Oct., 1951
EQUILIBRIUM OF CYSTINE AND DITHIOGLYCOLIC ACIDWITH SULFITE [CONTRIBUTION FROM
THE
SCHOOL OF CHEMISTRY OF
THE
4569
UNIVERSITYOF MINNESOTA]
Equilibrium Constants of the Reactions of Sulfite with Cystine and with Dithiodiglycolic Acid BY W. STRICKS AND I. M.KOLTHOFF The reaction between cystine and sulfite (RSSR
+ SO3'
e RS-
+ RSS03-) is shown to be reversible in alkaline medium
(PH varied between 8 and 13), the equilibrium being attained rapidly from both sides. Four equilibrium constants with reference t o the various charge types of the anions of cysteine, cystine and cysteine sulfonate have been determined a t 12, 25, and 37". Neglecting the heat of dissociation of the sulfhydryl group and taking +12,000 cal. for that of the h"~+ group, the heat of reaction was estimated t o be +7000 cal. between 12 and 37'. The reaction between dithiodiglycolate and sulfite was studied only at PH lower than 9.5 because of decomposition of dithiodiglycolate at higher PH. The equilibrium constant has been determined in the PH range between 9.4and 6.6 at 25". The equilibrium constant of the reaction between dithiodiglycolic acid and the anion HS03- (R,SSR, HS0,- e R,SH 4-R1SS03-) was also determined.
+
The reaction between sulfite and organic disulfides has been the subject of various investigations. After Hefterl had shown that cysteine is one of the products formed in the interaction between suEte and cystine, Clarke2 and Lugg* presented conclusive evidence that the reaction between cystine and sulfite proceeds according to the equation RSSR
+ Na2S03 = RSNa + RSSO3Na
where RSSR, RSNa and RSS03Na denote cystine, cysteinate and cysteine sulfonate, respectively. Clarke2 isolated the reaction products and described some of the properties of cysteine sulfonate. Later Schober14and Kassel, et u Z . , ~ reported on the behavior of various disulfide compounds of biological interest in the presence of sulfite. The reaction has been made use of by Folin6 in the colorimetric determination of cystine and cysteine and recently in the amperometric titration of traces of cysteine and cystine with silver nitrate or cupric copper as The reactions between sulfite and cystine, homocysteine lactone and thiazolin carboxylic acid, respectively, are also of biological interest in connection with the sulfite inactivation of the cobra venom neurot~xin.~A more complete understanding of the sulfite cystine system is desirable both from the theoretical and industrial (treatment of wool, hide) lo viewpoint. This paper deals mainly with a quantitative study of the reaction between cystine and sulfite a t various PH and temperatures. It is shown that this reaction is reversible in alkaline medium.
Equilibrium constants and the heat of reaction have been calculated from results of the polarographic method of analysis. The system dithiodiglycolate-suEte is simpler than the corresponding cystine-sulfite system since the glycolate does not contain amino groups. The equilibrium constant of the reaction between dithioglycolate and sulfite (and bisulfite) has been determined. Materials Cystine and sodium sulfite were C.P. reagent grade Merck products. Cysteine which was used in the form of its hydrochloride was a C.P. product from Pfanstiehl. Thioglycolic acid was an Eimer and Amend "pure" product which was purified by two vacuum distillations. The purification of thioglycolic acid and the preparation of pure dithiodiglycolic acid were carried out by D. Leussing in this Laboratory. All the other chemicals were C.P. reagent grade products. Stock solutions of cystine and dithiodiglycolic acid were 10-* M in RSSR and 0.1 M in hydrochloric acid. Stock solutions of sulfite were prepared with air-free water and were 0.2 t o 0.5 M in sodium s a t e . Only freshly prepared sulfite solutions were used. Cysteine and cysteine sulfonate solutions were prepared in the same way as described previ~usly.~*~~ The stock solutions used for the preparation of the buffers were: 0.2 M disodium phosphate, 0.2 M monosodium phosphate, M sodium hydroxide, 5 M and M ammonia, respectively, 4 M and M ammonium chloride, respectively. The ionic strength was adjusted by the addition of appropriate volumes of a 3 A l potassium chloride solution.
Experimental
Current-voltage curves were measured at 12, 25 and 37", with the manual apparatus and circuit described by Lingane and KolthofflS and automatically with a Heyrovsky selfrecording polarograph. The diffusion current of cysteine was measured manually. All reported values of i d were corrected for the residual current. All potentials were measured against the saturated calomel electrode (S.C.E.). (1) A. Hefter, Chcm. Zcnfr., 78, 11, 822 (1907). Oxygen was removed from the solutions in the cell with ni( 2 ) H.T.Clarke, J . Bioi Chcm., 97,233 (1932). trogen. During an experiment an atmosphere of nitrogen (3) J. W.H.Lugg, Biochcm. J., 36,2144 (1932) was maintained over the solution. (4) A. SchiSberl and E. Ludwig, Bcr., 70, 1422 (1937); A. Sch&berl The characteristics of the capillary used were: m = 1 .SO4 and F. Krumy, ibid., 71B,2361 (1938). mg. set.-', rn*/:P/a = 1.89 mg.*/a set.-% (open circuit); the (5) B. Kassel and E. Brand, J . Biol. Chem., U S , 131 (1938). height of the mercury column was 76 cm. (6) 0.Folin and J. M. Looney, ibid., 61, 421 (1922); 0.Folin and The PH was measured with a Beckman PH meter, LaboraA. D.Marenzi, ibid., 88, 103 (1929). tory Model G. (7) I. M. Kolthoffand W. Stricks, THISJOURNAL, 74, 1952 (1950). Glass electrodes made of the usual 015 type electrode (8)I. M. Kolthoff and W. Stricks, Anal. C k m . , 98, 763 (1951). glasses were used for solutions with pH below 9.5 while (9) F. Micheel and G. Bode, Ber., 71B,2653 (1938). measurements were made with the Beckman "Type E" (10) F. F. Elsworth and H. Phillips, Biochcn. J., 89, 837 (1938); hi h PH glass electrode at PH above 9.5. H. Phillips, J . Soc. Dyers ColourisLs, 64, 503 (1938), F. F. Elsworth $rocedure.-Apptop&te volumes of air-free stock soluand H. Phillips, Bwbhcm. J . , 86, 135 (1841); W.R.Middlebrookand H. tions of cystine or dithiodiglycolic acid and sulfite (or of Phillips, ibid., 86, 294,428 (1942); S. Blackburn, R. Consden and H. cysteine sulfonate, cysteine and sulfite) were added t o a given Phillipr, ibid., 88, 25 (1844): H.Lindberg and H. Phillips, ibid., 8B,17 yolume of an air-free buffer solution which had been placed (1945); E. 0. H.Carter, W. R. MiddIebmok and H. Phillip, J . Soc. in an electrolysis cell. The total volume df the mixture was DwrS Cdo*?&U, a,108 (1046); E.El6d .nd I% a h n , Md#laud Text& BO ml. in each experiment. The diffusion current of cys= b., 171W (1946); Ckm. Ench., i U , I, 880 (1947); R.Liadky m d H, phwipr, Eiach8m. J,, 4& 84 (1047); W. Wlndw -4 )rsQ. Trarlw, (11) I. M.S01tb8 .ad Wo 8ulO)laj TSSD fouann~, 011 1728 (1Qblb. 1. A n , k M k r C b n . A m . , 40, doI (1941), (14) 1. 1. L b r a r *ad t . Y, So)tbaff, &k, 11, )s6 (1989).
AWDIC DIFFUSION CURREXrS Or
TABLEI THIOGLYCOLIC AC~D SOLUrIONS
CYSTEINE
AND
\.AR\I\G
pbf
IN
MIXTURES!\'ITH
SUI.FITE A T
ANI) T R M P E R A f U R E h id a i
-0.4
volt
Buffer
Cysteine Cysteine Cysteine Cysteine Cysteine Cysteine Thioglycolic acid Thioglycolic acid
12 25 37 25
0.1 .05 .05
25 25 25 25
.05 .05 .06
(PA.)
0.1 A ' NaOH, 0.6 M KC1 . 1 df KaOH, 0.75 Af KC1 , 1 111 KaOH, 0.75 ill KCl .05 ill Xa2HPO4,0.075 iW NaOH, 0.475 M KCl ,075 M NatHPO,, 0.05 M NaOH, 0.015 M HCI, 0.57 M KCI ,075 ,If SatHPO,, 0.025 A-l XaOH, 0.015 M HCl, 0.6 KCI .VI SHr, 0.1 M NHdC1, 0.75 M KC1 .lI XI&, JT SI-I~CI
.lo
(15
teine or thioglycolic acid in the mixture was measured after various periods of time until a constant value was obtained. The diffusion current of cysteine was found to be well defined and proportional to the cysteine concentration in solutions of pH 8 t o 13. In agreement with the findings of Kolthoff and Barnurn's a true diffusion current of cysteine could not be measured in solutions of pH markedly lower than 8. Thioglycolic acid gives a well d e h e d diffusion current in the entire PH range (10.5 to 6.6) investigated. Diffusion current constants of cysteine and thioglycolate have been determined a t -0.3, -0.35 and -0.4 volt, respectively, in solutions of the same composition as the equilibrium mixtures but in the absence of the dithio- and sulfonate forms. The values obtained were used for the calculation of cysteine and thioglycolate concentrations in equilibrium mixtures. A few representative data at -0.40 v. only are given in Table I. The diffusion currents of both cysteine and thioglycolate decrease slightly with increasing pH. The temperature coefficient of the diffusion current of cysteine was found to be about?% per degree over the temperature range from 12 to 37 This is of the same order of magnitude as that of metal ions.14 The pH of each solution was determined a t the temperature specified in Table I.
.
Results and Discussion
I. The Reaction between Cystine and Sulfite.-A complete polarogram of an equilibrium mixture obtained by the addition of 2 ml. of 0.2 M sodium sulfite solution to 18 ml. of 2.2 X M cystine solution in an ammonia buffer of pH 10.32 is given in Fig. 1. The anodic wave is that of the cysteine formed in the reaction between cystine and sulfite. The diffusion current of this wave corresponds to a cysteine concentration of 8.3 X M . The cathodic waves are those of the residual cystine and of the cysteine sulfonate15
13.26 12.8 12.41 12.1 11.2 10.1 10.5 9 3
2.36 2.96 3.49 2.92 3.06 3.09 3.37 3.53
formed. Polarograms of the type given in Fig. I were also run in alkaline equilibrium mixtures of cysteine and cysteine sulfonate. Both sets of experiments indicated that reaction (1) is reversible RSSR
+ SO," +RS-
f RSSOS'
(I)
and that under the experimental conditions equilibrium is attained rapidly from both sides. Determination of the Apparent Equilibrium Constants and Heat of Reaction.-From the polarographic determination of the cysteine concentration the equilibrium constant in a given mixture can be calculated. The anodic cysteine waves in equilibrium mixtures are dif fusion controlled and no indication of kinetic currents was obtained. Experimental results and calculated values of the apparent equilibrium constant K in mixtures of various temperature, pH, ionic strength and concentration of reactants are listed in Table 11. Polarographic experiments with solutions of high PH (11-13) containing only cystine revealed that this amino acid is not decomposed in strongly alkaline medium during the first hour after preparation of the solution at temperatures between 12 and 37". This makes possible equilibrium measurements in solutions of PH as high as 13.4. The values of the apparent equilibrium constant K in Table I1 have been calculated by introducing total concentrations for RSSR, RS-, RSS03and SOa" in equation (2). Kupp
[Z RS- ] [Z RSSOS- ] = -[2 RSSR] [ZSOS']
(2,
Actually the charge types of cysteine, cystine, cysteine sulfonate vary with the pH. At a pH greater than 9 all the sulfite may be considered present as SOa", while at a pH greater than 8 the carboxyl groups of cysteine and cystine and the sulfonate group of cysteine sulfonate are present as the anions. On the other hand, the dissociation of the sulfhydryl group in cysteine and the amino groups in RS-, RSSR and RSS03- varies in the PH range between 8 and 11 and this accounts for Fig. 1.-Polarograms at 25" of an equilibrium mixture (30 minutes after preparation) which was originally 2 X lo-* the fact that the apparent constant Kapp.varies M in RSSR and 0.02 M in NatSO, in an ammonia buffer with $H. The acid-base constants of the various groups of ( M NHs, 0.1 M N H I C ~PH , 10.3). cysteine and cystine are known. In the PH (12) 1. EX. Kolthoff and C. Barnum, THISJOURNAL,6%,9061 (1940). range listed in Table I1 the carboxyl group is in (14) I. M. Kolthoff and J. J. Lingane, "Polarography," Interscience the ionized form and its constant need not be conPublishers, Inc., New York, N. Y., 1940. sidered. The variation of the acid-base constants (1.5) Cathodic waves of cysteine sulfonate have been described in a previous paper." of cysteine with temperature was estimated from
Oct., 1951
EQIJILIBRIUM OF CYSTINE AND ~ I T I I I O G I ~ Y C O I , I ACID C WIT11 SULFITE
TABLE I1 APPARENTEQUILIBRIUM CONSTANT OF EQUATION (2) IN CYSTINE SYSTEM AT 12', 25", AND 37'. ~ ~ s ~ 2x. 10-3 0 0M Initial concentration Temp. of sulfite oc. (M)
id Of
Buffer
cysteine
INITIAL CONCENTRATIOX OF
z: Cysteine
pa.
P
4571
M
No.
KSPP.
x
10%
of expt.
0 . 2 M NaOH, 0.3 M KCI 13.40 0.95 2.17 9.24 X 1O-l 0.60 38 0 . 1 M NaOH, 0.75 M KCl 13.23 0.65 1.0 1.55 6.58 X 37 0.1 M NaOH, 0.75 M KCI 1.0 2.34 7.91 X 12.78 1.04 13 0 . 2 M NaOH, 0.3 M KCI .I5 0.95 4.48 1.28 X 1.53 12.51 40 0 . 1 M NaOH, 0.75 M KC1 .05 39 1.0 12.41 1.64 3.25 9.30 X ,025 0.05 M NagHPO, 0.075 M KaOH, 0.7 M KC1 1.0 12.13 1.80 6.08 X 1O-l 1.06 in 25 .05 0.05 M NazHP04 0.075 iM NaOH, 0.6 M KCl 1.0 2.35 7.94 x 10-4 1.04 9 12.17 25 0.05 ill NazHPOl .10 0.075 M NaOH, 0.475 M KCI 1.0 2.96 1.01 x 10-3 1.03 11 12.11 12 .05 0.05 M NanHPOd 0.075 M NaOH, 0.6 M KCl 1.55 6.44 X 0.60 1.0 36 12.39 .05 0.075 M NazHPO4 25 0.05 M NaOH, 0.75 M KCI 2.45 8.17 X IO-' 0.98 1.0 15 11.20 .05 25 0.075 -44NarHPO, 0.025 M NaOH, 0.6 M KCI 3.22 1.04 X 1.32 14 10.20 1.0 M NHJ .05 25 0 . 1 M NHdCI, 0.75 M KCI 1.0 10* 22 3.26 1.05 X 18 1.35 .02 25 X NHd, 0.1 M NH4Cl 1.76 0.16 10.32 2.66 8.17 X 1O-l 6 25 .03 M NH3,O.l M "4'21 0.19 10.37 7 3.03 9.30 X 1O-l 1.70 25 .05 M "3, 0.1 M NHjCl 0.25 10.37 3.50 1.10 x 10-3 8 1.68 ,025 M "3, M NHiCl 25 2.19 17 1.07 9.32 1.06 x 10-3 3.39 25 .05 M "3, M NH4C1 1.96 1.15 4.04 1.26 X 9.34 16 ,025 M "3, M r\rH4N03 87 8.98 4.54 1.33 x 10-3 2.94 44 1.07 .05 M "3, M NHlCI 37 2.81 43 5.26 1.54 X 9.00 1.15 ,025 0.1 M "3, M NH'Cl 25 8.23 6.79 5.28 1.62 X 10-3 20 1.07 0 . 1 M NHa, M NHiCl 25 8.27 7.73 1.15 .05 19 5.83 1.79 X IO-* 0.1 M NH3,M NHaCl .05 37 7.75 41 8.9 6.52 1.91 x 10-3 1.15 .05* 25 0.05 M NalHPOd 0.075 M NaOH, 0.6 M KC1 12.22 (1,30)b 1.o 3.95 1-34 x 10-3 12 The PH was measured a t the temperature given in column 1. Components added: 2 X IOd3M RSSO3- and 2 X 10-3M RSH, value of K&,,,,. not reliable since concentration of RSSO3- and Na2S03 are not sufficiently accurately known. 12 12 25 37 37 25
0.15 .05 .05
the apparent heat of ionization A" for the sulfhydryl and amino group, applying the van't Hoff equation and assuming that AH' is independent of temperature, over the range of measurement. AH' for the sulfhydryl group is assumed to be very small and thus no temperature correction was applied for the dissociation constant of this group as suggested by Cohn16and Borsook, et al." A€€'for the NHa+-group ranges between S l 0 , O O O and $13,300 cal. as determined for a number of amino acids and peptides. Taking 12,000 cal. for AHf for the amino group of cysteine, approximate pK values of the acid-base constants a t 12 and 37" are obtained from values a t 25" reported by Ryklan and Schmidt.I9 (1) Constants of cysteine a t 12,25 and 37" : The concentrations of the various species are denoted by letters above the symbols. Equation (7) was used to calculate the concentration of each of the species of cysteine a t a given PH. (16) E. J. Cohn, Ergebnissc d . Physiol., 88,781 (1931). (17) H.Borsook, E.L. Ellis and H . M. Huffman, J . Biol. Chem., 117, 281 (1937). (18) E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides," Reinhold Publishing Corp., New York, N. Y., 1943. (19) I,. R . Ryklan and C. 1,. A. Schmidt, Arch. Biochem., 6, 89 (1944).
c
RSH
I
SHs+ c
RSH
1
--
-
d
RS&Ha+ b RSH
I
+
"3'
d
RS
I
+
XHI + h
RSH
NHz U
RS-
I
z"
+ H + , p K i = 8.66at 12, 25and37' 8.99 a t 12"
+ H + , pKfr
8.25 a t 37"
{
(4)
10.84 a t 12'
+ H + , p K & 10.45 a t 25'
10.10 a t 37'
(6)
n
RS-
+ H + , pKA = 10.51 a t 12, 25and37' z Cysteine = a + b + c + d = I
NHz
(3)
+
I
h'Hz
(6)
(2) Constants of cystine, K ; and K: at 12, 25 and 37" as obtained from data at 25" reported by Borsook, et al." The constants a t 12 and 37" were obtained in the same way as given for cysteine. From the concentration of the total cystine in the equilibrium mixture the concentration of the different species can be calculated. The equilib-
U'. STRICKS AND I. M. KOLTHOFF
4572
PKj
PK; Z: Cystine = al
+ hl + c1
\
4 \
8.39 a t 12" 8.00 a t 25; (8) 7.65 a t 37
10.64 at 12; 10.25 a t 25 (9) 9.90 at 37"
rium concentration of the total cystine is obtained by subtracting the equilibrium concentration of the total cysteine formed from the concentration of the total cystine in the original mixture. The total concentration of cysteine sulfonate is equal to the total concentration of cysteine in equilibrium mixtures prepared from cystine and sulfite. Only the following dissociation of RSS03- need to be considered in alkaline medium RSSO.1-
VOl. 73
From equations (16), (17) and (18) it is seen that the equilibrium constants KII, KIII and KIV can be expressed in terms of K I and Ks and of the known dissociation constants of cysteine and cystine. The constant KI which involves the species with uncharged amino groups is found directly from experiments with solutions of sufficiently high pH. As is to be expected, the value of the apparent constant in Table I1 remains practically constant in the pH range between 11.2 and 13.4. In this strongly alkaline medium Kapp.is approximately equal to K I . Application of slight corrections by use of various equations (example v.i.) yields values of K I of 0.0062, 0.0103 and 0.0159 at 12, 25 and 37", respectively. An example of the calculation of K I follows Expt. No. 13, original mixture: 2 X ,I4RSSR, 0.05[I. Na&O,; /H+] = 1.7 X 10-13,p = 1 id of cysteine in equilibrium mixture: 2.34 pA. a t - 0.4 volt id of 10-3?d cysteine in the same buffer: 2.96 p A . a t - 0.4 volt (see Table I) Total cysteine at equilibrium = 7.9 X
From equation (7) u = 7.83 x 1 0 - 4 ~ .~. .. .
with an equilibrium constant denoted as Ks. I t is now possible to calculate the equilibrium constants with reference to the various species of cystine, cysteine sulfonate and cysteine.
[E:+]
d = 3.65 X 10-6 M . . . . . . ZRSSOI- = total cysteine = 7.9 X lo-' M ZRSSR = 2 x 10-3 - 7.9 x 10-4 = 1.2 x 10-3 J I [so3-]= 5 x 1 0 - 2 - 7.9 x 10-4 = 4.9 x 1 0 - 2 M Kapp.= 0.0104 = K I a t 25'
I t is seen that d is negligibly small as compared to a, the latter being practically equal to the concentration of the total cysteine formed. Since the amino groups of cystine and cysteine sulfonate are practically all present in the uncharged form a t pH 12.8 the apparent constant can be considered to be equal to K I . The two dissociation constants of sulfurous acid are 0.017 and 6.24 X lo-* at 25°.20 Thus correction for the formation of bisulfite needs to be made only in solutions of pH lower than 9. Knowing K I the constant KS of equation (11) can be calculated by applying equations (10) and (12) at a lower pH. An example for the calculation of KSat 25' follows
iJs.63 (15) Introducing K s and combining equations (12) and (13) gives =
KII
=
K4
- KI K0
(16)
Equations (13) and (14) combined with equations (5) and (16) give A4
KIII = - KI
K:: Equations (14), (15) and (17) give
(17)
Expt. No. 20; original mixture: 2 X lo-* M RSSR, 0.025 M NazSOa, [H+] = 5.9 X id of cysteine in equilibrium mixture: 5.28 pa. a t -0.30 volt id of 10-3 M cysteine in the same buffer: 3.25 pa. a t -0.30 volt Total cysteine: 1.62 X M a = 2.02 X 10-6 M M d = 3.34 X 10-4 M , ZRS- = a d = 3.36 X ZRSS03- = 1.62 X M , ZRSSR = 3.76 X 10-4Af, [SOJ'] = 2 14 x 10-1 AI
+
From equation (10) bl
) .
- -
ZRSSR = 3 76 x 10-4 M 2.35 X 10-4 M , cl 2.24 10-8 M
(90) Norio Yut, BwU. In#. Phyb. Chrut. Rwarck, 18, 1828 (1840); €3. V. Taptar and H. H. Ouretwn, Twin JOUnNAb, 806 (1841).
EQUILIBRIUM OF C Y S T I N E
Oct., 1051
AND
DITHIOGLYCOLIC ACIDWlTH
TABLEI11 EQUILIBRKUM CONSTANTS, CHANGE IN FREEENERGY AF AND HEATOF REACTION AH Temp., OC.
KI
KII
KIII
12 25 37
0.0060 ,010 .016
0.00031 .00052 .00082
0.0095 .016 .025
KIV
0.086 .15 .23
From equation (12) bt = K1c1[S03s1= 2.45 X 10-4 a a2 = ZRSSOa- - bl = 1.38 X I O - S M
Ks = bz'H+l = 1.05 X 10-9 a2
The values for Ks at 25" obtained from experiments 16, 17, 19 and 20 are 1.50, 1.06, 0.85 and the average value of Ks is 1.1 X 1.05 X Values of Ks obtained from solutions of PH markedly greater than 10 show greater variations and are not reliable since the difference between a2 and b2 becomes too large as compared to the accuracy of the measurements. Values for KS a t 37") obtained from experiments 41, 43 and 44, are 3.3, 4.8 and 2.8 X lomgthe average of Ks a t 37" being 3.6 X From the KS values a t 25 and 37" a heat of dissociation AH' of 17,900 cal. for the NHa+-group of cysteine sulfonate is calculated. This value is higher but of the same order of magnitude as the corresponding values for AH' of other amino acids. Taking 12,000 cal. for AH' and 1.1 X 10-8 for KS a t 25" the constant a t 37" is calculated to be 2.5 X instead of 3.6 X From equations (16), (17) and (18) the equilibrium constants K I I , KIII and KIV can now be calculated. The values of these constants, the change in free energy ( A F = RT In K) and the heat of reaction A H for the reactions I, 11, I11 and IV are given in Table 111. Assuming that AH for the amino groups of RSSR, RSH and RSSOa- is the same, it is found that the heat of reaction of the four equilibrium reactions is the same (AH1 = AHII = AHIII = AHIV = AH). AH was estimated from the experimental values of KI a t 12, 25 and 37" and from the apparent constants (Table 11) a t 25 and 37" obtained with equilibrium mixtures of PH 9.3, 8.3 and 7.75. The average values for A H estimated from the results of various experiments are found to be the same (about +7,000 cal.) II. The Reaction between Dithiodiglycolic Acid and Sulfite.-Polarographic experiments with mixtures of dithiodiglycolic acid denoted as RlSSRl and sulfite a t PH 6 to 9 indicate that the reaction is reversible, the equilibrium being more rapidly attained in alkaline (10 to 15 minutes a t p H 9.4) than in acid solutions (about 2 hours a t PH 6.6). The anodic wave of thioglycolic acid and the cathodic waves of dithiodiglycolic acid and of thioglycolic acid sulfonate resemble those of cysteine, cystine and cysteine sulfonate, respectively, and polatograms obtained with equilibrium mi%tures prepared from RlSSRl and sulfite were similar to those given in Fig. 1. Experiments in solutions of PH markedly higher than 9.6 cannot be carried out with dithiodiglycolic
OF
4573
SULFITE
REACTIONS I, 11, 111
AND
A F I , cal.
AFII, cal.
AFIII,cal.
AFIV,cal.
AH,cal.
-2900 -2700 -2500
-4600 -4500 -4400
-2600 -2500 -2300
-1400 -1100 900
4-6900 +6700
-
Iv
acid since this acid, in variance with cystine, decomposes rapidly in strongly alkaline medium to form thioglycolic acid. Polarogram obtained with solutions of RISSRl in buffers of PH 10.3 and 12.3 a t 25", 30 minutes after preparation are shown in Fig. 2. The higher anodic wave in the more alkaline solution indicates that the hydrolysis of RISSRl proceeds faster with increasing PH. -4fter 30 minutes 12 and 78% of the RlSSRl was decomposed in solutions of pH 10.3 and 12.3, respectively.
Fig. 2.-Polarograms at 25' of solutions (30 minutes after preparation) which originally had the following composition : I, 1.04 X 10-8 M RzSSRj (0.1 M NH*CI, M "3, 0.9 M KCl), pH 10.3----; 11, 2.3 X M RISSRl (0.05 M NatHPO,, 0.075 M NaOH, 0.6 M KCl), pH 12.3-.
Schoberl, et aL121 who carried out extensive experiments on the hydrolytic cleavage of disulfide carbonic acids isolated and identified the hydrolysis products of dithiodiglycolic acid as thioglycolic acid, hydrogen sulfide and glyoxylic acid. In agreement with our findings he also reports that cystine in alkaline solution is more stable than CYdisulfide carbonic acids in the same medium. A polarographic study of the hydrolytic cleavage of various disulfide-amino acids and -peptides is planned as an aid in the understanding of the alkali denaturation of proteins. The equilibrium constants of the reaction between dithiodiglycolic acid and sulfite can be calculated from the measured diffusion current of thioglycolic acid in an equilibrium mixture of known over-all composition and PH. The dissociation constants of thioglycolic acid have been reported by Larson22a t 25" as KI = 2.14 X and K2 = 2.1 X lo-". In the pH range investigated the carboxyl groups of R1SSR1, RlSH (thioglycolic acid) and R1SSOs- (thioglyeolic acid sulfonate) as well as the sulfonate group of RISSOa- are present as anions. On the other hand, the dissociation of the SH group varies in the pH range studied while the sulfite is partly present as (81) A. ScbObul, ( I ai., Ana., 811, 97 (1986); 804, 310 (1988); M# ICI ( i e w . (93) P.Lamon,2,*mar#. Jl#rm, Ckm., In,878 (IOBD),
C!. R. SINGLETERRY AND LORRAINE ARKIN WRINRERGER
4574
EQUILIBRIUM CONSTANT K s
OF
2.08 x 1.04 x 1.04 x 2.08 x 1.04 x 2.08
1.04 1.04 1 n4
x x x x
10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-8
0.05 .10 .05 .05 .0125 ,0125 ,1025 ,028 ,0063
TABLE IV EQT'ATION (19) 4I' 23", IONICSrRENGTII APPROXIMATELY ONE id of thio-
Components added R1SSR.i NazSOa M M
Buffer
NHa, M NHICI 0 4 M "8, 0.4 KHaC1, 0 3 11 KC1 0 5 M "3, 0.5 L2.r XH4C1, 0 3 M KCI 0 1 M NHa, M SHdCI 0.1 ilf h " 3 , M NHdCI 0 1 Ai' Nt12HP04, 0.01 31 Sc H?POI, 0 65 il.I KCl 0 08 Jf Sa2HP04,0 01 1 .1 SJHLPOI,0 71 iM KCl 0 05 Jf Na2HPOI,0 0ij 3 L SaH2P04,0 73 -11 KCI 0 05 X SanHPOa, 0 05 If SnH?P04, 0 78 If KC1
SO3- and partly as HS03-. It is possible to calculate the equilibrium constants for the RISSR1sulfite system. a1
RiSSRi
+ SOd
-+ b
+-- R,S-
1) bhi + R,SSO,-, K, = aid I
(19) (1 I
R , SIZi
+
- + a HSOj- + RiSH
+ RiSSO,-,Kv bl
Vol. 7 3
=
bit (1,C
"0 1
Denoting the dissociation constant of the sulfhydryl group of thioglycolic acid as I
