STUDIES I N T H E ELECTROCHEMISTRY O F THE PROTEINS. VIII. THE DISSOCIATION OF SOLUTIONS O F T H E SULPHATE AND CHLORIDE OF PROTAMIN (SALMIN) BY T. BRAILSFORD ROBERTSON
(From the Rudolph Spreckels Physiological Laboratory of the University of California) 1. INTRODUCTION
In previous communications’ it has been shown: ( I ) That the caseinates and the serum globulinates of the alkalies and of the alkaline earths obey the Ostwald dilution law for a binary electrolyte, expressed in the form: n2 =
1.037 x P(U
IO-‘
+ v)
x
1.075 x IO-^ +K p ( u + v)’ X‘
where m = the equivalent concentration of the base neutralized by the protein, x = the conductivity of the solution in reciprocal ohms per cc at 30’ C, p = the number of equivalents of protein salt to which one equivalent of neutralized base gives rise, u v = the sum of the migration velocities, in cm per second per volt per cm potential full, of the ions into which the salt dissociates and K is the dissociation-constant of the salt. (2) The salts which ovomucoid forms with sulphuric and hydrochloric acids obey the Ostwald dilution law for a tertiary electrolyte. (3) Assuming the value of (u + u ) to be approximately the same for all protein salts (since they dissociate only into heavy ions) the value of p for the caseinates and serum globulinates of the alkaline earths is I , while €or the caseinates and serum globulinates of the alkalies it is 2 , and for the salts which serum globulin and ovomucoid form with acids it is 4.
+
T. Brailsford Robertson: Jour. Phys. Chem., 11, 542 (1907); 12, 473 (1908); 14, 528, 601, 709 ( 1 9 1 0 ) ;15, 166, 179, 387, 521 (1911). “Die physikalische Chemie der Proteine,” Dresden, 1912. Chapters 7-10.
Studies in the Electrochemistry of the Proteins
383
These deductions have been confirmed by cryoscopic determinations. (4) I t appears highly probable that the salts of casein and serum globulin with the alkalies are formed and dissociate in accordance with an equation of the type: R/coH,N\COH.N-
+ 2NaOH = R/
CON" ++
\CONa
+
H\/oH "N NN
. . . . . . . (A)
HAOH
the two anionic radicles being united to form a single ion, while the salts of casein and serum globulin with the alkaline earths form and dissociate in accordance with an equation of the type: dCOHN-\COH.N-
+ Ca(OH),
co = R/ \Ca ko.4++
H
I + PIN. . . . . . . . . . . .(B)
I
H/\OH
OH
and the salts which ovomucoid forms with acids form and dissociate in accordance with equations of the types: RCoH'N>R RC0H.N
RCOH .N
\R RCOH.N/
2
RC0H.N \R RCOH.N/
+ HC1 + H,O
=
++ H\/C1 NN
2RCOH
+
N N > R . . . . . . (c)
H/\OH
++
+ 2HC1 = 2RCOH +
H\/C1 ,"N l N > R . . . . . . . . . . . .(D) H/'\C1
+ H,SO, = 4RCOH +
. . . . . . . . . . (E)
HAOH
384
T . Brailsjord Robertson
Of the proteins hitherto investigated electrochemically, casein and serum globulin are predominantly acid, while ovomucoid, although predominantly basic, is nevertheless not markedly so, its power t o neutralize bases being only slightly less than its power to neutralize acids. It appeared of interest t o determine the mode of formation and dissociation of the salts which its strikingly basic protein forms with acids; accordingly, the following investigations were undertaken. 2. EXPERIMENTAL (a) The Preparation of Salmin Sulphate A member of the protamin group, namely salmin,l was prepared by the method of Kosse12 as follows: The ripe testicles of the Pacific salmon were minced and the macerated mass which was thus obtained was shaken up in tall glass cylinders with five or six times its volume of distilled water. The thick suspension of sperm which was thus obtained was syphoned off from the subnatant connective tissue and curdled by the addition of 80 cc per liter of N/IO acetic acid. The curdled mass of sperm was then washed in ten times its volume of 95 percent alcohol and this washing was repeated twice; i t was then washed once in the same volume of absolute alcohol and then in the same volume of ether. The powder, wet with ether, which was thus obtained, was spread out upon filter paper to dry in the air in a warm, dry place. Each 15 grams of the dried sperm was then stirred up in 350 cc of I percent by volume H,SO,, for about 6 hours. This mixture was then filtered through hardened filter paper and the filtrate obtained from the extraction of 15 grams of sperm was placed in a tall glass cylinder of about 4000 cc capacity which was then filtered with absolute alcohol. After allowing the precipitate t o settle, the supernatant fluid was According to Taylor: Univ. of Calif. Publ. Pathol., I , 7 (1904); the protamin which is contained in the sperm of the Pacific salmon is identical with the salmin found in the sperm of the European salmon. A. Kossel: Zeit. physiol. Chem., 25, 165 (1898).
Studies in the Electrochemistry of the Proteins
385
syphoned off, the precipitate contained in two cylinders was collected in one and this was filled with alcohol again.l The entire precipitate, suspended in alcohol, from the extract of 300 grams of sperm, was dissolved b y the addition of about 4 liters of hot water (about 80' C), the least soluble portion was filtered off, and the remainder reprecipitated by the addition of I O volumes of alcohol. This precipitate was washed once in the same volume of alcohol as that employed in precipitation and then in a like volume of ether. The final suspension of protamin sulphate in ether, obtained after syphoning off the supernatant ether, was collected in a hardened filter? dried over sulphuric acid at 40' for 2 days and then pulverized and sifted. The product is a friable white powder. The yield from 300 grams of sperm was 14.6 grams. The empirical formula of the substance which is thus obtained is, according to Kosse12 and Taylor, C,,H,,N,,O,. zH,SO,. It readily dissolves in water up t o about 2 percent at 2 0 ° . It diffuses through parchment paper (Taylor). Its solutions are very faintly acid in reaction. According to Taylor the acidity of a 1/2 percent solution, measured by the gas-chain, is N/3oo N H+; I found that I gram of my preparation in 1/4 percent solution required the addition of 9.6 cc of N/IO KOH to render the solution just alkaline t o rosolic acid, corresponding, in i / 2 percent solution, to an acidity of less than N/2oo; since an acidity determined by titration in protein solutions must obviously be considerably in excess of the true acidity it may be inferred that the acidities of solutions of my preparation were not appreciably in excess of Taylor's estimate cited above. Since a I percent solution of protamin sulphate contains 0.0424 equivalent of H,SO, per liter, it is evident that protamin sulphate does not, in It is necessary to avoid washing with alcohol too frequently, as on suspending the protamin sulphate in alcohol for a third or fourth time a very stable suspension is produced from which protamin is only deposited very slowly. LOC.cit.
3 86
T.Brailsford Robertson
aqueous solution, undergo hydrolytic dissociation to any very appreciable extent. According t o Taylor, a perfectly neutral preparation of protamin sulphate may be obtained by a special and lengthy process of preparation and purification. (b) The Preparation of Salmin Chloride Several attempts were made to prepare salmin carbonate according t o the method recommended by Taylor,1 in order to prepare the chloride from this substance. Many difficulties were found t o attend this procedure, however. If excess of Ba(OH), be added to a dilute solution of protamin sulphate great difficulty is encountered in removing this excess by means of CO, even a t 50' C. After several hours' passage of CO, clear filtrates can be obtained which contain barium, a fact which is probably attributable to the formation of the barium salt of a carbamino derivative of the p r o t a m h 2 Moreover, as Taylor points out, great difficulty is experienced in obtaining clear filtrates; indeed I have found the only successful method to consist in filtration under pressure through a Chamberland filter, a process which is attended by considerable loss of the protamin, since it is, t o some extent, retained by the filters3 Accordingly, salmin chloride was prepared directly from the sulphate in the following manner: To a carefully weighed amount ( I .48 grams) of protamin sulphate dissolved in IOO cc of water was added an exactly sufficient weight of carefully chosen barium chloride crystals, dissolved in about 20 cc of water, to precipitate the H,SO, in the protamin sulphate. This mixture was then set aside in a tall glass cylinder a t 50° for 24 hours a t the end of which time a compact precipitate of barium sulphate had settled to the bottom of the cylinder from which the clear supernatant fluid could readily be decanted. This fluid was filtered LOC.cit. Siegfried: Ergeb. d. Physiol., 9 , 334 (1910). This is true also of protamin chloride. A I percent solution of protamin chloride, after filtration through a porcelain filter under pressure, was found t o be reduced in concentration to about percent.
Studies in the Electrochemistry of the Proteins
387
through a hardened filter and the protamin chloride precipitated by the addition of 5-6 volumes of absolute alcohol. After allowing the precipitate to settle the supernatant fluid was syphoned off and the precipitate washed in I liter of absolute alcohol and twice in I liter of ether (ueber Natrium destilliert), and was finally collected on a hardened filter and dried over H,SO, a t 36' for 24 hours. I t was then pulverized and sifted and dried for another 24 hours. The yield was only about a third of a gram, which is attributable to the fact that the precipitate, after washing in alcohol, only settled very incompletely-a phenomenon which appears to be characteristic of very anhydrous (or, as Taylor believes, very highly purified) preparations of salmin. The empirical formula of this substance is, according to Kossel, C3,H,,N,,0,.4HC1. I t dissolves readily in water yielding very faintly acid solutions. (e) The Conductivity Determinations The methods employed in the conductivity estimations were those which I have described fully elsewhere.' The resistance-capacity of the conductivity-vessel employed was 0.1949. The conductivity of the distilled water employed was 3 x IO-' reciprocal ohms per cc at 30' C. The tabulated conductivities are the observed conductivities diminished by the conductivity of the distilled water. The following were the results obtained : TABLEI-SALMIN SULPHATE Grams&almin SUIphate in IOO cc. of solution
0.2500 0.1250 0.0625 0.0313 0.0156 0.0078
I I ,I
Equivalents of H,SO, in a litre of solution
0.01053 0.00526 0.00263 0.00132
j
0.00066 0.00033
1 i
Conductivity of solution in reciprocal ohms per cc. at 30° C
758 x Io-6 441 x IO-' 250 X IO-' 137 x IO-' 7 4 x Io-' 38 X IO-^
' T. Brailsford Robertson: Jour. Phys. Chem., 14, 5 2 8 ( 1 9 1 0 ) . "Die physikalische Chemie der Proteine," Dresden, I 9 I 2 . Anhang.
T. Rrailsford Robertson.
388
Grams salmin chloride in IOO cc of solution
Equivalents of HCl in a litre of solution
Conductivity 01 solution in reciprocal ohms per cc. at 30° C
I 686 X 365 X 194 x
0 . 005 5'8 0.00279 0.001 39 o.00070 0.00035
0.1250
0.0625 0.0313 0.0156 0.0078
Applying equation1
IO0
52
x x
IO-' IO-' IO-' 10-6
10-6
t o the above cited results and
(I)
enumerating the constants
1.037 P(U
x
IO-'
+ v)
and
1.075
x
Kp(u
IO-'
+ v)'
from all of the experimental data by the method of least squares we obtain, for salmin sulphate:
...............
m = 9.09% f 6 4 0 8 ~ ~
(2)
where m is the equivalent concentration of combined acid in the solution and x is its conductivity. For salmin chloride we obtain : m = 6.92 x + 1781 x 2 . .. . . . . . . . . . . . . ' (3) Applying, instead of equation (I), the equation : m =
1.037 x IO-' 1.115 x IO-' ~x + ~x3 . . . . . . . (4) P(U
+ VI
Kp(u
+v)~
which is the corresponding form of the dilution law'for an electrolyte which dissociates into three ions, we obtain, for salmin sulphate : m
=
10.58 x
+ 6.02 X
10'x3..
. . . . . . . . ..(j)
and for salmin chloride : m = 7.28 x
+ 1.82 X
10'
x 3 . . . . . . . . . . . . .(6)
In the following tables the experimental values of m and those computed from the observed conductivities with the aid of the above equations are compared: Cf. Introduction.
,,
T. Brailsford Robertson: "Die physikalische Chemie der Proteine," Dresden, 1912,p': 2 1 6 .
Studies irt the Electrochemistry of the Proteins
389
TABLEI I I ~ A L MSULPHATE IN Degree of dissociation of the salmin sulphate
Equivalents of H,SO, per litre of solution
Experimental
Calc. from Equation z
0.01053 0.00842 0.005 26 0.00263 0.00132 0.00066
0.00033
0.01057 0.00834 0.00527 0.00267 0.001 37 0.00071 0.00035
0.01065 0.00824 0 . 0 0 5 18 0.00274
65 69 76
0.00146
94
o.00040
Calc. from Eqn. s Percent
Calc. from Eqn. 2 Percent
Zalc. from Equation 5
76 80 89
85
1
I
IOO
\
IO0 IO0 IO0 100
Equivalents of HC1 per litre of solution
Experimental
Calc. Calc. from from Eqn. 3 Eqn. 6 Percent Percent
Calc. from Equation 3 ' Calc. from Equation 6 ~~
I
0.00558 0.00279
0.00139 o.00070 0.00035
0.00559
0.00276 0.00141 0.00071 0.00036
0.00558
0.00275 0.00143 0.00073 0.00038
85 92
95 97 IO0
89 97 99 100 IO0
The degrees of dissociation which are enumerated in these tables are computed from the ratios of
1.037.x IO-' .P (.U + v )
x to the
calculated values of m. The agreement between the experimental values of m and those calculated from equations 2.and 3 (dilution-law for a binary electrolyte) is highly satisfactory. The agreement
390
T . Brailsford Robertson
between the experimental values of m and those calculated from equations 5 and 6 (dilution-law for a tertiary electrolyte).is not so good. It would appear probable, therefore, that the sulphate and chloride of salmin yield 2 ions each (or a multiple of 2 ) on dissociation, arid not 3 or a multiple of 3 . The evidence afforded by conductivity determinations is, however, not sufficient, taken by itself, t o enable us t o determine the number of ions yielded by a molecule of an electrolyte;’ for this additional data, which may be derived from cryoscopic measurements, are requisite. (d) The Cryoscopic Determinations One-half percent or more concentrated solutions of salmin sulphate, on being cooled to the neighborhood of 0’ C, become diphasic, the salmin sulphate separating out in the form of an oil ; consequently solutions of salmin sulphate cannot be employed for the purpose of cryoscopic measurements. A percent solution of salmin chloride was investigated cryoscopically . Three successive determinations yielded the result: A = 0.04’ f 0.003 corresponding to a molecular + ionic concentration of M/46. The equivalent-concentration of HC1 neutralized by salmin in a percent solution of salmin chloride is M/45. Each molecule of neutralized hydrochloric acid yields therefore one molecule or one ion of salmin chloride. The equivalent conductivity of a solution of salmin chloride (or sulphate) does not increase more than 15 percent percent and at a dilution in the on dilution from 1/8-‘/128 percent approximates t o constancy. neighborhood of Consequently, these salts are highly ionized in solution and we may conclude, from the above-cited result, that two molecules of hydrochloric acid, in uniting with salmin, yield two ions of sahnin chloyide. One molecule of salmin chloride contains a t least four 1 cf. T. Brailsford Robertson: “Die physikalische Chemie der Proetine,” Dresden, 1912,p. 2 0 2 .
Studies
ilz
the Electrochemistry of the Proteitis
391
molecules of combined hydrochloric acid. Hence one molecule of salmin chloride must yield four ions or a multiple thereof. On the assumption, which is borne out by .the behavior of other protein salts,’ that each pair of these ions is capable of combining the true relationship between the conductivity and the dilution of a solution of protamin chloride is that expressed by equations I or 3, namely that which is characteristic of a binary electrolyte.
3. THEORETICAL The protein salts of inorganic acids and bases which have hitherto been investigated do not dissociate the inorganic radicle as such but bound up in a complex protein That this is also the case with salmin chloride may easily be inferred from the above-cited data. If each hydrochloric acid molecule bound up in a molecule of salmin chloride were to yield a chlorine ion to the solution then each molecule of hydrochloric acid would lead to the appearance of two, or a t any rate, more than one ion in the solution. I037
x IO-2 + v>
From the value of the constant in equation P(U
I as yielded by equation 3, we can estimate the value of the constant for salmin chloride. We thus find it to be: 150 x IO-^. The sum of the migration-velocities of two protein ions under unit potential-gradient a t 30’ C is from 30 x IO-^ cm per second to 40 X IO+ cm per second. Dividing the v) by 40 x IO-^ we obtain, in round above value of p ( u numbers, p = 4. Hence, if salmin chloride dissociates into two or a multiple of two protein ions, their valency must be 4. Summing up the inferences which we have drawn from the above data, therefore, we may conclude that each molecule
+
CE. Introduction. Cf. T.Brailsford Robertson: “Die physikalische Chemie der Proteine,” Dresden, 1912. Cf. previous articles of this series. Also “Die physikalische Chemie der Proteine,” loc. cit.
T.Brailsford Robertson
392
of salmin chloride yields fozir +valent
protein ions. Since salmin sulphate would also appear to obey the dilution law for a binary electrolyte rather than that for a tertiary electrolyte, we may assume with plausibility that salmin sulphate dissociates in an analogous manner These phenomena are obviously analogous t o those displayed by the caseinates of the alkalies' and correspond with those which would be exhibited by a salt which forms and dissociates in accordance with equations of the type: HvCl
/COH .N R'\CoH
.N
+ 2HCl=
COH++
R,/ \COH++
+
i i N > R z . . . . ..(F) , \
H'
C 'l
and ,COH.N\
)Rz
/
R'\COH.N
+ H,SO,
/COH++ =
R,
\COH++
H\ \ "N
+ SO,
R, . . (G)
A/
this process of combination and dissociation being repeated twice, i. e., a t four C 0 H . N linkages in each molecule of salmin. It will be obvious that the properties of a salt formed according t o these equations would in every way correspond with those of salmin chloride and sulphate, respectively, in so far as they have been observed. Every two molecules of neutralized acid would yield two 4-valent ions. None of the reactions illustrated by equations B, C, or D in the introduction would yield a salt exhibiting these properties, nor would any process of combination involving monoamino radicles, so far as we know at present, yield one quadrivalent ion for each molecule of neutralized acid. We may therefore conclude that the behavior of salmin chloride or sulphate in solution is such as to indicate that diamino-radicles are responsible for the neutralization of acids by salmin and not only that the anions of these salts are formed from diamino radicles, but that the cations, also, are either Cf. Equation A in the introduction.
S h d i e s in the Elecirochemistry of the Proteins
393
formed from diamino radicles or from monoamino radicles which remain linked in pairs. These inferences are in remarkable harmony with our knowledge of the structure of the salmin molecule. As is well known from the work of Kossel, the protamins as a group are characterized by their high diamino-acid content, salmin containing 87.4 percent of arginin.' According t o Kossel and Dakin the proportion of diamino t o monoamino radicles in salmin is z : I and, according to the same authors, salmin may be constructed from the union of I O mol. arginin + z mol. serin + I mol. aminovalerianic acid + z mol. prolin or else from the union of 1 2 mol. arginin + z mol. serin + I mol. aminovalerianic acid 3 mol. prolin. If we were t o accept the latter formula, that of salmin chloride z mol. serin I mol. aminowould be: 1 2 mol. arginin 1 2 mol. HC1. Recalling valerianic acid + 3 mol. prolin the fact that each diamino-radicle can neutralize two molecules of hydrochloric acid, it will b.e evident that in neutralizing twelve molecules of hydrochloric acid only half the arginin radicles would be used up. It is inviting to suppose that the remainder form the corresponding cations. Assuming the valencies of the ions produced by salmin sulphate and chloride to be 4, we can compute, from the values of the constants in equations z and 3, the values of u + ZI and of K (the sum of the specific ionic migration velocities and the dissociation-constant, respectively) for these two salts. The results are enumerated in the accompanying table : TABLEV
+
'
~-
1
Salt
Salmin sulphate Salmin chloride
,
+
+ +
+
v ) in cm per set. per unit potential gradient
(u
2 8 . 5 X IO-^ 3 7 . 5 x IO-'
K = dissociation constant
0.0516 0.1076
A. Kossel and H. D. Dakin: Zeit. physiol. Chem., 41, 4 1 2 (1904).
T.Brailsford Robertson
394
4. CONCLUSIONS Solutions of the chloride and sulphate of salmin obey the Ostwald dilution law for a binary electrolyte. (2) The depression of the freezing point o$ water which is brought about by the presence of dissolved salmin chloride is equal (in I/, percent solution) t o the depression which would be produced by a dissolved substance of the same molecular (or molecular ionic) concentration as that of the combined acid. Each molecule of combined acid, therefore, produces one ion (or molecule) in the solution of salmin chloride. (3) It is probable that each multiple of C,,H,,N ,,O6.4HCi in the molecule of salmin chloride dissociates yielding four quadrivalent ions. (4) It is suggested that the formation and dissociation of salmin chloride takes place in accordance with equations of the type: (I)
+
R,
