STUDIES I N THE ELECTROCHEMISTRY OF THE PROTEINS. IV. THE DISSOCIATION I N SOLUTIONS O F THE GLOBULINATES O F THE ALKALINE EARTHS
tration of NaOH neutralized to phenolphthalein by serum globulin X 105
x = Conductivity of the solution in ’
3’25 I563
I044 627 353 189 54
78 2
391 98
reciprocal ohms per cc X IO^
* T. Brailsford Robertson: Part I1 of these studies, Journal of Physical Chemistry. W. B. Hardy: Jour. Physiol., 33, 251 (1905).
Studies in the Electrochemistry of the Proteins
167
The proportion of alkali t o globulin in each solution was 18 X IO-^ equivalents per gram. Such solutions are neutral t o phenolphthalein, accordingly m, the equivalent molecular concentrations of alkali neutralized by the globulin is, in these solutions, equal t o the total concentration of sodium. I n previous communications1 I have shown that the Ostwald dilution-law for a binary electrolyte may be written m=
1.037 v1
x
IO-’
+ v,
x
x + 1.075 G(v,
IOd4
+
v2)z x 2 . .
.......
where m is the equivalent-molecular concentration of the electrolyte, x its conductivity in reciprocal ohms per cc, G its dissociation-constant and v, and v, are the ionic velocities in cm per sec. per potential gradient of I volt per cm. , In applying equation I to solutions of salts of proteins we have no guarantee that m, the equivalent-molecular concentration of salt, is the same as m, the equivalent-molecular concentration of the alkali neutralized in its formation. But if not one, but p equivalent gram-molecules of globulinate are produced by the neutralization of one equivalent grammolecule of NaOH then we may write equation I in the form
in which m is now identical with the equivalent-molecular concentration of the alkali neutralized in the formation of the globulinate. a Applying equation (2) to Hardy’s results, enumerated in Table I and computing the constants
1.037 x IO-’
+
and P(% 4 1.075 x IO-‘ from all of the observations by the method of pG(v1
+ 4’
least squares we obtain m = 17.65 x
+ 0.0115 X
IO^,^^.
Inserting, in this equation, the observed values of x, we IT. Brailsford Robertson: Jour. Phys. Chem.,
11,
542 (1907);
(1908).
Cf. number I1 of these studies, Ibid., 14,601 (1910).
12,
473
T . Brails f ord Robertson
I 68
can compute the “theoretic‘al” values of m, that is, the equivalent concentration of NaOH neutralized by the globulin which should, provided the sodium globulinate dissociates into two ions, coirespond to the observed conductivities. In the following table the observed and calculated values of m are compared : TABLEI1 mX
106
observed
312.5 I563 782
391 98
.
I
1I
m X IO^ calculated
I I
3095 I559 767 375 99
It is evident that the correspondence between the experimental results and those which are indicated by the Ostwald dilution-law for a binary electrolyte is very close. It appeared t o be of importance to ascertain whether the alkalineearth salts of globulin, like those of casein, also obey the dilution-law for a binary electrolyte. Accordingly the following experiments were undertaken 11. Experimental
(i) T h e Preparation of the globulin. The globulin employed by Hardy in the experiments cited above was the “ insoluble ” globulin of ox-serum’ prepared by precipitation from ‘dilute serum through the cautious addition of acetic acid. I have employed the same globulin, precipitated, however, by passing a stream of CO, through the diluted serum, as recommended by Quinan. The following was the complete procedure. Three liters of ox-serum were diluted with ten times their volume of distilled water and CO, was bubbled through the mixture for about half an hour. The globulin which was thus precipitated was allowed to settle in tall glass cylinders, the supernatant fluid being syphoned off after settling. The Cf. Quinan: Univ. of California Publ. Pathol.,
I, I
(1903)
Studies in the Electrochemistry of the Proteins
169
precipitate was then washed with about 60 liters of distilled water, in two washings. The globulin was then dissolved in a minimal quantity of N/IO KOH. This precipitate, after settling and the decantation of the supernatant fluid, was washed in 60 liters of distilled water in 6 successive washings, the precipitate, after each agitation with distilled water, being allowed to settle for 24 hours in the presence of toluol, after which the supernatant fluid was drawn off and the globulin suspended in a fresh quantum of distilled water. The thick suspension of globulin which was thus obtained after the final washing was kept, in the presence of toluol, in a stoppered bottle and used in this form, since globulin, if ,washed with alcohol and ether and dried, is redissolved only with difficulty. The suspension was, of course, always well shaken before withdrawing a sample. Twenty-five cc samples of this suspension were placed in small and accurately weighed beakers: the fluid was then evaporated to dryness on a water bath and the residue was dried at 70' over H,SO, until its weight became constant. Three determinations yielded the following results : Determinations
I
Grams globulin jn IOO cc of suspension
I 2
1.49 1.47 I .48
3
1
Average,
I
.48
I n all, about 14 grams of globulin were obtained. (ii) Experimental results. The experimental procedure was the same as that described in previous communications. Since Hardy's experiments were conducted upon sodium globulinate and at 1 8 O and all my determinations were made at 30°, for purposes
:
Part I of these studies, Jour. Phys. Chem., 14,5 2 8 (1910).
170
T . Brailsford Robertson
of comparison, I have not only measured the conductivities of solutions of the globulinates of calcium, barium and strontium, but also those of solutions of the globulinate of potassium. According to Hardy bases dissolve globulin in molecular, not equivalent-molecular . proportions, about I O x IO-' equivalents of an alkali and 2 0 X IO-^ equivalents of an alkaline earth being required to dissolve I gram of globulin. The latter solutions are neutral t o phenolphthalein, the former to litmus; but if I gram of globulin be dissolved in 20 X IO-^ equivalents of an alkali then the resultant solution is also neutral to phenolphthalein. That is t o say, globulin combines with bases in equivalent molecular proportions to form solutions neutral to phenolphthalein, but in molecular proportions when the base is combined with the maximum quantity of globulin which it can hold in solution. The solutions employed in the following experiments (except the solutions of strontium globulinate) were all prepared in the following manner. To IOO cc of the globulinsuspension (containing I .48 grams of globulin) were added 29.6 cc of a hundredth-normal solution of the base and the resultant solution was diluted t o 2 0 0 cc. One hundred cc of this solution was then diluted to 200, one hundred of the new solution to 2 0 0 and so on. The solutions of strontium globulinate were made up by adding to 25 cc of the suspension 7.4 cc of hundredth-normal Sr(OH),, diluting t o 200 cc and then proceeding as described above. All of these solutions were practically neutral to phenolphthalein, one-tenth of a cc of N/IO KOH sufficing t o render IOO cc .of the most concentrated solutions alkaline to this indicator. The resistance-capacity of the conductivity-vessel employed was 0.1949. The conductivity of the distilled water (4.0 x IO-^) has been subtracted from each of the observed conductivities. The following were the results obtained (temperature 30') :
Studies in the Electrochemistry of the Proteins
_
_
_
~
171
TABLEI11 Potassium globulinate
m = equivalent-molecular concentration of KOH neutralized by globulin X 105
I
= conductivity in reciprocal ohms per cc X 106
2 96
I 16
148 74 37 18
64
33 I8 9
TABLEIV Calcium globulinate m = equivalent-molecular concen-
= conductivity in reciprocal ohms per cc X 106
tration of Ca( OH), neutralized by globulin X 105
45
296 148 74 37
26 I5
9 4
I8
i ,
f n = equivalent-molecular concentration of Ba( OH), neutralized by globulin X 105 I
296 I48 74 37
74 37 I8
ohms per cc X
106
49 27 16 9 4
18
m = equivalent-molecular concentration of S r ( OH), neutralized by globulin X 105 .
= cpnductivity in reciprocal
!
= conductivity in reciprocal ohms per cc X IO^
I7
9 5
172
.
mX
,
T.Brailsford Robertson
m X
10)observed
105 calculated
-I 296 148 74 37 I8
297 I47 70 37 18
Applying equation 2 in the same way to the results enumerated in Table IV’ we obtain: m = 43-25x
+ 0.502 + I o ” x 2 .
The observed values of m and those calculated from this formula are compared in the following table:
TABLEVI11 Calcium globulinate m
x ios observed 2 96
148 74 37 18
I
1
m
X IO^ calculated
296 146 76 43 18
Only the Ist, znd, 3rd and 5th determinations in Table I V were employed in the computation of the constants, the 4th being obviously somewhat in error.
Studies in the Electrochemistry of the Proteins Applying equation Table V we obtain:
2
I73
to all of the results enumerated in
m = 42.61 x
+ 0.369 X
IO'
x2
The observed values of m and those calculated from this formula are compared in the following table.
TABLEIX Barium globulinate mX
I
106 observed
m X io5 calculated
2 96
297
148 74 37
142
78
4'
I8
18
.
-
nt X ~dobserved
m X io6 calculated
~
~~
74 37
74
36
It is evident that the Ostwald dilution-law for a binary electrolyte holds good for the globulinates of the alkaline earths as well as for the globulinates of the alkalies; that the dependence of the conductivity of solutions of the globulinates of the alkaline earths upon their concentration, like that of the conductivity of solutions of the caseinates of the alkaline earths, is such as would be anticipated if they dissociated into two ions. If we compute, from the values of the constants in equa-
T.Brailsford Robertson
I74
tion 2, the values of p(v, t v2) for these globulinates we obtain. For For For For
+ vz) = 5 1 9 x + v,) 24.4 x + vz) '248..35 xX p(v1 + v2)
potassium globulinate calcium globulinate barium globulinate strontium globulinate
p(v1 p(v, p(vl
105 105
106
IO^
+
The relation between the values of p(v, v,) for the globulinates of the alkalies and of the alkaline earths is very similar t o that which subsists between the corresponding values for the caseinates' and a t once suggests the ratio I
:
2.
+
The above values of p(v, v,) for the globulinates of the alkaline earths are 50 percent less than the velocities of the metal ions themselves. As in the similar case of the caseinates, the only feasible explanation of this is that the globulinates dissociate into two complex protein ions and p is twice as great for the globulinates of the alkalies as for the globulinates of the alkaline earths.' Since, a t neutrality t o phenolphthalein ,globulin neutralizes bases in equivalent molecular proportions, the molecule of calcium globulinate must, a t this hydroxyl concentration, be twice as heavy as that of potassium globulinate. Hence representing the mode of dissociation of potassium globulinate (neutral t o phenolphthalein) by the various schematic formulae : I.
KX++
+ X(OH)i
2.
K,X++++
+ X(OH):
3.
+
K3X++++++ X ( O H ) F
according to the number of -COOH groups concerned in the neutralization of bases, then the calcium globulinate (like calcium caseinate) must be represented by the corresponding schematic formulae : Cf. Part I1 of these studies. Journal of Physical Chemistry. The full discussion of this interpretation will be found in 'Part I1 of these studies, referred to above. I refrain from unnecessary reiteration of the argument.. , . a
I
Studies in the Ebctrochemistry of the Proteins
X+++
ca3eIII
‘X+++
I 75
X(OH);
+Ill
X(OH):*
Assuming that schematic formulae ( 2 ) represent the true state of affairs, as they do in the case of the caseinates, we can at once understand why bases dissolve globulin in molecular and not equivalent molecular proportions. Evidently the molecule K H X + + + + X(OH): can exist in solution, but the molecule: Ca = X++ X(0H):
+
II
+I1
H, =X++ x(ow: splits off H,XX(OH), which is the insoluble free globulin and two residual halves, derived from molecules which have decomposed in this manner, unite to yield the calcium globuh a t e , represented in schematic formulae ( 2 ) , which is neutral to phenolphthalein : The same assumption obviously accounts for the fact that the combining capacity of globulin for the alkalies a t neutrality to phenolphthalein is twice as great as it is when the alkali in solution its maximum capacity of globulin. Assuming that the value of p for the globulinates of the alkaline earths is I , while for those of the alkalies it is 2 (cohesponding with any of the above pairs of schematic formulae), then the values of v, v, at 30° for the above globulinates would be :
+
For For For For
potassium globulinate calcium globulinate barium globulinate strontium globulinate
26.5 X IO-^ 24.4 x IO-^ 2 4 . 3 x IO-^ 2 8 . 5 X IO-^
Hardy has shown, b y direct observation’ that the specific, velocity, per volt per cm potential gradient, of the globulin I
W. B. Hardy: LOC.cit.
T.Brailsford
176
Robertson
ion a t 18' lies between I O X IO-^ and 2 0 X IO-^ cm sec. Increasing these values by 2 . 7 percent per degree centigrade rise in temperature (which, according to the same observer, is the temperature coefficient of the conductivity of globulinates) at 30' they bedome, respectively 13 X IO-^ and 26 x The above results, therefore, point to the lower value as being, more probably, the correct one. Calculating, on the assumption that p = 2 , the values of G, the dissociation constant for these globulinates at 30' we obtain : __
I
Salt
Potassium globulinate Calcium globulinate Barium globulinate Strontium globulinate
I
G
I
0.01470
I
0.00360
1
0 ' 00493 0.0021I
I
1
As in the case of the caseinates, the dissociation-constants of the globulinates of the alkaline earths are considerably smaller than those of the globulinates of the alkalies-this is not improbably connected with the greater size of the molecules of the protein salts of the alkaline earths.
Conclusions ( I ) The serum-globulinates of the alkalies, in solutions neutral t o phenolphthalein ( I gram globulin = 2 0 X IO-^ equivalents of base), obey Ostwald's dilution-law for a binary electrolyte. (2) The serum-globulinates of the alkaline earths, in solutions neutral to phenolphthalein, also obey Ostwald's dilution-law for a binary electrolyte. (3) The value of p(v, v,) where p is the number of equivalent gram-molecules of globulinate which is formed by one equivalent gram-molecule of base, and v, and v, are ionic velocities of the globulinate ions, in cm sec. per potential gradient of I volt per cm, is twice as great for the globulinates
+
'
Studies in the Electrochemistry of the Proteins
177
of the alkalies as it is for the globulinates of the alkaline earths. (4) It is concluded that at neutrality to phenolphthalein the globulinates of the alkalies and alkaline earths dissociate into two protein ions, each possessed of twice as many valencies as there are molecules of base bound up in one molecule of globulinate. (5) On the basis of this conclusion the values of vl v2 (sum of the ionic migration-velocities, or molecular conductivity at infinite dilution) and those of the dissociationconstant are computed for the various globulinates a t 30'. ( 6 ) It is probable that at neutrality to phenolphthalein each molecule of globulinate contains two atoms of base, the globulinate of an alkaline earth containing two molecules of globulin linked together, and that the molecule so formed dissociate, in solution, into two quadrivalent protein ions, in one of which the metal is bound up in a non-dissociable form. When an alkali is combined with the greatest amount of globulin which it can hold in solution, the globulin neutralizes only half the amount of the alkali which it will neutralize at neutrality to phenolphthalein. I n such solutions, therefore, the molecule of globulinate probably contains only one atom of base.
+
