POLAROGRAPHIC P ~ W A VOF ES CYSTINEAND DITHIODIGLYCOLIC ACID
Sept. 20, 1955
teine, glutathione and thioglycolic acid are practically the same. Acknowledgment.-This investigation was supported by a research grant (C-721, C5) from the [CONTRIBUTIONFROM
THE
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National Cancer Institute, U. S. Public Health Service and by an institutional grant from the American Cancer Society. MINNEAPOLIS, MIXN.
SCHOOL O F CHEMISTRY O F
THE
UNIVERSITY
OF
MINNESOTA]
The Polarographic Prewaves of Cystine (RSSR) and Dithiodiglycolic Acid (TSST) and the Oxidation Potentials of the Systems RSSR-RSH and TSST-TSH BY I. M. KOLTHOFF, WALTERSTRICKS AND NOBOYUKI TANAKA’ RECEIVEDMAY4, 1955 The two-step reduction wave of cystine has been reinterpreted. Evidence is given that the prewave is determined by the 2RSH where RSSR and RSH are cystine and cysteine. The prewave is over-all reversible reaction RSSR ZeZH+ found to be kinetic in nature. The electrode reaction is catalyzed by mercury which reacts with cystine to form mercury cysteinate, the latter being reduced rapidly to mercury and cysteine. The potential of the cystine-cysteine system a t the dropping mercury electrode was found to correspond to the true oxidation potential. The same was found true for the thioglycolic-dithiodiglycolic acid couple but not for the glutathione system.
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+
No satisfactory explanation has been offered for the two-step waves observed with cystine a t the dropping mercury electrode in the p H range between 3 and 9.2.2 The first and steeper part of the double wave, called the “prewave,” has been attributed2 to a mixed-potential effect. I n the present paper i t is shown that this interpretation is inadequate. Conclusive evidence is presented that the potential of the cystine-cysteine system measured a t the dropping electrode after correction for the residual current corresponds to that of the couple RSSR + ZH+ + 2e- I_ ZRSH (1) in which RSSR denotes cystine and RSH cysteine. A t a platinum electrode this reaction is highly irreversible because of the slow rate of electroreduction of cystine. This reaction is catalyzed a t the mercury surface which reacts rapidly with cystine according to RSSR
+ Hg
Hg(RS)*
(2)
This reaction may occur in steps, e.g., as RSSR
+ Hg
HgRS
+ RS
--+
Hg(RS)2
(Za)
Materials.-Thioglycolic acid and dithiodiglycolic acid were prepared and purified by methods described previously.s All other chemicals were C.P. reagent grade products.
Experimental Current-voltage curves were measured with the manual apparatus and circuit described by Lingane and Kolthoff and automatically with a Heyrovsky type self-recording Sargent polarograph, Model XII. All potentials were measured against the saturated calomel electrode (S.C.E.). Oxygen was removed from the solution in the cell with 99.99% pure Linde nitrogen. The characteristics of the capillary were m = 2.53 mg. set.+, t = 3.51 sec. (open circuit) a t a height of the mercury column of 60 cm. The pH was measured with a Beckman Model H-2 PH meter.
Results Characteristics of the Cystine Wave. Effect of pH.-The effect of pH on the current-voltage curves of 5 X M cystine solutions is illustrated in Fig. 1. From the curves which were taken between p H 3 and 9 it is seen that the potentials a t which the waves are shifted to more negative values with increasing pH, the shift for the prewave being approximately 60 millivolt per pH unit. The height of the prewave is little affected by p H and attains a maximum value a t pH 8 while the height of the total wave (diffusion current) is constant over the pH range investigated.
but the experimental results do not allow further insight into the mechanism of this reaction. The mercuric cysteinate formed is rapidly reduced a t the electrode r r Hg(RS)2 Ze ZH Hg 2RSH (3) Thus equation 1 represents the over-all reaction which is the sum of equations 2 and 3. According to this interpretation the prewave of cystine should be kinetic in nature and the analysis of the wave should correspond to that of the reversible reaction (1). The effect of various factors upon the height of the prewave and the characteristics of the entire cystine wave are described in the experimental part. The conclusion is arrived a t that the potential of 0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -1.4 -1.6 the cystine-cysteine system measured a t the dropE d . e . ,v. us. S.C.E. ping mercury electrode corresponds to the oxidation 144 RSSR a t Fig. 1.-Current-voltage curves of 5 X potential of the system. In this connection the potentials of the thioglycolic-dithiodiglycolic acid pH (A) 3.3, (B) 4.7, (C) 8.0, ( D ) 9.2 and of ( E ) 5 X lo-‘ M and reduced and oxidized glutathione systems are TSST a t pH 5.0. briefly discussed. (3) D.L.Leussing and I . M. Kolthoff, J. Electrochem. SOC.,100,334
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(1) Tohoku Uni\ersit% Sendai, Japan (2) I \I Kolthoff a n d C Barnurn THISJ O U R N A L , 63, 520 (1941)
~
(1953). (4) J. J . Lingane a n d I . M . Kolthoff,
THISJ O U R N A L , 61,825 (1939)
1. M. KOLTHOFF, W. STRICKS AND N. TANAKA
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Effect of Cystine Concentration.-Polarograms were run over a concentration range from 5 x loV5 to 8 X M in buffered solutions a t PH 9.2; the results are listed in Table I. The plateau of the prewave becomes ill defined a t large cystine concentrations (larger than 3 X l o p 3 M RSSR). TABLE I PREWAVE ( i p ) AND TOTAL WAVE ( i d ) O F CYSIINE AT VARIOUS CYSTINE CONCENTRATIONS AT PH 9.2; h = 58 CM. Concn. CYS-
tine,
Buffer 0.05 M borax-0.1 M KCI .06 M borax- . 1 M KCl .06 M borax- . 1 M KC1 . 1 M NHs-O.1 M NHiCl . 1 M N H r . 1 M NHiCI .1 M N H r . 1 M NHiCl . 1 M N H r . l M NH4Cl .1 M N H r . l A4 NHiCl . 1 M N H r . 1 M NHiCl .1 M N H r .1 M NHiCI .1 M N H r .1 M "4'21
kl 101 0.50
x
,167 ,067 8.7
5.00 2.50 0.91 .48 ,246
.10 ,050
Prewave poorly defined. a maximum a
ip (pa.)
at -0.7 v. 1.01 0.361 0.16 (8.8)a 6.80' 3.60
1.26 0.74 .37 .15 ,074
id
at
ip/C 2.03 2.16 2.40 (1.01) 1.36 1.40 1.40 1.50 1.50
1.50 1.50
- 1.3 v.
3.21 1.10 0.448 57.1b 33.66 16.88 6.18 3.29 1.705
id/C 6.42 6.59 6.69 6.60
6.73
6.75 6.80 6.85 6.9 0 696 7 . 0 0.354 7 . 0
Main wave is preceded by
Considering that the prewave quite generally is not sharply defined, i t may be concluded from the results in Table I that the height of the prewave as well as the diffusion current are proportional to the cystine concentration a t a given PH. However, the height of the prewave appears to be markedly affected by the composition of the buffer; in an ammonia buffer it is some 25% smaller than in a borax buffer of the same $H. Effect of Temperature.-Polarogram obtained with a 5 X M cystine solution in a borax buffer (PH 9.2) a t 25, 3.5 and 4.5' gave temperature coefficients of 1 and 1.6y0per degree for the heights of the prewave and total wave, respectively. Effect of Height of Mercury Column.-The change in height of the prewave in a 5 X M RSSR solution (0.05 M borax, 0.1 M KC1) with height h of the mercury column is listed in Table 11. As expected the diffusion current is found to be inversely proportional to the square root of the height of the mercury while the prewave increases only 10% when h is increased 2.5 times. Experiments carried out in 5 x M and 5 x 10-5 M cystine solutions in an ammonia buffer of pH 9.2 yielded results similar to those in Table 11.
Vol. 77
fect on the potential a t which this wave occurs. Cysteine, which is also capillary active, was found to have no effect on the height of the prewave but to shift it to more negative potentials. The main wave of cystine was hardly affected by the presence of cysteine. Values of the oxidation potential of the system cystine-cysteine and also of TSSTTSH as measured a t the dropping electrode are given in Table 111. TABLE I11 OXIDATIONPOTENTIALS Eo OF THE CYSTINE-CYSTEINE ACID SYSTEMS (RSSR-RSH) ATD OF THE THIOGLYCOLIC FROM ZERO-POTENTIALS E, (TSST-TSH) CALCULATED AT THE DROPPING ELECTRODE Concn. RSSR,
Concn. RS.H, M X 103
Buffer
pH
M X loa
Acetate" Acetate Acetate Acetate Acetate Acetate Boraxb Borax
4.98 4.98 4.88 4.98 4.96 4.85 9.2 9.2
0.50 .50 .52 .80 .81 .82 .50 .50
1.0 5.0 10.0 1.0 5.0 10.0 1.02 5.0
Concn. TSST,
Cnncn. TSH,
x!4' 10% x1M103
Acetate" 4 . 8 2 Acetate 4 . 8 5 Acetate 4 . 8 8 Boraxb 9.00
5.00 LO0 2.50 5.00
4.93 1.97 4.93 1.37
a0.06 1Z.I CH3COOH, 0.14 borax, 0.1 M KC1.
AM
Ez (RSSR), v . 85
S.C.E.
-0.380 - ,420 - ,433 - ,375 - ,415 - ,426 - ,620 - ,658 Av. E , (TSST)
Eo (RSSR), v . ws. N.H.E.
$0.076 .077 ,076 ,075 ,075 ,075
+ + + + + + +
,067
,070 4-0.074 (TSST)
-0,382 +0.075 - ,353 .077 ,393 ,078 - ,618 ,063 Av. + 0 . 0 i 3 CHaCOONa. b 0 . 0 5 AI
-
+ + +
Some Characteristics of the Wave of Dithiodiglycolic Acid.-Current-voltage curves of dithiodiglycolic acid have been described previously. The polarogram of a 5 X AT TSST solution a t pH 5 is shown in Fig. 1E. It is seen that the prewave is much smaller than that of cystine under the same conditions and that the main wave is more drawn out than that of cystine. The height of the prewave was found to be markedly affected by the PH. Thus the prewave of l o w 3Jf TSST in an acetate buffer (pH 5) was 0.50 Fa. and in a borax buffer (pH 9.2) 0.14 pa. The height of the prewave was practically proportional to the concentration of TSST. When the height of the mercury column was TABLEI1 CYSTINE I S 0.05 31 BORAX, increased 2.5 times the height of the prewave of 5 (IYSTISE XYAVES O F 5 x M TSST in acetate p H 5 increased by about 0.1 M ECCl (pH 9.2) AT \*ARIOUS HEIGHTSh OF MERCURY X 25%. The temperature coefficient between 25 and COLCMS 5' was found to be about 2y0 per degree. Gelatin Prewave ( a t -0.7 v.) Total wave (at -1.6 v . ) a t a concentration of 0.0025y0 in 5 X 10-3 AI TSST 18, cm." 7 , pa. 1 4 a , ra. i/
