Mediators for the oxidation of glutathione and other biological thiols

131

J. Electraunal. Chem., 245 (1988) 131-143 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

MEDIATORS BIOLOGICAL

RANDALL

D. THACKREY

Department (Received

FOR THE OXIDATION THIOLS

and THOMAS

OF GLUTATHIONE

L. RIECHEL

AND OTHER

*

of Chemistry, Miami University, Oxford, OH 45056 (U.S.A.) 18th June 1987; in revised form 24th November

1987)

ABSTRACT The oxidations of glutathione, cysteine, cysteine methyl ester, and penicillamine do not occur readily at electrode surfaces without the use of a mediator. We report here the use of one vanadium compound and two ruthenium compounds as solution mediators for the oxidation of such biological thiols. The vanadium compound, Amavadine, is an effective mediator for these substrates in aqueous solution at a glassy carbon electrode. The ruthenium compounds were used in DMSO. In each case, the substrate is oxidized at the potential of the mediator itself. These are far positive of the literature values for homogeneous solution oxidation, but this effect is common for such catalyzed reactions.

INTRODUCTION

Glutathione has recently been suggested to be the reducing agent responsible for keeping vanadium(V) levels in animal systems low and thus preventing the inhibition of the sodium pump by vanadate [l]. Specifically, vanadium(V) has been shown to be a potent inhibitor of (Na+, K+)ATPase, which regulates the sodium level in cells. It has been hypothesized that when a significant amount of naturally occurring vanadium(W) is oxidized to the (V) state, the enzyme may be inhibited sufficiently to lead to hypertension [l-4]. The electrochemistry of glutathione is thus of great interest as it may be the key reducing agent necessary to prevent excessive levels of vanadium(V). As a reducing agent, glutatbione is oxidized to the disulfide (2 GSH + GSSG + 2 e- + 2 H+) but there are conflicting reports in the literature concerning the standard potential for this reaction. Early direct electrochemical studies were complicated by the formation of sulfur-metal complexes between thiols such as glutathione and cysteine with the electrode. Kolthoff and Barnum [5] found that the

* To whom correspondence

0022-0728/88/$03.50

should be addressed.

6 1988 Ekvier

Sequoia S.A.

132

anodic wave of cysteine at the dropping mercury electrode occurred near 0.0 V vs. SCE and corresponded to the formation or mercurous cysteinate rather than the dimer, cystine. However, at a platinum electrode the anodic wave of cysteine did correspond to oxidation to cystine and was found at a potential about 0.6 V more positive than at the DME. Polarographic studies by Stricks and Kolthoff [6] reported the similar formation of a mercurous glutathione compound. Using polarography at platinum electrodes, Davis and Bianco [7] studied the oxidation of cysteine to cystine and suggested that a small amount of cysteic acid might also be formed by subsequent oxidation. They also concluded that adsorption of the reactants at the electrode was taking place. Koryta and Pradac [S] later concluded that cysteine is most likely oxidized directly to cystine at gold electrodes, but at platinum the thiyl radical is adsorbed. Thus, the electrooxidation of glutathione and cysteine is complicated by adsorption and reactions with the working electrodes themselves. Based on these results, Jocelyn [9] concluded that direct determination of the standard redox potentials for such thiols was not practical. Thus, the approach of this and other workers was to estimate E o values for thiols by mixing their disulfides with a reference thiol and finding the equilibrium point in the thiol-disulfide exchange reaction which resulted. Using this approach, E Q for glutathione is reported to be -0.24 V [lO,ll] and that for cysteine is -0.22 V [12,13]. These values are consistent with the observed reducing capabilities of these molecules in homogeneous solutions, but are far negative of the various values reported from direct electrode measurements. As part of our study of biological reducing agents and vanadium model complexes for inhibition of the sodium pump, we have developed three mediator-titrants for the oxidation of biological thiols. We report the use of Amavadine, a vanadium natural product [14,15], and two bipyridine complexes of ruthenium(I1) as mediator-titrants in the electrooxidation of glutathione, cysteine, and similar thiols. This is only the second [16] report of a vanadium species as a mediator-titrant, while numerous ruthenium mediators have been reported [17-191. By use of a glassy carbon electrode we have avoided the formation of metal-thiol complexes on the electrode surface. EXPERIMENTAL

Instrumentation Cyclic voltammetry

experiments were carried out with a Princeton Applied Research Model 173 three-electrode potentiostat and a Model 175 Universal Programmer. The voltammograms were recorded on a Houston Instruments Model 2000 Omnigraphic X-Y recorder. Controlled-potential electrolysis was performed with the above potentiostat and a Princeton Applied Research Model 179 Digital coulometer. The working electrode for the non-aqueous studies was either a Bioanalytical Systems glassy carbon electrode or a Bioanalytical Systems platinum electrode. All

133

aqueous studies employed the glassy carbon electrode. A platinum mesh electrode was utilized for controlled-potential electrolysis. The auxiliary electrode was made from platinum mesh and separated from the cell solution by a fine porosity frit. The reference electrode consisted of a Ag/AgCl electrode in aqueous tetramethylammonium chloride (Aldrich) with the concentration adjusted to make the electrode potential 0.000 V vs. SCE. The reference electrode junction was a platinum wire sealed in a Pyrex tube. The electrode was positioned in a luggin capillary in the cell assembly. Prepurified nitrogen was bubbled through the cell prior to each experiment and a septum-covered port allowed the addition or removal of solution aliquots by syringe. A Corning Model 12 pH meter and combination electrode were used for pH studies. Spectrophotometric measurements were made on a Hewlett Packard Model 8450A spectrometer. Reagents

High-purity dimethyl sulfoxide (DMSO) (0.017% water) was obtained from Burdick and Jackson Laboratories. Tetraethylammonium perchlorate (TEAP) was prepared from tetraethylammonium bromide (Aldrich) and perchloric acid as previously described [20] and was used as supporting electrolyte. Potassium chloride, obtained from MCB, was used as supporting electrolyte when deionized water was the solvent. Glutathione, penicilIamine, cysteine, and cysteine methyl ester were obtained from Sigma Chemical Company and used without further purification. Solutions were prepared immediately prior to use. Preparation of compounds

Amavadine, or bis( N-hydroxy-cu,cu-iminodipropionato)oxovanadium(IV)monohydrate, was synthesized according to the method of Nawi and Riechel [15]. Ruthenium complexes were also prepared according to literature methods. Bis(bipyridyl)chloro(tri-n-butylphospine)ruthenium(II)hexafluorophosphate, {[(bpy), Ru(tri-n-Bu)P]Cl}PF,, was made by following the procedure of Sullivan et al. [21]. Bis(bipyridyl)pyrazolylpyrazoleruthenium(II)hexafluorophosphate, [(bpy) ,Ru(pz) (pzH)]PF,, was synthesized according to Sullivan et al. [22]. RESULTS AND DISCUSSION

A cyclic voltammogram of 11 mM glutathione in 0.1 M KC1 is shown in Fig. la. Using a glassy carbon electrode, the scan exhibits no redox behavior. However, when a small aliquot of Amavadine is added to the cell solution, the subsequent scan (Fig. lb) shows a large, irreversible oxidation wave at about 0.82 V vs. SCE. The molar ratio of this solution is 0.125 : 1, Amavadine to glutathione. Figure lb demonstrates the mediator-titrant effect when glutathione becomes oxidized in the presence of the Amavadine redox couple. Figure lc is a voltammogram of a 1.3 mM Amavadine solution in 0.1 M KC1 which exhibits a reversible couple whose

134

I

I

I

I

I

I

I

I

b

I

20j~A

I

I

0.8

I

I

0.0

I

I -0.8

I

1

-1.8

E vs.SCE/V

Fig. 1. Cyclic voltammograms in aqueous 0.1 M KC1 at a glassy carbon electrode. Scan rate 200 mV/s. (a) 11.0 mM glutathione; (b) solution (a) with 1.3 mM Amavadine; (c) 1.3 mM Amavadine.

oxidation peak is at 0.47 V. Since the concentration of Amavadine is the same in Figs. lb and lc, but the current sensitivities are different, it is clear that the large oxidation current in Fig. lb is due solely to glutathione. Also, although the peak potential is considerably more positive than that for Amavadine, the foot of the large oxidation wave is nearly the same as that for Amavadine. These facts indicate that ghttathione is being oxidized irreversibly, at the potential of the mediator. The results of a ratio study appear in Fig. 2 where a voltammogram was recorded after each of several additions of Amavadine to a glutathione solution. The solution was buffered at pH 3.9 with an acetate buffer. The results were the same as those

135

1.0

0.5

0.0

-0.5

-1.0

E vs.SCE/V

Fig. 2. Cyclic voltammograms in aqueous 0.1 M KC1 and pH 3.9 acetate buffer at a glassy carbon electrode. Scan rate 200 mV/s. (a) 6.0 mM glutathione; and additions of Amavadiue to solution (a) giving ratios of Amavadine to glutathione of: (b) 0.125 : 1, (c) 0.250 : 1, (d) 0.500 : 1, and (e) 1: 1.

obtained in unbuffered 0.1 M KC1 at about the same pH. The current and potential data are summarized in Table 1. Note the appearance of the oxidation wave at 0.62 V for an 0.125 : 1, Amavadine to glutathione solution (Fig. 2b). As the ratio of Amavadine to glutathione increases, the oxidation wave is first shifted positively. Then, as the ratio approaches unity the potential shifts negatively toward that of

136 TABLE

1

Current

and potential

Amavadine glutathione

data corresponding

to ratio

O.lOO:l 0.125 : 1 0.167 : 1 0.250 : 1 0.500 : 1 l.OO:l

to Fig. 2

Fig. No.

&/PA

J%Ot /v

E*,/v

2b 2c 2d 2e

6.9 12.5 20.0 36.3 38.8 58.1

0.38 0.38 0.38 0.38 0.38 0.38

0.56 0.62 0.66 0.76 0.68 0.63

Amavadine alone (Fig. 2e). The ratio in Fig. 2c is 0.250 : 1 and appears to be where the maximum mediator effect occurs. That is, the maximum oxidation current is observed without the reduction wave of Amavadine appearing. Here, the oxidation

bd-

2d

I

20pA

J--

0.8

0.0

-0.8

E vaSCE/V Fig. 3. Cyclic voltammograms in aqueous 0.1 M KC1 at a glassy carbon electrode. Scan rate 200 mV/s. (a) 6.0 mM Amavadine; and additions of glutathione to solution (a) giving ratios of Amavadine to glutathione of: (b) 0.500 : 1, (c) 0.385 : 1, and (d) 0.286 : 1.

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potential for glutathione is 0.76 V, the most positive value observed. In short, the foot of the oxidation wave does not vary and is indicative of Amavadine, but the peak potential is sensitive to the mediator-substrate ratio. Figure 3 represents the ratio study done in reverse. When small aliquots of concentrated glutathione were added to an Amavadine solution, the voltammograms change from the Amavadine reversible couple to a broad oxidation wave which shifts positively. As before, a 0.250 : 1 Amavadine to glutathione ratio gives the maximum mediator effect at 0.70 V (Fig. 3d). Spectra were recorded on the solutions of Fig. 3 in an attempt to discern whether or not complexation was occurring between glutathione and vanadium. That is, glutathione might be replacing the N-hydroxy-cw,cw-iminodipropionic acid ligand of Amavadine to form a new complex. The solution spectrum of Amavadine exhibits absorbance peaks at 564 and about 760 nm. When glutathione is added, giving the voltammograms of Fig. 3, the resulting spectra are virtually identical, differing only by slight decreases in absorbance of the Amavadine peaks due to dilution. This spectral data and the 0.250 : 1 optimum ratio provide evidence that a true mediator effect is occurring, not the oxidation of a newly-formed vanadium-glutathione complex. A CV scan rate study of a 0.167 : 1, Amavadine to glutathione solution was carried out to determine whether or not the observed oxidation process was diffusion controlled. A plot of i,, vs. u112 (u = 5, 10, 20, 50, 100, and 200 mV/s) was not linear as would be expected for a diffusion controlled process, but rather was a curve. This suggests a more complicated process, such as catalytic cycling of the mediator. The results of controlled potential electrolysis on a 0.083 : 1 Amavadine to glutathione solution are shown in Fig. 4. The mediator effect is shown in Fig. 4a with an oxidation wave at 0.74 V, while Fig. 4b is the voltammogram taken after exhaustive oxidation of the solution at 0.9 V. For this process, a large charge (0.90 electrons per molecule based on the sum of glutathione and Amavadine) was obtained which is consistent with the one-electron oxidation of both glutathione and Amavadine. The initial positive scan after electrolysis (Fig. 4b) shows no oxidation wave, indicating that oxidation was complete. The scan does show a cathodic peak for reduction of the oxidized Amavadine at 0.33 V and subsequent oxidation at 0.58 V on the second scan. This solution was then exhaustively reduced at 0.0 V and only a small charge (0.92 electrons per molecule based on Amavadine alone) was obtained, which corresponds to reduction of the mediator only. Figure 4c demonstrates that, following this reduction, only the Amavadine redox couple appears. Thus, coulometry suggests that the glutathione was irreversibly oxidized. Figure 5 shows the resulting voltammogram when an 0.125 : 1 Amavadine to glutathione solution is scanned continuously for three complete cycles. The oxidation wave for glutathione decreases markedly, while the reduction peak corresponding to oxidized Amavadine increases slightly. Thus, since the solution is unstirred, it appears that the glutathione is being consumed, leading to a final voltammogram that resembles Amavadine alone.

138

I

I

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b

1 OJJA

I

1 1.0

I 0.5

I

I

0.0

-0.5

I

-1.0

E vs.SCE/V

Fig. 4. Cyclic voltammograms in aqueous 0.1 M KC1 at a glassy carbon electrode. Scan rate 200 mV/s. (a) 0.083: 1 ratio, Amavadine to glutathione, solution; (b) solution (a) after controlled potential electrolysis at 0.9 V; (c) solution (b) after controlled potential electrolysis at 0.0 V.

The effect of changing the pH of the Amavadine + glutathione solution was also studied. A 0.167 : 1 Amavadine to glutathione solution has an initial pH of 2.9 and shows a typical mediator effect. When the pH was lowered to 2.0 by addition of HCl, the voltarmnogram shows only the Amavadme couple. Raising the pH back to 4 results in the reappearance of the mediator effect, although the oxidation wave has broadened significantly. This suggests that in order for it to act as a mediator for glutathione, Amavadine must be deprotonated *. Furthermore, increasing the pH * Each of the two ligands of Amavadine has a carboxylic group which does not bind to the metal. Apparently, it is these groups which must be deprotonated for the mediator effect to occur.

139

32-

l-

I

I

I

I

I

1 .o

0.5

0.0

-0.5

E vs.

SCEl

I -1.0

V

Fig. 5. Three continuous cyclic scans of an 0.125 : 1 Amavadhe KC1 at a glassy carbon electrode. Scan rate 200 mV/s.

to glutathione

solution in aqueous 0.1 M

slightly in this system tends to increase the size of the glutathione oxidation wave. A pH of about 4 was found to be optimal. Beyond this point, the oxidation wave broadened and became distorted. Amavadine is also an effective mediator for other biological thiols. A ratio study of the oxidation of cysteine produced voltammograms analogous to those for glutathione. As with glutathione, a voltammogram of cysteine alone in 0.1 M KC1 at the glassy carbon electrode is flat. Upon additions of Amavadine the positive scan produces a large oxidation wave near 0.62 V for the 0.250 : 1 optimum mediator to substrate ratio. This peak gradually shifts negatively as more mediator is added and the mediator couple appears. Similarly, cysteine methyl ester exhibits no redox behavior when alone in aqueous solution. Yet, adding Amavadine produces an irreversible oxidation wave at 0.54 V. Comparable results were also obtained using penicillamine whose oxidation peak appeared at 0.62 V. In each of these examples, the optimum mediator to substrate ratio was 0.250 : 1. Two ruthenium complexes were used as mediator-titrants in non-aqueous solvents. Bis(bipyridyl)chloro(tri-n-butylphosphinm(II)hexafluorophosphate and bis(bipyridyl)pyr~olylpyr~oleruthenium(II)hexafluorophosphate both

140

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I

1.0

0.5

0.0

I

1 -0.5

E vs SCE/V

Fig. 6. Cyclic voltammograms in 0.1 M TEAP+ DMSO at a glassy carbon electrode. Scan rate 200 mV/s. (a) 6.0 mM glutathione; and additions of ([(bpy)zRu(tri-n-Bu)P]Cl)PF~ to solution (a) giving ratios of ruthenium to glutathione of (b) 0.167 : 1 and (c) 0.500 : 1.

reversible couples in the positive potential range in 0.1 M TEAP + DMSO solution at a platinum electrode. The respective oxidation peak potentials for these species are 0.90 and 0.75 V which agree with previous reports [21,22]. A 6 mM solution of glutathione in 0.1 M TEL4P + DMSO showed no distinct oxidation wave (Fig. 6a). Additions of {[(bpy)2Ru(tri-n-Bu)P]Cl}PF, produced a distinct oxidation wave at about 0.90 V (Fig. 6b), while further additions produced a voltammogram resembling the mediator couple (Fig. 6~). Similar results were obtained when [(bpy),Ru(pz)(pzH)]PF, was added to glutathione in 0.1 M TEAP + DMSO. The oxidation wave for glutathione was found at 0.80 V. The first ruthenium mediator, {[(bpy)2Ru(tri-n-Bu)P]Cl}PF,, is also useful for observing cysteine in DMSO. As in water, cysteine alone in DMSO shows no oxidation peak. When the ruthenium complex is added, the cysteine oxidation appears at 0.80 V. Although a reduction peak for the ruthenium compound does appear, the mediator effect is clear, due to the significantly larger oxidation peak for cysteine. The second ruthenium complex, [(bpy),Ru(pz)(pzH)]PF,, is an effective mediator for both cysteine (0.74 V) and cysteine methyl ester (0.70 V). In these cases the thiol exhibit

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oxidation substrate peaks are broad, and no reduction peak for the oxidized mediator appears. (Penicillamine exhibited an oxidation peak in DMSO without a mediator present. Further studies of this system will be reported later.) CONCLUSION

Table 2 summarizes the oxidation potentials (E,,) for the various substrates observed using the three mediators. In a mediator system it is the oxidation or reduction of the mediator which is observed directly, while a homogeneous electron transfer occurs between the mediator and substrate in solution [23]. Thus, the oxidation waves recorded here are due directly to the vanadium or ruthenium mediators, but the peak currents and shapes are affected by the substrates. Specifically, while the potential at the foot of each oxidation wave is near that of the mediator alone, the peak potentials vary with mediator to substrate ratio. When the relative concentration of mediator is low, the peak potentials shift positively, and the peaks broaden. When the mediator concentration becomes high (1: 1 mole ratio) the peak potentials shift back negatively and approach those of the mediators alone. The peak currents are related to the concentration of substrate, but not linearly. This is because a significant amount of substrate is consumed during a cyclic scan (Fig. 5). On the basis of the known electrochemistry of Amavadine [15] and the data presented above, this system can be described as a “catalytic reaction with charge transfer”, case VII, discussed by Nicholson and Sham [24]. Their equations cannot be applied rigorously though, because glutathione is not in high enough concentration (relative to Amavadine) to be considered constant. Once again this is demonstrated by Fig. 5 where the Amadavine couple becomes evident as glutathione is consumed. Recently, Halbert and Baldwin [25] developed a carbon paste electrode modified with cobalt phthalocyanine which catalyzed the oxidation of sulfhydryl species including glutathione, cysteine, and penicillamine. As in our work, they reported

TABLE Oxidation Mediator

2 potentials (E,,/V

for biological

thiols using mediators

vs. SCE)

Amavadine (0.47) { [(bpy) zRu(tri-n-Bu)P]Cl)PF, Kbpy) 2 R~W(PZH)IPF,

(0.75)

Substrate

(0.90)

E&V

vs. SCE

Glutathione

Cysteine

Cysteine methyl ester

Penicillamine

0.7 a 0.90 b 0.80 b

0.62 0.80 b 0.74 b

0.54

0.62

a Values range from 0.54 to 0.82 V depending b Values obtained in DMSO.

on the mediator

0.70 b

to substrate

ratio.

142

that the voltammograms of these thiols measured with unmodified carbon paste electrodes showed no oxidation waves. However, using the modified electrode, these substrates produced large irreversible oxidation waves at about 0.80 V vs. Ag/AgCl. Since these results parallel our findings using vanadium and ruthenium solution mediators, we believe that the redox chemistry is basically the same in these two cases. That is, the thiol oxidations occur at the potential of the mediator or modified electrode and thus can be monitored readily. By comparing the oxidation potentials for glutathione in Table 2, it can be seen that the applied potential at which glutathione is oxidized can be adjusted by the choice of mediator. In summary, we have developed mediators to enable us to observe biological thiols electrochemically. As we proceed to study vanadium model complexes for the inhibition of the sodium pump we will be able to monitor the presence of these species. As in Halbert and Baldwin’s work [25], these mediators could also be the basis of a voltammetric detection system. ACKNOWLEDGEMENTS

We wish to thank Mohd. Asri Nawi and Alex Reiter for their help with some preliminary experiments. Funding for this project was provided by the National Institutes of Health under Grant No. l-R15-HL37252-01, the Faculty Research Committee of Miami University, and Sigma Xi, The Scientific Research Society. REFERENCES 1 LG. Macara, K. Kustin and L.C. Cantley, Jr., Biochem. Biophys. Acta, 629 (1980) 95. 2 L.C. Cantley, Jr., L. Josephson, R. Warner, M. Yanagisawa, C. Lechene and G. Guidotti, J. Biol. Chem., 252 (1977) 7421. 3 L.C. Cantley, Jr., J.H. Ferguson and K. Kustin, J. Am. Chem. Sot., 100 (1978) 5210. 4 L.C. Cantley, Jr. and P. Aisen, J. Biol. Chem., 254 (1979) 1781. 5 I.M. Kolthoff and C. Barnum, J. Am. Chem. Sot., 62 (1940) 3061. 6 W. Stricks and I.M. Kolthoff, J. Am. Chem. Sot., 74 (1952) 4646. 7 D.G. Davis and E. Bianco, J. Electroanal Chem., 12 (1966) 254. 8 J. Koryta and J. Pradac, J. Electroanal. Chem., 17 (1968) 185. 9 P.C. Jocelyn, Biochemistry of the SH Group, Academic Press, New York, 1972, p. 55. 10 J. Rost and S. Rapoport, Nature (London), 201 (1964) 185. 11 E.M. Scott, I.W. Duncan and V. Ekstrand, J. Biol. Chem., 238 (1963) 3928. 12 P.C. Jocelyn, Eur. J. B&hem., 2 (1967) 327. 13 G. Gorin and G. Doughty, Arch. B&hem. Biophys., 126 (1968) 547. 14 M.A. Nawi and T.L. Riechel, Inorg. Chim. Acta, 93 (1984) 131. 15 M.A. Nawi and T.L. Riechel, Inorg. Chim. Acta, 136 (1987) 33. 16 K.A. Rubinson and H.B. Mark, Jr., Anal. Chem., 54 (1982) 1204. 17 R. Szentrimay, P. Yeh and T. Kuwana in D.T. Sawyer (Ed.), Electrochemical Studies of Biological Systems, ACS Symposium Series, No. 38, American Chemical Society, Washington, DC. 1977, p. 143. 18 M.L. Meckstroth, B.J. Norris and W.R. Heineman, Bioelectrochem. Bioenerg., 8 (1981) 63. 19 J.M. Johnson, H.B. HaIsalI and W.R. Heineman, Anal. B&hem., 133 (1983) 186. 20 T.L. Riechel and D.T. Sawyer, Inorg. Chem., 14 (1975) 1869.

143 21 22 23 24 25

B.P. Sullivan, D.J. Salmon and T.J. Meyer, Inorg. Chem., 17 (1978) 3334. B.P. Sullivan, D.J. Salmon, T.J. Meyer and J. Peedin, Inorg. Chem., 18 (1979) 3369. R. Szentrimay, P. Yeh and T. Kuwana in ref. 17, Ch. 9. RS. Nicholson and I. Shain, Anal. Chem., 36 (1964) 706. M.K. Halbert and RP. Baldwin, Anal. Chem., 57 (1985) 591.