11.KOLTHOFF configuration would lead to a correspondingly larger decrease in the stability of the naphthylazo compounds. The two dyes which contain an amino group in ortho position to the azo group again show a double band spectrum, probably as a result of the two possible paths of resonance, as described above. These compounds also show some phototropism (although their rate o f reversal is also high), indicating that the hydrogen bond which is possible between the amino group and the azo nitrogen in these compounds is not as strong as that in dyes of Type 111and V. The normal bathochromic effect of a methyl group is not discernible from the spectra of these two coinpounds. The spectra of the four azo dyes derived from &naphthol (Type I-) are not affected by irradiation of this type, probably because of the exist-
[COSTRIBUTION FROM
THE
Vol. 71
ence of a strong hydrogen bond in these molecules, similar to that shown in Fig. 8. The strong displacement of the main absorption band toward longer wave lengths and the absence of a second band in the spectra of these compounds is consistent with this structure, since the formation of an additional chelate ring would be expected to cause a large bathochromic shift24and to provide a single preferred path for the resonance in the molecule. Acknowledgment.-The authors express their appreciation to Miss Iva Halow who carried out a large part of the experimental work, and to the Bureau of Ships for financial support of the investigation. (24) U', R. Brode a n d G h1. Wyman, THISJOURNAL. 73, 4267 (1931).
~VASHIXGTON25, D. C.
SCHOOL OF CHEMISTRY OF
THE
UNIVERSITY OF MIXNESOTA]
Polarography of Glutathione BY W. STRICKS AND I. M. KOLTHOFF RECEIVED MARCH15, 1952 The polarography of glutathione in the reduced (GSH) and oxidized form (GSSG) has been studied. Reduced glutathione piucs two anodic waves a t the dropping mercury electrode. The normal wave is well defined and corresponds t o the formation of a mercurous compound (GSHg). The current-voltage curve in the PH region from l to 10.5 has been found to obey GSHg f H + e. Yormal diffusion currents have been observed with GSH the equation for t h e reaction: H g f GSH over the entire pH range investigated. The diffusion coefficient of reduced glutathione is calculated t o be 5.6 X 10" and 4 ,i' X set.-' a t pH 1 and 10.82, respectively, a t an ionic strength 1 and a t 25". The characteristics of the second \~;~i:egreatly depend upon the ionic strength of the medium. At an ionic strength of 1 it is fairly well defined and its height is of the same order of magnitude as that of the normal wave. It is suggested that the second wave corresponds to the formation of GSHg( 11). Oxidized glutathione gives well defined reduction waves. Surface active substances like gelatin or thymol a t low concentrations hardly affect the GSSG wave. Larger concentrations of thymol shift the waves to more negative potentials. This is accounted for by the electro-capillary behavior of thymol and GSSG. GSH is more capillary active a t the dropping electrode than GSSG. The characteristics of the GSSG waves in the presence and absence of an excess of GSH are accounted for quantitatively by the sequence of reactions: (14) (15) = (12) in which reaction (14) is the rate a t i d potential determining step and equation (12) is the over-all reaction. The diffusion coefficient of oxidized glutathione a t iunic strength 1 and pH 10.3 is calculated to he 1.5 X 10V6em.* sec.-I a t 25".
+
+
polarographic study of glutathione in the reduced (denoted as GSH) and oxidized form (denoted as GSSG) is described in this paper. The results are compared with those obtained with the amino acids cysteine and cystine.',* Catalytic polarographic waves obtained with glutathione have been described by Brdicka. and Tachi, More recently Reiser,4 Coulson, et et u Z . , ~ reported on diffusion-controlled currentvoltage curves of glutathione.
catalyst. The progress of the oxidation was followed polarographically. The air bubbling was continued until the GSH wave had disappeared. Toward the end of the reaction ammonia was driven out from the solution with air. The solution, which was stored in a refrigerator, was found to be stable for several months. Cysteine which was used in the form of its hydrochloride was a Pfanstiehl product. Cystine, C.P., was from Merck and Co., Inc. Stock s o h tions of cysteine and cystine were prepared in the same way as described in a previous paper.* All the other chemicals reagent grade products. used were commercial C.P.
Materials.-~lutathione in the reduced state was a Pfnnstiehl product. The purity of this product was 99% :is determined by titration with cupric copper.' Stock solutions 0.01 a n d 0.1 M i n GSH were prepared in air-free wat m . Only freshly prepared stock solutions were used. A .\I stock solutioii of oxidized glutathione (GSSG) was preiM GSH p a r d h y passing purified air through a 2 X solution in an ammonia buffer (0.1 M in NH4Cl, 0.1 .If in Sli.i)which contained a trace of copper (2 X lo-' Ai) as a
Current-voltage curves were measured a t 25.0 i 0.1" with the manual apparatus and circuit described by Lingane and KolthoffB and automatically with a Heyrovsky selfrecording polarograph. All potentials were measured against the saturated calomel electrode (S.C.E.). Oxygen was removed from the solution in the cell with a stream of oxygen-free nitrogen which was purified by bubbling through vanadous sulfate. lo During an experiment an atmosphere of nitrogen was maintained over the solution. Corrections were made for the residual current. The characteristics of the capillary used were: 9% = 1.56 mg. sec.?, t = 4.82 sec. (open circuit); nzl'at'/s = 1.748 .2/asec.-'/z; h = 80 cm. mgFhe pH was measured with a Beckman pH meter, Laboratory Model G . A glass electrode made of the usual 015 type electrode glass was used for solutions with pH below
.
( 1 I I. h i . Kolthoff and C. Barnurn, TEXIS JOURNAL, 62, 3061 (1940). ibid., 63,520 (1041). 3 K. BrJi?ka, Cdleclio?i Czechoslou. Chem. Communs., 6 , 118
) ? ) I. LLI Kultl~offand C. Bnmum,
(1033; (4) K. G. Reiser, L'niv. Pitlrbiug B d I . , 40, 220 (1944). #.5, D.?.f. Coulson, W. I K.%nil \V Sri-icks. I r i o i . i i r c m . , 23,7tid ilR.ill.
Experimental Methods
-
(8) I. hl. Kolthoff and W.Stricks, THISJOURNAL., 72, 1952 (1950) (9) J. J. Lingane and I. M. Kolthoff, ibid., 61, 825 (1939). (10) I.. M e i t r s a n d T. Meites, A n a l . Chem., 2 0 , 9% ( I n d S i .
POLAROGRAPHY OF GLUTATHIONE
Sept. 20, 1952 -3
-4
-5
-6
v; W
K
w
P I
a
-
0 a V
-I
-0'
MLT
I
E (VOLT). Fig. 1.-Polarograms of M GSH in various buffers phosphate, NaN03, PH 10.82; (2) borate, XaSO,, pH 6.93; Xai'ioa, PH 5.13; (4) phosphate, NalL'03, pH 5.22. 9.5 while measurements were made with the Beckman "General Purpose" glass electrode a t pH above 9.5. The ionic strength of the supporting electrolyte was adjusted by the addition of appropriate quantities of sodium nitrate or potassium chloride.
''
Results and Discussion
4647
'
monia buffers the half-wave potential is found to be more negative than that observed in phosphate or borate buffers of the same p H and ionic strength. I n the latter two buffers as well as in dilute perchloric acid solutions reversible waves were obtained a t glutathione concentraM or less. At higher GSH tions of concentrations the wave exhibits a flat maximum in borate, phosphate, ammonia or acetate buffers but has a normal appearance in dilute perchloric acid solutions. Surface active substances like gelatin (0.005%) or thymol (saturated) have no effect on the shape of the anodic wave a t various pH. Reduced glutathione markedly reduces the surf ace tension of mercury as is evident from the electrocapillary curves (drop time against applied potential) obtained in an ammonia buffer of p H 10.3 ( p = 1) and in 0.1 M ( p 1): (1) perchloric acid (1 A l in NaN03) (see (3) acetate, Fig. 2), the effect being greater a t higher pH. At $H 10.3 the electrocapillary maximum is shifted from -0.5 to about -0.65 v. (GSH is anion) while a shift from -0.6 to about v*& found at PH (GSH is cation). Figure 2 illustrates that the electrocapillary curves in the mesence of GSH exhibit a discontinuity on both' the positive and negative side of the halfwave potential (-0.502 and -0.028 v. a t p H 10.3 and 1.0, respectively), indicating that the product of the anodic reaction (GSHg) as well as GSH are electrocapillary active. I n fact, the electrocapild l 1 (GS),Hg lary curve obtained with a 5 X solution was found to be identical with that of loe3 M GSH (see Fig. 2A). It is shown below that the diffusion current of the GSH wave corresponds t o a transfer of one electron per molecule of GSH. The following electrode reactions were
Reduced (GSH)'40me 50 polarograms were taken with solutions of various concentrations in glutathione in buffers from PH 1 to For the sake of brevity the results are not tabulated but in connection with the discussion essential data are presented in the form of graphs. All GSH solutions gave a well-defined anodic wave and another relatively poorly definedsecond wave (see curve 4, Fig. 1) a t potentials more positive than that where the normal diffusion current of GSH was observed. The shape of these waves GSH B +e was- found to be affected by PH, nature of the buffer, and concentration of glutathione. I n solutions with a p H considerably greater than 9 the normal i wave becomes drawn out v) P indicating an irreversible z 0 electrode process (compare 0 w curves 1 and 2 in Fig. 1). v) This was confirmed by analW ysis of the waves. A spe3c. cific buffer effect can be a seen from a comparison of 0 K the c--21 curves obtained P with an acetate and phosphate buffer of practically the same $H (5.13 and 5.22, respectively) and of the *4 -2 0 -.2 -4 -6 -8 -10 -2 -4 -6 -8 -10 -12 -14 -1s same ionic strength, as illusE (VOLT). E f VOLT). trated in curves 3 and 4 of Fig. 1. The analysis indi- Fig. 2.-Effect of GSH, (GS)ZHg and GSSG on the electrocapillary curve: A, (1) 0 M GSH; (3) cates that the electrode supporting electrolyte (0.1 hl HC104, M NaN03, pH 1); (2) h with with 5 X lo-' M (GS)ZHg. B, (1) 0 Supporting electrolyte (0.1 M NH,CI, M "I, process is irreversible in acetate buffers. I n am- 0.9 M KCI, PH 10.3); (2) h with lo-* M GSH; (3) El with 5 X lo-' M GSSG.
-
\\-. STRICKS .WD I. 11. KOLTHUFE'
-lMh
+ 2e
i'ol. i l
dropping electrode. If reaction (4) is reversible If the electro oxidation is reversible and occurs the mercury in GSHg should be reversibly reducible according to equation (1) the plot of log (id - i) i a t the dropping mercury electrode. Experiments with glutathione and mercurous uersus the potential should yield a straight line with a slope of 0.059. If equation ( 2 ) represents the nitrate indicated that GSHg is not stable and de(reversible) reaction a plot of log (id - i)? i 11s. composes in solution with formation of mercury and (GS)pHg. .i borate buffer (pH 6.94) which was I< should be a straight line with a slope of 0.0295 2.5 X IO-* -11 in (GS)2Hg and 3 X lo-' A 2 1 in glutathione was polarographed. The composite wave obtained with this solution was analyzed and found t o have the same characteristics as the anodic wave observed in a solution of -11GSH alone in the same buffer. Figure 4 gives the c-E curve of this wave and the plot log (i - (id)a/(id)c - ii V A . If, where (&ja is the anodic and (id), the cathodic diffusion current. i is taken positive for the 2 GSH
B
(2)
I O
011 0 6 0 4 0 2
0 0
ci
-0 2
-1
-0 4
- 30
.34
E p
- 38
- 42
(VOLT),
'0 6
Fig. 3---Aiialysi> of wave of 10 .li C;SH :it pH tj,!j:j, 1 : A, plot log (i,i - i) . E; B, plot log (iLt - i)* i
i'S.
-0 8
E.
-1 0
An example of the plots is given in Fig. :j corresponding to a solution 10 -a -11 in GSH in a borate buffer of p H 6.93. The plot ot' log (id - i j i i's. is gave a straight line with a slope of 0.035. This value is close enough t o the theoretical slope of 0.0.59 to allow the conclusion that equation I 1 I) represents the electrode reaction. Siinilar results were found upon analysis of waves obtained in 1 0 .: .I2 GSH solutions over the PH range between 1 and 9.8. The equation of the wave is given by
-1
2
- 1
4
-20
-24
-28
-32
-36
E
-44
-48
-52
-56
(VOLT)
curve A , .tnd plot log [ I - ( I ~ I J ~ ; ]( / , t i c - i , i: U,o l ,t rni\ture of .7 X IO 11 GSH, 2 5 X 10 - I -If .Hg < t t />H f i 9,~1 = 0 2
TIg i
-40
i -i+
cathodic and negative for the anodic current. The experiment was repeated in 0.1 X perchloric acid, This equation holds in perchloric acid solutions, the solution being 1 h i in sodium nitrate. The and in buffers containing phosphate, borate and log plot has the same slope as a t pH 8.9. The aniinonia a t concentrations of ammonia eyual to or cathodic wave obtained in a borate buffer (PH 6.9) -21 in mercuric glutathionate smaller than 0. I . The equation is not satisfied which was 3 X i n the absence of GSH was also analyzed and i i i acetate buffers and i n buffer solutions with a P H greater than ! I . > . -41~0in dilute perchloric acid found to have the same characteristics as the COTIIsolutions the eyuatioii does not hold when the posite wave (plot log i (%,I - i) LIS. E was a straight GSH concentratioii is equal to or greater than 2 line o f slope 0.031). ;in experiment carried out X .I/. Ikiuatic~ii( 2 ) was I K J t ioulItl tcJ 1 ) ~ with '1 solution .5 X I O -11 i n (GS),Hg in the pres~ i i c e~l '111 C X C ~ S S uf iiiercuric acetate (-5 X 10 - 4 satisfied in m y C J the ~ sulutious tested. Kolthoff and Barnuni' concluded that the aiiodic AI) in a borate buffer (PH 6.9) did not give consistcysteine waves correspond to the formation of ent results since the diffusion current of both the slightly dissociated mercurous cysteinate according excess mercury and the (GS),Hg were found to decrease on standing. A detailed study of the reto the equation actions of glutathione with mercury and other RSII -1-IIg RSIIg - ? I T ' ; Y (1, met& is planned. .ipparently a similar reaction is involved in the oneThe results of these studies allow the conclusion c,lcctroii aiiodic I)roc.rss ( i f filutathionc at thc that GSII i i not beiiig oxidized to G S S G at thc E
=
El,%- (i.0.59 log ( i d - ii i'
(3)
+
cL
Sept. 20,. 1932
POLAROGRAPHY OF GLUTATHIONE
dropping electrode but that it depolarizes the mercury with formation of (unstable) GSHg. The reduction wave of (GS)zHg corresponds to the reduction of a univalent cation. Apparently the following reaction is very rapid (GS)zHg
+ Hg
2 GSHg
4649
-.s
'1.5
(5)
-A the GSHg being the compound which is reduced. In this connection it may be men- 2 tioned that the reaction between mercuric chloride and mercury has been found to be 2 -,: very rapid a t the dropping electrode." . +, The equation of the anodic wave corre- w sponding to reaction (4) is
E = E"
+ R T / n F I n [GSHg]' [ H + ] a / [ G S H ] " ( 6 )
where E is the potential of the electrode, E o the s$andard potential of reaction (4) and the exmessions in the brackets are concentration's (activity coefficients are neglected) of the reacting substances a t the surface of
-.2
_,I
1
1
,
I
~
~
the Considering thatacid thewith sulfhyI t 3 4 5 6 7 (I 9 IO I1 12 13 dryl electrode. group in GSH is a weak an P H. ionization constant K it is easily derived from the previous discussion that E at 250 Fig. 5.--Half-wave potential9 of GSH and cysteine (RSH) us. PH: GSH, 1.1 1 : A , HC10a-SaS03; O ' ., acetate; 0 , phosphate;, D, amis given by rnonia; X , borate, 1.1 0.15 and 0.2: C3, borate. RSH, 1.1 1: -0, phosE
=
E'
+ 0.059 log k / k i + 0.059 log { [ H + l + K i - 0.059 log
(id -
i)/i ( 7 )
phate; !$ , 0.1 .1P NaOH; NH4XO3.
where k and kl are constants which are proportional to the square roots of the diffusion coefficients of glutathione and the mercury glutathione compound (GSHg), respectively. I n equation (7) [H+] is written instead of [H+]", since we consider buffered solutions only. The half-wave potential is expressed by El:, = E' + 0.059 log ( [ H + ] K ] (8) where the constant E' is equal to E o 0.059 log k/kl a t 25". The half-wave potentials which were taken from the plots log (id - i ) / iversus the potential are plotted versus pH in Fig. 5. By extrapolation of the GSH-curve of Fig. 5 the value of E' which corresponds to the halfwave potential a t "pH 0" is found to be f0.033 v. us. S.C.E. The experimental data agree well with equation (8). pK: ( K of the sulfhydryl group) of glutathione is 9.12.12 Thus K;becomes negligibly small as compared to the hydrogen ion concentration a t a pH less than 8. I n acid region the plot is a straight line with a slope of 0.038 as compared to the theoretical value of 0.59 a t 25' (eq. 8). -4t a p H greater than 9.t5 the hydrogen ion concentration becomes negligibly small as compared to K: and the half-wave potential becomes practically constant and independent of pH (see Fig. 5 ) . Between pH 7.6 and 9.2 the line is curved as is theoretically to be expected. The data plotted on Fig. 5 also show the specific buffer effect. I n acetate and ammonia buffers values of El/%deviate from those on the drawn line. With cysteine (RSH) in ammonia and phosphate
+
+
(11) I. h l , Kolthoff and C. S.Miller, THISJOKTRXAI., 63, 1405, 2732 (1941). f l 2 ) I
