STUDIES I N THE ELECTROCHEMISTRY OF THE PROTEINS. 111. THE DISSOCIATION O F THE SALTS OF OVO-MUCOID I N SOLUTIONS O F VARYING ALKALINITY AND ACIDITY '
BY
T. BRAILSFORD ROBERTSON
(From the Rudolph Spreckels Physiological Laboratory of the University of California)
I. EXPERIMENTAL (a) The Preparation of the Ovo-mucoid The whites of eggs were beaten up to a froth and allowed t o stand in shallow vessels overnight. The subnatant fluid was then poured off, the froth being rejected. This fluid was diluted to five times its volume with distilled water, and t o every liter of the diluted fluid was added 130 cc of approximately N / I O acetic acid (made up by diluting I O cc of glacial acetic acid t o 1750 cc). This mixture was heated slowly to boiling-point, being rapidly and uniformly stirred meanwhile, and, after being allowed to boil for about 3 to 5 minutes, was put aside in rather shallow vessels for about 1 2 hours. A t the end of this time most of the coagulum had floated to the top, and the subnatant fluid was filtered through hardened filter-paper. Filtration was very rapid, and the filtered fluid, when boiled, either with or without the further addition of acetic acid, remained perfectly clear. The fluid which was thus obtained was now slowly evaporated t o 1/5 of its volume; the temperature of the fluid never being allowed t o rise above 55' C. After allowing this fluid to cool, the protein was precipitated from it by the addition of ten volumes of 99.8 percent alcohol (Kahlbaum's) and was allowed t o settle in tall glass cylinders. The supernatant fluid was then syphoned off and the precipitate was washed in the same volume of alcohol as that employed in the precipitation. This washing was repeated, again employing
710
I
T . Brailsford Robertson
the same volume of alcohol, and the precipitate was allowed t o steep in this alcohol for about 24 hours, in order, if possible, t o remove all adherent or combined acetic acid. The alcohol was then siphoned off and the precipitate was washed in the same volume of ether (Kahlbaum’s ueber Natrium destilliert). This washing was repeated. The ether was then siphoned off and the thick suspension of protein in ether which was thus obtained was rapidly poured into a hardened filter, the filter and the contained suspension of protein in ether being at once transferred to an incubator and the filtration continued over H,SO, a t 40’ (to avoid condensation of atmospheric moisture on the filter. It goes without saying that throughout the processes of washing, settling, etc., possible introduction of atmospheric moisture was avoided by keeping the glass cylinders closed with ground glassstoppers). After the completion of filtration, the ether which had filtered off was removed from the incubator and the precipitate allowed to dry for 24 hours. The protein is then obtained in the form of chalky cakes which are very readily broken up into fine, impalpable powder. This powder was passed through a fine sieve and kept in a glass-stoppered bottle. I t was found not advisable to work with fewer than 6 dozen eggs at one time as, otherwise, the amount of precipitate which is finally obtained is so small that the danger of excessive caking and partial decomposition, in drying, due to the deposition of moisture upon the filter, is very great. Not quite 40 grams of the powder were obtained from 24 dozen eggs. The protein which is thus obtained has been identified by M6rner1 as a mucoid and is termed by him ovo-mucoid. About a gram of the ovo-mucoid thus obtained was dissolved in about IOO cc of N/2 HC1 andithis solution was boiled until 30 cc of fluid had distilled over. This distillate was C. Th. Morner: Zeit. phys. Chem., 18, 5 2 5 (1894). Cf. Hammarsten: “ A Text-book of Physiological Chemistry,” Translated by Mandel, New York, 19049 P. 431,
Studies in the Electrochemistry of the Proteins
71 I
then tested for acetic acid. It contained a trace of an acid of the fatty series, sufficient to yield a slight coloration with ferric chloride, but insufficient to yield a precipitate of ferric hydrate on boiling or t o yield the ethyl acetate test.
(b) The Experimental Procedure The experimental procedure was, with the simplifications which were rendered possible by the fact that ovo-mucoid dissolves readily and rapidly in solutions of all reactions, identical with that employed in a previous investigation, upon the dissociation of potassium caseinate. To each of the solutions introduced into the gas-chain 0.01 KC1 was added in order to attain a favorable conductivity in the chain. This KC1-solution was itself very faintly acid; its acidity was estimated by determining the potential between it and 0.01N KOH (dissolved in 0.01 KC1) and was found to be 6.95 X IO-’”+. The solutions which were employed in the conductivity determinations did not, however, contain added KC1, but were simply prepared by dissolving the ovomucoid in solutions of known HC1 or KOH concentration. the depression in the conductivity of the acid Hence A ( 5 or alkaline solution resulting from the addition of the protein) was estimated by subtracting the observed conductivity of the ovo-mucoid solutions from that of the HC1 or KOH solution in which it was dissolved-the latter being estimated by interpolation from the tables of Kohlrausch and Holborn.2 It may be mentioned, in passing, that none of the difficulties which are encountered in casein solutions, arising from precipitation at the electrodes are met with in solutions of ovo-mucoid. Ovo-mucoid would also appear to be much less readily hydrolyzable than casein. In the accompanying tables the symbols employed have the following significance : Part I of these “Studies:” Jour. Phys. Chem., 14,528 (1910). Kohlrausch and Holborn: “Leitvermogen der Elektrolyte,” Leipzig, Pp. 160 and 199 (1898). For the mode of employing these tables, cf. Part of I these “Studies.” e
T . Bruilsford Robertson
712
e The
concentration of the KOH solution in which the ovo-mucoid was dissolved. a, E The concentration of the HC1 solution in which the ovo-mucoid was dissolved. zr F The potential of the chain in volts. I n solutions of b,
ovo-mucoid in alkali in acid b
=
=
0.0601
log,,^b ;
in solutions
a1 0.0601 log,, -.
a
e The
hydroxyl concentration of the solution (in alkali) of ovo-mucoid. a E The hydrogen ion concentration of the solution (in acid) of the ovo-mucoid. ni The concentration of alkali (or of acid) neutralized by the ovo-mucoid. xT The conductivity, in reciprocal ohms per cc of the solution containing no ovo-mucoid. (At 30’ C!. x The conductivity in reciprocal ohms, of the solution containing ovo-mucoid. (At 30’ C). 2 xI - - x ~ The alteration of the conductivity of the alkaline or acid solution which is brought about by the addition of ovo-mucoid. It should be observed that since free, uncombined ovo mucoid is soluble in water it is not possible, without additional data, to evaluate the “apparent” molecular conductivity of the ovo-mucoid salt as it is in solutions of the caseinates. (Cf. Part I of these “Studies.”) TABLEI I
percent ovo-mucoid dissolved in KOH solution 77
oooj 0,0755 0.0010 0.0475 0.0020 0.0500 0.0030 0.0367 o.ooj0 0.0356 0
O.OIO0
0.0185
6
1
“
2.77 x IO-’ 10.00047 1.62 X IO-^ 0.00084 2.95 x 1 0 - 4 10.00171 7.34 x IO-^ 10.00227 1.28 x IO-3 10.00372 4.92 x IO-^ I 0.00508
14.5
I
41.2 44.9 57.6 I 56.5 86.0 I 70.3 142.0 103.8 28.9
__
1
I
,
-
-26.8 -16.0
+
1.1
t-15.7 +38.2 -
Studies in the Electrochemistry of the Proteins
I
713
TABLEI1 percent ovo-mucoid dissolved in HCl solution
6.95 X IO-^ 0.10871 1.08 X IO-^ 0.2261 8.65 X IO-* 0.0005 0.0010 0.21071 3.12 X I C 7
1
,
6.84 X
IO-’
0.00050 0.00100
I
-
22.6 I 45.1 ~
60.6 68.4
+ 74.4 +III.I
It will be observed that in many of the least alkaline and acid solutions ,I is negative, in other words, that the addition of ovo-mucoid to the solution actually raises the conductivity instead of depressing it. This is undoubtedly because, in these solutions, the conductivity of the free ovomucoid itself more than makes up for the depression in the conductivity due t o the neutralization of the small amount of acid or alkali which is present. 11. THEORETICAL
The combining capacity for acid of ovo-mucoid a t absolute neutrality (neutrality t o litmus) can be estimated (cf. part I of these “Studies”) by determining the abscissa of the point of intersection of the experimental curve: 7T =
with the curve: 7T =
0.4107
/(a,)
+ 0.0601 log,, a ,
The point of intersection of these curves was determined by the graphic method described in a previous paper and found to be a t : a, = 0.0007 hence I gram of ovo-mucoid neutralizes 7 . 0 x IO-^ equivalentgram-molecules of acid in forming an absolutely neutral solution. It will be observed that ovo-mucoid is more basic than it is acid. The contrary is the case with the proteins,
714
T . Brailsford Robertson
the electrochemistry of which has hitherto been studied. Thus I gram of casein, at absolute neutrality, binds 50 X IO-^ equivalent-gram-molecules of base’ and I gram of “insoluble” serum globulin neutralizes, at neutrality to litmus, I O x IO-^ equivalent-gram-molecules of base. The relation between m, the amount of acid or of alkali neutralized by the ovo-mucoid, and the acidity or alkalinity of the solution in which it is dissolved is shown graphically in the accompanying figure. It will be seen as the proportion
Fig. I b, is the cnncentration of KOH in a, is the concentration of HCl in which I percent ovomucoid is dis- which i percent ovomucoid is dissolved; m is the number of gram- solved; in is t h e nurnber of gramequivalents of KOH which is neutral- equivalents of HCl which is neutralized per litre. ized per litre.
of acid or of base to ovo-mucoid declines, the combiningcapacity of the ovo-mucoid tends t o become directly proportional t o the concentration of the base, but that as the proportion of acid or of base to ovo-mucoid (and the excess of unneutralized acid or base) becomes large, the combining capacity of the ovomucoid tends to attain a maximum. The behavior of ovo-mucoid is therefore strictly analogous to that of casein (cf. Part I of these “Studies”). The m a x i m u m combining capacity of ovo-mucoid for Soldner: Landw. Versuchs., 35, 351 (1888). Lacqueur and Sackur: Beitr. chem. Physiol. und Pathol., 3 , 1 9 6 (1903). Van Slyke and Hart: Am. Chem. Jour,, 33, 461 (1905). T. Brailsford Robertson: Part I of these “Studies,” Joiir, Phys. Chem., 14, 528 (1910). W. E. Hardy: Jour. Physiol., 33, 251 (1905-6).
Studies in the Electrochemistry of the Proteins
715
acid was evidently not attained in any of the solutions investigated-but it is obviously in excess of 90 X IO-^ gram-molecules per gram, probably in excess of IOO X IO-‘ equivalent-gram-molecules per gram. The maximum combining-capacity of ovo-mucoid for alkali would appear t o be in the neighborhood of 50 X IO-^ equivalent-gram-molecules per gram. For purposes of comparison it may be recalled that the maximum combining capacity of casein for alkali is about 180 X IO-j equivalent-gram-molecules per gram. It is an interesting fact, which a glance at Table I1 reveals, that when I percent ovo-mucoid is dissolved in 6.95 X IO-’ N acid, although the protein is still acting as a base, yet this solution is actually more acid than the solution which is obtained by dissolving I percent ovo-mucoid in 0.0005 N HC1. A similar phenomenon was observed by W. B. Hardy in solutions of serum g1obulin.l The interpretation of this phenomenon is probably as follows. The acid-capacity of the protein increases with the acidity of its solution and that very rapidly in the neighborhood of the neutral point where a proportion of the amphoteric molecules are probably behaving as acid molecules, although the majority are behaving as basic molecules. With decreasing acidity of the solution in which the protein is dissolved, therefore, the acid-combining capacity of the protein may actually diminish so rapidly as t o leave uncombined a greater quantity of acid in a less than in a more acid solvent. I n a previous communication (Part I of these “Studies”) I have shown that the relation between the depression of the conductivity of an alkaline solution which is brought about by the introduction of casein and the alkalinity of the solution to which the casein is added may be expressed b y the equation A
x
105 =
P ?
abl - C bl - YC,
where 1 is the depression of the conductivity of the solution, b, its alkalinity, C the percentage concentration of the ~ _ _ _ _ _ _ _ .
’ Loc. cit.
.
T . Brailsford Robertson
716
casein and a, /3 and y are constants. For I percent solutions this becomes A X IO’ = ah, - j’b, ’- y. I find that the same law holds both for solutions of ovomucoid in acid and for solutions in alkali, save that in the acid solutions we must replace b, by a,, the acidity of the solution to which the ovo-mucoid is added. Applying this equation t o the results enumerated in Table I we obtain R X 1 0 5 = 20850 b,- 1250000 b:-35.6; in the accompanying table the values of A X 1 0 5 calculated from this formula are compared with those which were obtained experimentally. TABLE I11 ~ _ ~ _ ~
_
_
_
~
~
_
A X
_
_
105
Experirnen tal
~
______~.____. ~~~
~~
I
1
I
+ I 1
I
+I5.7 $38.2
I
105
Calculated
I
-26.8 -16 o
x
-25.5 -16.0
+
1.1
+I5 7 $38.4
Applying the corresponding equation t o the results enumerated in Table I1 and evaluating the constants from all of the observations by the method of least squares we obtain A x 1 0 5 = 41990 a, - 852700 a: - 41.4; in the following table the calculated and observed values of A X 105 are compared. TABLEIV ____________-
A’
x
105
Experimental
~~
~
i
A’ x 105 Calculated
2.0
- 20.6 - 0.3
$146.2 $228.9 +291.8
39.2 76.9 +113.0 $147.2 $225.5 $293.2
- 17.7
+
+ 37.7 + 74.5 +III.I
+ +
Studies in the Electrochemistry of the Proteins
.717
The agreement between the observed and calculated values is all that could be desired and it is evident that the equation I X 1 0 5 = ab, - Pb: - y expresses a general relation (for a given concentration of the protein) which subsists between the alkalinity or acidity of a solution to which protein is added and the depression (or increase) of its conductivity which results. Unfortunately lack of material has prevented me, for the present, from ascertaining the influence of the concentration of the ovo-mucoid upon the values of the constants which define this relation for ovomucoid. Regarding the theoretical significance of this relation the following may be tentatively suggested. In my previous communication referred t o above I have pointed out that I = (U V)m - (v, vz v, . . . .)c, where U V is the molecular conductivity of the neutralized acid or base vl, vz, v3, etc., are the velocities in cm per sec per cm potential gradient and c is the equivalent-molecular concentration of the ions derived from the protein and its salts. Comparing the equations
+
+
I
=
(U
+
+ +
+ V)m - (v, + v2 + v, . . . . . ) c
and
and recollecting that in the less alkaline solutions mi very nearly equal t o b,, it would appear probable, from considerations of symmetry, that in solutions of low alkalinity: a ~~~
I o5
=
u + v.........................
and (v,
+ zJ2 + v 3 . . . ) c *
= - b:
105C
re + -
