequavalents

E&-

ml.

1.360 Anode 20.1 18.8 0.775 Anode 0.775 Cathodc 18.6

c. Gelatin

":::

I

EDESTIN CHANQE

N

= 150

T'edestin

- - - ~ - - 1.68 1.00 0.99

"8.

QELATIN CONCENTRATION

COULOMETER CURRENT

g. liter per

milliequivalents

COMPART-

milliequivalenls

-121 -0.189 -76 -0.1175 f75f0.117

-Average ........................................ hO+edestin. ......................................

2;;-

T'edeatin

ii = 640

0.14 0.15 0.15 _.

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

0.15

1 1 I::,":~ 1 VOLUME OF COMPARTMENT

TITRATION

-0.161 0.12 -0.1015 0.135 $0.1000 0.13 ___ 0.13 7.7

-

GELATIN CAANGE I N COMPARTMENT

COMPARTMENT

N

= 960

T+selstin

-~ 1.27 1.27

Average.. Aofgeisiin..

Anode Cathode

m'.

milliequivalents

mg.

milliequivalents

17.8 16.4

0.554 0.562

-153 $144

-0.160 +0.150

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

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

0.125 0.12 0.12 7.2

675


TABLE 6-Concluded

d. Deaminized gelatin

EXPERIMENT NO.

GELA- COULO:M-i : :M ;E

I

- -I

RENT

1I

~~

DEAMI-

COMPARTMENT

DEAMIVOL- NITROGEN NIZED NIZED UME OF TITRATION T+deamiGELATIN COMCHANGE nised PARTOFCOM- CHANQE CHANGE gelatin N = 1705 MENT PARTMENT -:: ,"I MENT

TION

-I

DEAMINIZED QELATIN CHANGE

N = 1616

-

1-

T'deami. nized gelatin

~

-

mizzi-

16.8 18.8

6 { -

equivalents

I l l 0.444 0.419

-121 -0.071 0.056 $127 +0.074E 0.059

-

............................................... A0+deaminized golatin ....................................... Average..

0.08 0.063 0.084 0.066

~ 0.064 3.5

0.058

TABLE 7 Electromotive force cells and transference of sodium formate in formic acid Temperature = 25°C.; reference concentration = 0.147 molal sodium formate E

CONCENTRATION OF SODIUM FORMATE

(MEASURED)

per cent

moles per liter of solvent

volts

0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

0.0147 0.0220 0.0294 0.0367 0.0441 0.0514 0.0688 0.0662 0.1470

0.0286 0.0236 0.0197 0.0164 0.0142 0.0123 0.0110 0.0090 0 .om0

1.oo

,

,

Average. .................................................

0.241 0.241 0.238 0.232 0,230 0.236 0.236 0.220

./

0'.236

ion mobility in formic acid is astonishingly low, but it is quite concordant with the figures for the mobility of the cations of the alkali and alkaline earth elements in formic acid obtained by Schlesinger and associates. It was also attempted to determine the transference numbers of formic acid solutions through the use of electromotive force cells with transference. Since Hall and Conant (6) have shown that certain oxidation-reduction couples can be used to measure the hydrogen-ion activity in such nonaqueous solvents as acetic acid, it seemed not unlikely that the quinhydrone electrode could be used for this purpose in formic acid. Tests showed that this electrode worked well in the presence of the alkali formates, but unfortunately, not, with solutions of proteins in formic acid. This may be due to an interaction between quinone and the proteins,

-

676


similar to the reaction which occurs in the quinone leather tanning process (see ref. 23). Although no valid results were obtained with the proteins, the data obtained with sodium formate will be presented, since the method offers an independent means of obtaining such information that has hitherto, as far as we are aware, not been employed for non-aqueous solutions. The cell set-up for the measurements is given by the equation,

Pt I Quinhydrone, [N~OOCHIII [NaOOCH12,Quinhydrone I Pt E8

E1

(1)

E2

The electromotive force of this cell (5), as can be readily shown, is given by equation 2 below, provided the mass law relationship, [H+][HCOO-] = IC holds in formic acid.

E = EZ - El

+ E3 = 2T, RT - In [NaOOCHIs F [NaOOCH]1

where T,is the cation transference number. At 25"C., using numerical values of the constants, the equation becomes

E = 0.118 T 1

Og

[NaOOCHla [Na00CHI1

(3)

I n carrying out the measurements, the liquid junction in the cell was established by plugging a stopcock which connected the two a r m of the cell with cotton wool so that only a slow diffusion of liquid obtained, With this arrangement it was found that after a few minutes to allow for the attainment of diffusion equilibrium, a very steady and reproducible electromotive force was established. Since quinhydrone is quite soluble in formic acid, it was introduced just before the start of an experiment in amounts required to make the concentration in each arm of the cell the same. The results obtained with the sodium formate solutions are recorded in table 7. With the concentrations of sodium formate employed of between 0.0147 and 0.066 molal, the transference numbers found for sodium ion are reasonably constant. The average value of 0.236 for the sodium ion transference number, while not identical with the value of 0.22 obtained by Schlesinger and Bunting by the Hittorf method, is perhaps in as good agreement as might reasonably be expected, since no great degree of refinement marks the results by either procedure. SUMMARY

1. Measurements of the conductivity of solutions of proteins in the glacial carbon acids, lactic, acetic, and formic have been carried out. I n lactic and acetic acids this is increased but little. On the other hand, a marked

PROTEIN SOLUTIONS IN GLACIAL ORGANIC ACIDS

677

conductivity increase is produced in anhydrous formic acid, so that the solutions become nearly as good conductors as are solutions of the alkali and alkaline earth formates in formic acid. 2. On the basis of certain plausible appearing assumptions the equivalent conductivity of the proteins casein, edestin, gelatin, and deaminized gelatin in formic acid has been calculated. 3. Transference numbers of the protein formates have been determined by the Hittorf method. The results of the conductivity and transference measurements lead to the picture that the proteins in formic acid form ionizable salts having electrochemical properties which would be expected of formic acid salts of rather high valency. 4. Measurements of the transference numbers of sodium formate in formic acid have been carried out by means of an electromotive force cell with transference. 5 . The solvent action of the three glacial acids used in this work for proteins does not appear to be particularly dependent upon their ability to form electrically charged protein ions. REFERENCES (1) BURK,N. F., AND GREENBERQ, D. M.: J. Biol. Chem. 87, 197 (1930). (2) BURIC,N. F.: J. Biol. Chem. 98,353 (1932). (3) CHAPMAN, L. M., GREENBERQ, D. M., AND SCHMIDT, C. L. A.: J. Biol. Chem. 72, 707 (1927). (4) COHN,E. J., AND CONANT, J. B.: Z. physiol. Chem. 169,93 (1926). (5) CREIGHTON, H. J.: Electrochemistry, 2nd edition. John Wiley and Sons, New York (1928) (6) HALL,N. F., AND CONANT, J. B.: J. Am. Chem. SOC.49,3047, 3062 (1927). R. O., AND KOBEL,M.: Z. physiol. Chem. 134, 296 (1924). (7) HERZOG, (8) HITCHCOCK, D. I.: J. Gen. Physiol. 4, 733 (1921-22). C. E., AND GREENBERQ, D. M.: J. Am. Chem. SOC.66, 2798 (1933). (9) LARSON, (10) LOISELEUR, J.: Bull. SOC. chim. biol. 14, 1088 (1932). (11) MCBAIN,J. W.: Third Report on Colloid Chemistry, Brit. Assn. Adv. Sci., 1920, p. 2. (12) MARDLES, E . W. J.: Biochem. J. 18, 215 (1924). W. F.: J. Am. Chem. SOC.a8, 1482 (1906). (13) MATHEWSON, (14) OSBORNE, T. B.: Am. Chem. J. 16, 671 (1892). (15) PAULI,W., AND VALKO,E.: Elektrochemie der Kolloide, p. 152. Julius Springer, Vienna (1929). (16) ROBERTSON, T. B.:.J. Phys. Chem. 16,387 (1911). (17)SCHLESINGER, H. I., AND BUNTINQ, E. N.: J. Am. Chem. SOC.41, 1934 (1919). (18) SCHLESINGER, H. I., AND COLEMAN, C.: J. Am. Chem. SOC.38,271 (1916). H. I , AND MARTIN,A. W.: J. Am. Chem. SOC.36, 1589 (1914). (19) SCHLESINQER, (20) SCHLESINGER, H. I., AND MULLINIX,R. D.: J. Am. Chem. SOC.41, 72 (1919). N., AND SCHMIDT, J.: Z. physiol. Chem. 133, 116 (1924). (21) TROENSEGAARD, (22) VAN SLYKE, L. L., AND BAKER,J. C.: J. Biol. Chem. 36, 127 (1918). (23) WILSON,J. A . : The Chemistry of Leather Manufacture, Am. Chem. SOC.Monograph, 2nd edition, Volume 11, p. 748. The Chemical Catalog Co., New York (1929).

ELECTROCHEMICAL PROPERTIES OF PROTEIN

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