The electrochemistry of l -cystine and l -cysteine

Journal of Electroanalytical Chemistry, 375 (1994) 1-15

Review

The electrochemistry

of L-cystine and L-cysteine

Part 1: Thermodynamic

and kinetic studies

T.R. Ralph Johnson Matthey Technology Centre, Blount’s Court, Sonning Common, Reading RG4 9NH (UK)

M.L. Hitchman Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow Gl IXL (UK)

J.P. Millington EATechnology, Capenhurst,

Cheshire CHl 6ES (UK)

F.C. Walsh * Applied Electrochemistry Group, Chemistry Department, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT (UK)

(Received 23 November 1993; in revised form 22 February 1994)

Abstract Thermodynamic and kinetic aspects of the electrochemistry of mercury and a range of solid electrodes are considered. In the solid electrodes, reactant adsorption is a complicating factor. At transport of reactant can become an important rate-determining the electrosynthesis of L-cysteine are highlighted.

1. Introduction Electrochemical studies of r_-cystine and L-cysteine have been performed principally in two areas, namely investigation of the electrosynthesis of L-cysteine by the electroreduction of r_-cystine, which is the major industrial route to the thiol, and electroanalysis for these biologically important compounds. Many of the electroanalytical studies are linked with the use of the L-cystine/L-cysteine system as a model for the role of disulphide linkages and thiol groups in, for example, proteins in a variety of biological media. This review is aimed principally at the electrosynthesis of r_-cysteine and is divided into two complementary parts. The first

l

To whom correspondence

0022-0728/94/$7.00 SSDI 0022-0728(94)03407-T

should be addressed.

L-cystine reduction and L-cysteine oxidation are reviewed. Both former case, cysteinate complex formation occurs; in the case of both types of electrode, the kinetics of electron transfer and mass step. The implications of thermodynamic and kinetic studies on

part considers fundamental studies of L-cystine and L-cysteine, and spans the electroanalytical and the electrokinetic literature, while the second part highlights the practical electrosynthesis of L-cysteine in both laboratory- and pilot-scale cells.

2. Chemical background Cystine and cysteine are sulphur-containing a-amino acids which are members of a group of only about 20 amino acids to be found commonly in natural proteins, all exclusively as the L-isomer. Two other stereoisomers are possible for cystine and one for cysteine as shown in Fig. 1. In aqueous solutions, ionization of the amino acids depends upon pH, as shown in Fig. 2. At the isoelectric 0 1994 - Elsevier Science S.A. All rights reserved

2

T.R. Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part I

pH of the compounds, the molecules possess no net charge and are considered to have a zwitterion structure. Below or above this pH, the molecules are predominately cationic or anionic, respectively. Seven acidic dissociation constants have been identified from pH titration data [l], four for cystine and three for cysteine. In the case of cysteine, identification of pK, with the thiol group and pK, with the charged amino group is an oversimplification since the two groups are acids of comparable strength with the thiol group slightly stronger [2]. Cystine is anomalous among the common amino acids in showing a low solubility in all but strongly acidic or alkaline aqueous media (e.g. 0.46 mmol dmm3 in water at 25°C [l] compared with 0.77 mol dme3 in 2.0 mol dmd3 hydrochloric acid [31X Conversely, cysteine has a solubility of 2.30 mol dmP3 in water at 25°C [ 11. Consequently, in aqueous media, electrosynthesis of L-cysteine from r_-cystine is restricted to strongly acidic or alkaline solutions. In alkaline electrolytes, however, both amino acids are decomposed [21 to a variety of products and cysteine is rapidly oxidized [2] to cystine by air or oxygen, especially in the presence of a small amount of catalyst, such as iron(II1) chloride. Hydrochloric acid is often the favoured electrolyte for the electrosynthesis since solvent evaporation yields L-cysteine hydrochloride, which is a common starting material for many applications. It is also possible to

pKf’1.92

7”“”

H2N-Y-H HrC-S -s-c-I2 L-(-) CYSTINE

COOH

COOH H-kNHr

H-k-NH2

FOOH

H2N-Y-H a2

L-(-) CYSTEINE

700” HT-NH2 AH

D-(+) CYSDNE

0~

coo-

coo-

COOH

H&k-H

H&-#-H

H&+-H

H2&-S-S--cH

a2

2

JH Zwinerion pH = 5.02 + yoo-

coo-

HsNT-H

pK2=8.37

H+

OH 11

H&--H

COO’

H2C-S-s-&2

H&b-H

ZwirterionpH = 5.03

+32

S

pK3=10.70 H+ H2NJz

H&j:

11 IC OH

I

H&S-S-CH

2

700‘yoo HsNT-H s-2

Fig. 2. Ionization of rxystine and L-cysteine in aqueous solutions of different pH. The pK values quoted are at a temperature of 35°C [501.

&I

(352

H2C-S -S-A-I,

H+ 11

H2C-SCOO”

H&&-H

H2&-S-S-&

HrNT-H

HINT--H

COOH

COOH H&-&-H

produce the L-cysteine free base directly by electrolysis of the disulphide in ammonia electrolytes. Once again, solvent evaporation to dryness yields the product. In contrast to the case of the acid salt (and as suggested by the chemistry of the thiol), the free base is prone to decomposition during long periods of storage. 3. Thermodynamic

studies

D-(+) CYSTElNJ3

A reliable value of the standard formal electrode potential for the L-cystine/ L-cysteine redox couple, according to the equation 70”

F””

H2N-Y-H H-Y-W H2C-S-S-CL+

lneso-

CYSTINE

Fig. 1. Stereoisomers of cystine and cysteine.

RSSR + 2H++ 2e-o

2RSH

(1) is not available in the literature, although many workers [4-111 have attempted to determine the value. Table 1 lists reported values of the standard formal potential (E”) at pH 0 and pH 7 (the latter condition often being used to define the standard state for bio-

T.R. Ralph et al. / Electrochemiwy of L-cystineand L-cysteine:Part 1 TABLE 1. Standard formal electrode potentials of the r_-cystine/r.-cysteine Method

From the equilibrium constant for the reaction between the r_-cystine/L-cysteine couple and various dye redox couples From the equilibrium constant for RSSR + 2Hf- 0.33 + 2Fezfo 2RSH + 2Fe3+ For all measurements,

redox couple at 25°C

E” (vs. SHE)/V

Calculated from thermal data Potentiometric measurements at a mercury electrode

3

pH7

PHO

- 0.39 - 0.333 - 0.336

+ 0.02 + 0.080 + 0.077

- 0.222

+0.191

- 0.33

+ 0.08

pH range

Electrolyte

Not applicable 7.5-8.6 8.5-9.2

Not applicable Various buffers

4 5

Phosphate and borate buffers

10

Ammonia buffers

11

7.1-9.2 10.03-10.12

Ref.

[RSSR] = [RSH12.

chemical redox couples). The E” values were obtained by extrapolation of a linear plot of the equilibrium potential versus pH, which was assumed to have a slope of 0.0591 V over the entire pH range. Borsook et al. [41 used thermal data to calculate the standard Gibbs energy of reaction (1) and, by incorporating values for ionization constants, they evaluated equilibrium electrode potentials at intervals of one pH unit from pH 0 to 12. The calculations involved differences between large numbers, each uncertain to some degree, and a choice of slightly discordant ionization constants which the authors estimated could have produced an error of up to 43 mV in the equilibrium electrode potential (although the probable error was considered to be much smaller). Kinetic studies at a mercury electrode (Section 4.1) have indicated that E" values measured by Ghosh et al. [5] and Green 161are erroneous. L-cysteine reacts with the mercury electrode to form mercury cysteinate, as indicated later in reactions (12) and (13). Potential measurements at platinum or gold electrodes [7-91 are slow to equilibrate and do not give true values of the reversible potential for the couple. In addition, the equilibrium electrode potential does not respond to changes in L-cystine concentration. As discussed in Section 4.7, there is evidence of strong adsorption of both amino acids at these electrodes. A method free from the objections of the action of the amino acids on electrodes involves determination of an equilibrium constant for reaction between the L-cystine/ r_-cysteine redox couple and another couple of known standard electrode potential. Fruton and Clarke [lo] equilibrated the amino acids with various dyes of known redox potential and measured the dye concentrations spectrophotometrically. Tanaka et al. [ill claim to have established the equilibrium constant for reaction with the iron(III)/ion(II) couple. The technique does not appear to have been used to evaluate E" in hydrochloric acid solutions. Identification of

a suitable redox couple in hydrochloric acid media would be valuable, since it has been shown [3] that the extrapolated E” values in Table 1 are not applicable for this electrosynthesis medium with E” being more positive than +0.24 V (vs. SHE) at pH 0 in hydrochloric acid. 4. Kinetic studies 4.1. Mercury

Kolthoff and Barnum studied RSH oxidation [12] and RSSR reduction [131 as in the early 1940s. The electrochemistry of thiol oxidation and disulphide reduction is known to complicated by the adsorption of the amino acids at the electrode surface. Kolthoff and Barnum [121 suggested mercury(I) cysteinate (RSHg) as the product of L-cysteine oxidation: RSH + Hg + RSHg(,,,, + H+ + e -

(2)

The mercury(I) ion was postulated to be Hg+ rather than Hgl at the low concentrations (typically 1 X 10p2’ mol dme3) of mercury ions present. Polarograms showed a diffusion-controlled wave with a half-wave potential (E1,2) of -0.04 V (vs. SCE) in 0.1 mol dme3 hydrochloric acid and - 0.5 V (vs. SCE) in aqueous 0.1 mol dmv3 sodium hydroxide. The change of 38 mV per pH unit is unexpectedly low for electrode reaction (2). In buffer solutions of pH 2.0-9.0, at L-cystine concentrations above 0.2 mmol dmW3, rather than the diffusion-controlled wave a small limiting current was obtained which was almost independent of the amino acid concentration. This was attributed to mercurous cysteinate film formation, which Grubner [14] suggested inhibited further oxidation of r_-cysteine. Evidence for the film has been obtained by Kolthoff et al. [15], who found that a negative sweep from high positive potentials produced a small reduction wave at a potential immediately negative of that of the oxidation

T.R. Ra&h et al. / Electrochemistry of L-cystine and L-cysteine: Part 1

4

waves. They postulated that this reduction current was due to reduction of the film. From the charge passed, the film thickness was estimated to correspond to a monolayer of mercury(I) cysteinate. At potentials more positive than the limiting current region, a series of poorly reproducible anodic waves, with several breaks in the current typical of adsorption, were ascribed in later studies [15-181 to further diffusion-controlled oxidation of r_-cysteine, either after film breakdown or by reactant diffusing through the film [16-191. Kolthoff and Barnum [13] also tried to elucidate the mechanism of L-cystine reduction. They found a single, diffusion-controlled, irreversible wave on polarographic reduction in acid or alkaline solutions with an additional small prewave in buffer solutions of pH 3.0-7.0. The main wave was attributed to the reduction of L-cystine to L-cysteine:

cury(I1) cysteinate to r_-cystine of 6 x 10m4. The standard formal potential values on the left-hand side of this equation correspond to the conditions [RSSR] = [RSHI’ and [(RS),Hg] = [RSHl*, respectively, at pH 0. A number of workers [22-241 have essentially confirmed the findings of Kolthoff and co-workers [12,13,15,211. The stoichiometry of the mercury cysteinate complex remains uncertain; RSHg, (RS),Hg, and (RS),Hg have each been proposed. Miller and Teva [25] used cyclic voltammetry to investigate L-cystine reduction in nitric acid solutions at a hanging mercury drop electrode (HMDE). Two reduction peaks were found. A small, apparently reversible peak at about -0.25 V (vs. SCE), corresponding to the prewave found in polarography, was postulated to be due to the following series of reactions: RSSR + Hg w (RS),Hg,,,,,

(7)

RSSR f 2H++ 2e-+

(RS),Hg,,,,

+ Hg W 2RSHg,,,,,

(8)

w)2Hg(,&,

+

2RSH

(3)

Reduction commenced at - 0.1 V (vs. SCE) in 0.1 mol dmP3 hydrochloric acid (a polarographic maximum makes it difficult to quote a precise El,* value) and at a potential of -0.6 V (vs. SCE) in aqueous 0.1 mol dmP3 sodium hydroxide. The shift of 42 mV per pH unit is again low for electrode reaction (3). Kalousek et al. [20] and later Kolthoff et al. [21] examined the prewave in detail. Both groups of workers concluded that reaction of L-cystine with the electrode produced a mercury compound which was reduced at the prewave potentials. Kolthoff et al. [Zl] suggested that reaction of L-cystine with the mercury electrode surface produced mercuric cysteinate ((RS),Hg) as the rate-determining step (RDS) in the following reaction: RSSR + Hg + ( RS)2Hg

(4)

The cysteinate was reversibly reduced at the prewave: (RS),Hg

+ 2H++ 2e-o

2RSH + Hg

(5)

This was based upon the near independence of the limiting current on mercury head height, which indicated the prewave was kinetically controlled, and upon its proportionality to L-cystine concentration. From the E” value of the L-cystine/ r_-cysteine ( + 0.08 V vs. SHE; see Table 1 and refs. 5 and 11) and mercury(I1) cysteinate/L-cysteine (+ 0.175 V vs. SHE) redox couples 1211 and the nernstian relationship between the couples:

(6) reaction (4) was estimated to produce a ratio of mer-

RSHg,,,,

2H++ 2e-o

+ H++ e-o

2RSH + Hg

RSH + Hg

(9) (10)

Evidence was proposed for a slow, rate-determining surface reaction between r_-cystine and the electrode according to reactions (7) and (8) which preceded the electrode reactions (9) and (10). A second peak at ca. -0.7 V (vs. SCE) was ascribed to the irreversible, diffusion-controlled reduction of r_-cystine to r_-cysteine desribed by reaction (3). After the initial potential scan, at the highest r_-cystine concentration used, twin peaks were obtained for the reversible wave. These were attributed to the presence of both mercury(I) and mercury(I1) cysteinate on the electrode surface. Stankovich and Bard 1261clarified the electrochemistry of both amino acids principally in a buffer solution of pH 7.4. Cyclic voltammograms in solutions of L-cystine (Fig. 3) essentially agreed with those reported by Miller and Teva [25]. In the initial forward potential scan, two peaks were observed at potentials of approximately -0.5 and -0.9 V (vs. SCE). In agreement with the literature, the peak at -0.9 V (vs. SCE) was attributed to the reduction of dissolved L-cystine to L-cysteine, as shown by reaction (3). The first peak, however, was attributed to the reduction of adsorbed L-cystine: RSSR,,,,, + 2H++ 2ee-t

2RSH

rather than the reversible reduction of mercury teinate by the route proposed by Miller and Teva in reactions (7)-(10). Strong evidence for reaction (11) was provided constant peak area in voltammograms at r_-cystine centrations above 0.01 mmol dmm3, attributed

(11) cys1251 by a conto

T.R. Ralph et al. / Electrochemistry of L.-cystine and L-cysteine: Part 1

monolayer coverage of the electrode by L-cystine rather than mercury cysteinate. By the method of Kolthoff et al. [21] only 5 X 10m5 mm01 dme3 of mercury cysteinate was calculated to form from the spontaneous reaction between L-cystine and the electrode, a concentration too small to give monolayer coverage. Conversely, construction of a model of L-cystine and determination of the area occupied with the disulphide bond orientated on the electrode surface suggested that the charge passed corresponded to monolayer coverage by the disulphide. In the initial reverse potential scan, a peak at approximately -0.5 V (vs. SCE) was associated with the reversible oxidation of L-cysteine (formed in the forward scan) to mercury(I) cysteinate by reaction with the electrode material: 2RSH + 2Hg = (RS),Hg,,,,,, 2RSH + Hg = (RS),Hgo,

+ 2H++ 2e+ 2H++ 2e-

W,W

(12) (13)

The second potential scan showed a reversible cathodic peak corresponding to the reduction of the mercury cysteinate complexes to L-cysteine (see the dashed line in Fig. 3). The current spikes shown on the reversible wave appeared at L-cystine concentrations above 0.4 mm01 dmp3. These spikes were assigned to the formation of a tight mercury cysteinate film, formed after

UW)

E(vs.

5

SCE)/ v

(12,W Fig. 3. Cyclic voltammetry at an HMDE of N,-purged 0.46 mmol dmm3 L-cystine in a buffer solution of pH 7.4. Potential sweep rate 200 mV s-l [26]. The solid line shows the first and the dotted line shows the second potential scan.

(12,131 Fig. 4. Cyclic voltammetry at an HMDE in N,-purged 0.3 mmol dmA3 L-cysteine in a buffer solution of pH 7.4. Potential sweep rate 200 mV s-l [26].

monolayer coverage, via lateral interaction between adsorbed molecules. The spiked response clearly differentiated the cathodic peak current for reaction (11) from that corresponding to reactions (12) and (13). These peaks were confused in the earlier literature as the peak potentials for these two sets of reactions are within 10 mV. Cyclic voltammograms of solutions of L-cysteine (Fig. 4) showed the reversible wave corresponding to reactions (12) and (13), with the spiked response evident at L-cysteine concentrations above 0.3 mmol dme3. Stankovich and Bard [26] proposed an order of increasing strength of adsorption on mercury of weakly adsorbed L-cysteine, r_-cystine :and mercury cysteinate. This was based on cyclic voltammetry of a mixture of L-cystine and mercury cysteinate, with r_-cystine in large excess, which showed the spiked behaviour of mercury cysteinate reduction on the initial potential scan. These studies have been confirmed and extended by others [3,27,28]. Pena and Alarcon [27] examined the amino acids in buffered media of pH 4.6-9.3. They found that electrode reactions (3) and (ll)-(13) occurred in all media although at pH 9.3, mercury cysteinate reduction (reactions (12) and (13)) was diffusion controlled since the complex(es) were formed in solution. Bianco and Haladjian 1281modified the surface of an HMDE

6

T.R. Ra&h et al. / Electrochemistry of L-cystine and L-cysteine: Part 1

with surface-active agents (i.e., Triton X-100, cytochrome c and cytochrome c,). In cyclic voltammograms the peak for reduction of adsorbed L-cystine was diminished. It was argued that L-cystine adsorption occurred either through holes in, or at preferential sites on the film of surface active agent. In addition each of the surface active agents increased the barrier for electron transfer to dissolved L-cystine with the main diffusion-controlled, irreversible wave for reduction of r_-cystine to r_-cysteine being shifted to more negative potentials. Clearly, the electrode kinetics of the synthesis reaction are strongly influenced by the double-layer structure. More recently, studies in our laboratories [3] have examined conditions more closely resembling those used in the electrosynthesis. In hydrochloric acid media, peaks in cyclic voltammograms, corresponding to electrode reactions (3) and (ll)-(131, could be identified. For the main reduction wave corresponding to reaction (31, steady-state measurements at a static mercury dropping electrode (SMDE) showed a diffusionlimited current starting at approximately -0.5 V (vs. SCE). With disulphide concentrations between 0.4 and 4 mmol dmw3, which are much higher than those investigated at the SMDE, a study at a mercury-plated copper rotating disc electrode (RDE) showed that the reduction wave was shifted to progressively more negative potentials as the reactant concentration was increased. This can be attributed to strong adsorption of r_-cystine hydrochloride at the electrode surface through which the reduction reaction (3) has to occur. From a cyclic voltammetric peak for reduction of the more weakly adsorbed r_-cystine hydrochloride to L-cysteine hydrochloride at a mercury-plated copper RDE (cf. Fig. 3, reaction (11)) the maximum surface coverage of L-cystine hydrochloride can be calculated as 3.1 X lo-’ mol m-*. This value is achieved at a bulk reactant concentration of 4 mmol dmP3. At bulk disulphide concentrations above this level, there is no further shift in the position of the convective diffusion-controlled wave. At these higher reactant concentrations, the wave is shifted to such negative potentials that the limiting current is hidden by simultaneous hydrogen evolution at the RDE (cf. Fig. 51, in contrast to the well defined plateau observed at the SMDE [3]. In the Tafel region, slopes of - 180 mV per decade can be measured for the reduction of L-cystine hydrochloride [3]. This high value can be attributed to adsorption of the disulphide rather than to an effect caused by simultaneous hydrogen evolution. Controlled-potential coulometry at -0.95 V (vs. SCE), corresponding to the mixed kinetic-mass transportcontrolled region of the current vs. potential curve, confirmed that L-cystine hydrochloride was reduced to

j / A me2 -160

-120

-80

-40

0 -0.6

-0.8

-1.0

-1.2

-1.4

E(vs. SCE)/ V Fig. 5. Steady-state voltammograms at a mercury-plated copper RDE in deoxygenated 10 mm01 dmm3 L-cystine hydrochloride and 0.1 mol dmm3 HCI at 25°C [3]. Rotation rates: (1) 5; (2) 10; (3) 20; (4) 30; (5) 40; (6) 50 Hz.

L-cysteine hydrochloride with 100% current efficiency in the Tafel region. Further, electrochemical reaction orders of + 1 for the disulphide (at concentrations above 4 mmol dmM3) and + 1 for protons between pH 1.1 and 2.5 were obtained and the mechanism below can be proposed, with the first electron transfer to the disulphide molecule being the RDS and proton transfer either preceding or occurring at the RDS: RSSR + H++ e-+ RS + H++ e-o

RSH + RS

RSH

( 14a) (14b)

4.2. Lead The kinetic studies of L-cystine hydrochloride reduction in our laboratories [3] have been extended to a lead RDE. In order to measure the disulphide reduction currents, the RDE was maintained at a potential more negative than -0.7 V (vs. SCE). At more positive potentials, lead chloride formed on the RDE surface as a result of the corrosion of the electrode and the reduction current for the film completely masked that due to r_-cystine hydrochloride reduction. The reduction was found to be electrochemically irreversible and, as at mercury, a convective diffusioncontrolled limiting current for disulphide reduction via

T.R. Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part I

j I A rn-* -160

I

7

separation for the two reactions (ca. 1.0 V> showed the L-cystine reduction reaction (1) was electrochemically irreversible at these electrodes. Both reactions were diffusion controlled. From the range of transition metal TSPs, only at a Co-TSP/OPG rotating disc electrode was a diffusioncontrolled limiting current obtained for L-cysteine oxidation (Fig. 8). Electrochemical reaction orders of + 1 for L-cysteine and - 1 for protons below pH 8.5 and 0 for protons above this pH were measured. The authors noted that pH 8.5 is close to the pK, of 8.37 [l] for

-80

-40

I/mA (1)

0 -0.8

-1.0

-1.2

-1.4

-1.6

-0.4 E(vs.SCE)/ V Fig. 6. Steady-state voltammograms at a lead RDE in deoxygenated 10 mmol dmm3 L-cystine hydrochloride and 0.1 mol dmm3 HCl at 25°C. Rotation rates: (1) 5; (2) 10; (3) 20; (4) 30; (5) 40; (6) 50 Hz.

-0.8

-1-4

reaction (3) was masked by simultaneous hydrogen evolution (see Fig. 6). There was no direct voltammetric evidence of adsorption of L-cystine hydrochloride at lead, although there was indirect support from Tafel slopes of - 180 mV per decade which are comparable to those obtained with mercury. Constant-potential coulometry at - 1.1 V (vs. SCE) confirmed that Lcystine hydrochloride was reduced to r_-cysteine hydrochloride with essentially 100% current efficiency as long as the current was below the mass transport-controlled value. From electrochemical reaction orders of + 1 for r_-cystine hydrochloride and + 1 for protons, between pH 1.1 and 2.5, the reduction mechanism appears to be similar to mechanism (14) for mercury. 4.3. Transition metal macrocycles Zagal and co-workers [29-321 investigated L-cystine reduction and r_-cysteine oxidation in a variety of electrolytes at various transition metal tetrasulphophthalocyanines (Co-TSP, Fe-TSP, Mn-TSP, Cr-TSP, Ni-TSP, Cu-TSP and Zn-TSP) adsorbed on ordinary pyrolytic graphite (OPG). Cyclic voltammetry (Fig. 7) indicated that L-cystine reduced to L-cysteine in the negative potential scan was regenerated by L-cysteine oxidation in the reverse anodic scan. The large peak potential

E(vs. SCE)/ V

(a> I/mA

-l!z+&&++4 E(vs.SCE)/

V

@I Fig. 7. Cyclic voltammetry of the L-cystine/L-cysteine redox couple at (a) Fe-TSP/OPG and (b) Co-TSP/OPG electrodes. Potential sweep rate 200 mV s-l. Background electrolyte 0.2 mol dme3 NaOH containing 0.5 mmol dm-’ L-cystine [30,31]. (A) Fe(I)TSP 0 Fe(II)TSP + e-. (B) Fe(II)TSP 0 Fe(III)TSP + e-. (C) Co(I)TSP 0 Co(II)TSP + e-.

8

T.R. Ralph et al. / Electrochem&y

I/mA

Ni-TS I

/

l’i’

Cu-TS

Co-TSP

-0.4

0

-0.2

0.2

0.4

E(vs. SCE)/ V Fig. 8. Voltammograms in N,-purged 10 mmol mm3 L-cysteine chlorhydrate in aqueous 0.2 mol dme3 Na,C03 and 0.6 mol dm-3 Na,SO, at transition metal TSP/OPG rotating disc electrodes. Electrode rotation rate 33 Hz. Potential sweep rate 2 mV s-i. Temperature 25°C [30,31].

of L-cystine and L-cysteine: Part I

electrolyses. High Tafel slopes of - 240 mV per decade were reported. Zagal and co-workers concluded that both r_-cystine reduction and L-cysteine oxidation could only proceed at reasonable rates if the reactant molecules were adsorbed at the electrode surface. Adsorption at transition metal TSP was believed to be closely linked to the ability of the central metal ion to bind extra-planar ligands. Preliminary studies with non-sulphur-containing amino acids indicated that they were much less reactive. This suggested adsorption occurred between the metal centre and the sulphur atoms of r_-cystine and L-cysteine. For L-cystine, adsorption through only a single sulphur atom was proposed. The authors argued that, if the macrocylic complexes were adsorbed with the ring parallel to the OPG surface [33], then the metal centres in the TSP would be too far apart to adsorb L-cystine via both sulphur atoms. In the absence of reliable heats of adsorption for the amino acids on transition metal TSPs, the number of d electrons on the metal was used to correlate with the electrocatalytic activity for the reactions. Calculations indicated that the energy of the highest occupied molecular orbital of the transition metal TSPs decreased almost linearly as the number of d electrons increased. At electrode potentials of -0.1 and - 1.05

I/mA

L-cysteine and the following mechanism involving sulphide radical formation was proposed: RSH=RS-+H+ RS-+

RS’+ e-

2RS’ -+ RSSR

( 15a) (RDS)

( 15b)

( 15c) Tafel slopes of 140 mV per decade in alkali and 120 mV per decade in acid suggested that the first electron transfer either preceded or occurred at the RDS. Constant-potential electrolysis with paper chromatographic analysis of the electrolyte confirmed L-cystine as the major oxidation product in acidic media, with trace quantities of cysteic acid the only other product. In alkaline solutions many unknown products were obtained. For L-cystine reduction, a sloping plateau at CoTSP/OPG was the only response which resembled a limiting current from the range of RDEs (Fig. 9). At Co-TSP/OPG detailed kinetic parameters for the reaction were not quoted, probably because of interference from hydrogen evolution. Certainly, the catalysts were gradually removed from the OPG surface by copious hydrogen evolution during constant-potential

-2.4 2 -1.6

-0.8

-0.6

-1.0

-1.4

-1.8

E(vs.SCEl/ V Fig. 9. Voltammograms for N,-purged 0.5 mmol dm-3 t_-cystine in aqueous 0.2 mol dmm3 Na,CO, at transition metal TSP/OPG rotating disc electrodes. Electrode rotation rate 33 Hz. Potential sweep rate 1 mV s-l. Temperature 25°C [30,31].

T.R. Ralph et al. / Electrochemistry of xystine

-

Mn(II)

1

I

1

I

I

I

I

1

3

4

5

6

7

8

9

10

d electrons

(a>

1.6 1.2

.,

0.8 0.4 0

-\-

-’

Cu(I1) II I 8 10

&I)

I 4

6 d electrons

W Fig. 10. Volcano plots for (a) the reduction of L-cystine and (b) the oxidation of L-cysteine 1321.

V (vs. SCE), the currents for L-cysteine oxidation and L-cystine reduction, respectively, produced volcano plots when correlated with the most likely metal oxidation state(s) in the TSPs (see Fig. 10). No attempt was made to account for the different number of d electrons required to produce the maximum rate of L-cystine reduction (i.e. five d electrons; Mn(I1) TSP) and L-cysteine oxidation (i.e. seven d electrons; Co(B) TSP). The coulombic interaction between the sulphur atoms in the amino acids and the metal in the TSP perhaps helps to explain the effect. For r_-cysteine oxidation occurring by reaction mechanism (151, the volcano plot suggests RS’, rather than RS-, is the adsorbed species. If RS- was adsorbed, then on a coulombic basis the metal-sulphur bond strength would have decreased with increasing number of d electrons, and a volcano plot would not have

and L-cysteine: Part 1

9

resulted. With RS’, however, the metal-sulphur bond strength increases with increased d electrons. An optimum bond strength was obtained with seven d electrons, with the bond being weaker being below this value and too strong above it. For L-cystine reduction it is unlikely the disulphide bond dissociated before adsorption. Since the bond has eight delocalized electrons, on a coulombic basis it might be expected, as observed, that the optimum metal-sulphur bond strength would be obtained with fewer d electrons than in the case of the RS’ adsorption. Above the optimum value of five d electrons the metal-sulphur bond strength is weaker owing to coulombic forces. Of course, this explanation ignores the possible influence of steric effects. Wang and co-workers [34-361 examined heat-treated cobalt(B) tetramethoxyphenylporphyrin (Co-TMPP) adsorbed on graphite. As in the case of transition metal TSPs, cyclic voltammetry indicated that reaction (1) was electrochemically irreversible, with the oxidation and reduction reactions being diffusion controlled. Both electrode reactions, at a given pH, occurred at similar potentials on Co-TMPP and transition metal TSP. Adsorption of the amino acids at the cobalt(B) metal centre was advanced. In support of disulphide adsorption at the metal centre, Wang and Pang [37] noted a similar electrocatalytic effect for L-cystine reduction at a glassy carbon electrode using water-soluble cobalt(B) tetrakis(4-trimethylammoniumphenyl)porphyrin(Co-TTAPP) or cobalt(B) tetrakis(4-sulphophenyljporphyrin (Co-TSPP) dissolved in sulphuric acid electrolyte. No such homogeneous effect was observed, however, with H,‘TTAPP. Cobalt(B) complexing L-cystine in the N, internal ring of the porphyrin was proposed. The similar performance of Co-ITAPP and Co-TSPP was highlighted as indicative of the lack of an electrocatalytic effect from substituents on the porphyrin ring. At a Co-TMPP/ graphite RDE, mechanism (15) was proposed for r_-cysteine oxidation [36]. Tafel slopes and electrochemical reaction orders were identical with those reported for the oxidation at Co-TSP/OPG [29321. Constant-potential electrolysis in acidic media produced L-cystine with current efficiencies in excess of 90%. At a Co-TMPP/graphite RDE a well defined, diffusion-controlled, limiting current was not obtained for L-cystine reduction in electrolytes below pH 12 [34-351. Sloping plateaus, attributed to simultaneous hydrogen evolution, were recorded. At more positive potentials, Tafel slopes of 120 mV per decade were measured in all media and electrochemical reaction orders of + 1 for L-cystine and + 1 for protons below pH 4 and 0 above pH 9 were recorded. Reaction mechanism (14)

10

T.R. Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part I

was proposed at pH < 4 whereas the following mechanism might apply at pH > 9: RSSR + e--+ RS+

RS’

(RDS)

( I6a)

RS’ + e--+ RS-

( I6b) At a large Co-TMPP/ graphite electrode, constantcurrent electrolysis of r_-cystine in 2.0 mol dmw3 hydrochloric acid at 200 A m-* yielded exclusively L-cysteine. The current efficiency was 60% with current losses ascribed solely to hydrogen evolution. 4.4. Polymer-modified electrodes Arai et al. [38] examined r_-cystine reduction at glassy carbon electrodes (GCE) coated with conductive polymers containing fixed metal (Pt, Pd, Cu, Ag and Hg) thiolate sites. With the exception of mercury, the metal thiolate films were electrocatalytic for r_-cystine reduction, in comparison with the corresponding bulk metal electrodes. The films also appeared to hinder hydrogen evolution, which is the major secondary reaction. In steady-state voltammograms (see Fig. ll), Lcystine reduction waves were evident at the metal thiolate electrodes (in contrast to the predominant hydrogen evolution shown at metal surfaces), although limiting currents for the disulphide reduction were masked by hydrogen evolution. Constant-potential coulometry at - 0.85 V (vs. SCE) in a buffer solution of pH 7.0 confirmed that reaction (1) proceeded with a current efficiency of 90-95% at the copper thiolate electrode and at 97-99% at the other polymer-modified electrodes. In contrast, the platinum and palla-

dium wire electrodes gave rise to current efficiencies of less than 3% and silver electrodes produced a value of only 10%. 4.5. Carbon In contrast to the high electrocatalytic activity at macrocyclic and polymer-modified carbon electrodes, bare carbon surfaces show very poor activity. Indeed, r_-cystine reduction did not occur within the working potential range of vitreous carbon [39,40] or wax-impregnated spectroscopic graphite (WISGE) [41] electrodes. Both L-cystine and L-cysteine were, however, oxidized. At a vitreous carbon RDE, r_-cystine oxidation was electrochemically irreversible and diffusion controlled. Comparison of the limiting current with that obtained for hexacyanoferrate(I1) oxidation indicated that five electrons were transferred. Oxidation to cysteic acid was proposed: RSSR + 3H,O + RSO,H + 5H++ 5e-

(17) From Tafel slopes of 120 mV per decade the authors concluded the first electron transfer was the RDS. Since the El,* value of approximately + 1.4 V (vs. SCE) was constant from pH 1.7 to 6.2 proton involvement, either at, or preceding the RDS was precluded. The oxidation of L-cysteine produced an ill-defined current at potentials more positive than +0.2 V (vs. SCE). From ac voltammetry it was deduced that a little r_-cysteine was adsorbed on the electrode. r_-cystine was not adsorbed. In cyclic voltammograms at a WISGE, L-cysteine and r_-cystine were oxidized at peak potentials of about + 1.1 and + 1.3 V (vs. SCE), respectively, in pH 4.0 buffer solution. Both oxidations were electrochemically irreversible and diffusion controlled. 4.6. Silver

Watanabe and Maeda 1421reported quasi-reversible reduction of r_-cystine to L-cysteine according to reaction (18) on the surface of a silver electrode with the disulphide adsorbed form aqueous 2.0 mol dme3 hydrochloric acid: RSSR,,,,, + 2e-o

E(vs. AgfAgCI I/ V Fig. 11. Voltammograms for L-cystine reduction in a N,-purged, pH 7 Britton-Robinson buffer solution on metal thiolate polymer-modified glassy carbon electrodes (solid curves) and on bare metal electrodes (broken curves). Potential sweep rate 2 mV s-l [38]. (1) Pt; (2) Pd; (3) Ag; (4) Cu; (5) Hg; (6) GCE.

2RS;,,,

(18)

The redox reaction occurred in the potential range between -0.35 and +0.25 V (vs. SCE) and was followed by surface-enhanced Raman spectroscopy (SERS). The change in the intensity of the S-S stretching peak at 502 cm-’ was followed during a potential sweep experiment and the absence of an S-H stretching peak at 2565 cm-’ suggested that r_-cysteine was adsorbed as RS’. Voltammetric data were not presented by these authors and it is likely that the redox

T.R. Ralph et al. / Electrochemistry

(a)

of L-cystine and L-cysteine: Part I

of papers [39,43-491 on the of both amino acids. Davis and Bianco [43] voltammogram for L-cysteine sulphuric acid at a platinum the oxidation was considered the formation of a platinum reaction of the type

11

electrochemical

oxidation

reported a peak-shaped oxidation in 1 mol dme3 RDE. At high potentials, to be inhibited owing to oxide film according to a

Pt + H,O + PtO + 2H++ 2e(E”=

(Oxldatm

-x-+ (b)

Fig. 12. Surface adsorption of L-cystine and L-cysteine at sjlver. The atoms are scaled referring to the Ag-Ag distance (2.88 A) for the silver (III) plane [42]. (a) Favoured orientation; (b) hindered orientation.

reaction could not be separated from hydrogen evolution in the 2.0 mol dmm3 background electrolyte (see, for example, Fig. 11). When L-cysteine was adsorbed on the silver electrode, reaction (18) did not occur. This was rationalized in terms of the model for the adsorbed layer illustrated in Fig. 12. In view of a fairly intense S-S stretching peak obtained, it was believed that the disulphide bond was tilted to some extent against the surface (Fig. 12(a)). On electrochemical reduction it was converted into two r_-cysteine molecules which (because of strong Ag-S bonding) were trapped on neighbouring silver atoms, in spite of some steric hindrance between the amino acid molecules. Anodic oxidation readily formed the disulphide bond between the closely sitting sulphur atoms. Conversely, for a silver electrode in contact with an r_-cysteine solution the steric hindrance was believed to operate first, at the time of adsorption, and the adjacent siting of two r_-cysteine molecules was unfavourable (Fig. 12(b)). The strong Ag-S bonding prevented surface diffusion, and Lcystine formation by electro-oxidation was strongly hampered. Some indirect evidence for the model was provided by the quasi-reversible redox reaction of the ethanethiol/ diethyl disulphide couple, which was detected by SERS with either member of the couple adsorbed on the electrode. It was argued that there was little steric hindrance from the small ethyl group attached to the sulphur atom. 4.7. Platinum and Gold There appears to be no published work on t_-cystine reduction at these electrodes, but there are a number

+0.88Vvs.SHEatpHO)

(19)

Constant-potential electrolysis at +0.9 V (vs. SCE) with infrared spectroscopic analysis of the electrolyte, identified L-cystine according to the following reaction: RSH + RS’ + H++ e-

(2Oa)

2RS’ -+ RSSR

(2Ob)

as the major oxidation product. During electrolysis the current rapidly fell to about 3% of the initial value owing to formation of the oxide film. Pradac and co-workers [44,451 presented cyclic voltammograms for solutions of r_-cystine (Fig. 13) and L-cysteine (Fig. 14) in 0.5 mol dme3 sulphuric acid at platinum and gold electrodes. r_-Cystine completely suppressed the hydrogen adsorption peak(s) at both metals. This suggested the disulphide was adsorbed on the electrode surface. Radiometric analysis, with 35S, confirmed strong adsorption, according to reaction (21), with a smaller quantity of the amino acid being adsorbed at gold. (21)

RSSR,,,,, * 2RS;,,,,

At platinum electrodes (Fig. 13(a)), L-cystine oxidation to cysteic acid via reaction (22) and an adsorbed RSO, radical by reaction (23) was proposed, together with surface oxide formation, from a potential of approximately +OS V (vs. SCE) into the oxygen evolution region. A cathodic current, between +0.45 and 0 V (vs. SCE), was attributed to reduction of the adsorbed RSO, radical. 2RS;ads, + 2RS;ads,

+

3H,O + RSO;+

6H++ 5e-

(22)

2H,O + RSO; + 4H++ 4e-

(23)

At gold electrodes (Fig. 13(b)), r_-cystine oxidation to cysteic acid via reaction (22) and simultaneous surface oxide formation occurred from a potential of +0.65 V (vs. SCE) into the oxygen evolution region. There was no evidence for production of the adsorbed RSO, radical. L-Cysteine was oxidized to L-cystine at both platinum and gold electrodes. At platinum electrodes (Fig. 14(a)), oxidation occurred at a peak potential of +0.8

12

T.R. Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part 1

j / A m-*

gold electrodes (Fig. 14(b)), oxidation of r_-cysteine via reaction (24) occurred at a peak potential of + 1.0 V (vs. SCE), with the r_-cystine produced oxidized via reactions (21) and (22) at a peak potential of + 1.2 V (vs. SCE). At both noble metals, Pradac and co-workers [44,45] reported that currents due to r_-cystine oxidation were independent of electrolyte stirring, whereas those for L-cysteine oxidation were enhanced by faster stirring

A

j/Am-* 0.6 -

(22,23)

(a>

0.4 -

j / A me2

0.2 -0.2L

(a> jl

A me2 0.4 (24)

(b) Fig. 13. Cyclic voltammetry in N,-purged 5.0 mmol dmW3 L-cystine in 0.5 mol dmm3 H,SO, at (a) platinum and (b) gold electrodes 144,451. (A) surface oxide formation; (B) surface oxide reduction; (C) hydrogen adsorption; (D) desorption of hydrogen. The dashed line shows voltammetry in the background electrolyte.

0.2 -

0

0.26

... ... 1 ...... .. .. ..... 0.76 :: ;'" -0.24

V (vs. SCE) as described sequence:

by the following reaction

RSH = RSH(,,,,

(24a)

RSH,,,,, + 2RS&,,, + H++ e-

(24b)

2RS;xl,, = RSSR

(24c)

The r_-cystine produced was oxidized via reactions (21)-(23) at a peak potential of + 1.0 V (vs. SCE). At

hi

1.26

E(vs. SCE)/ V

-0.2

(b) Fig. 14. Cyclic voltammetry of N,-purged 0.5 nun01 dmm3 L-cysteine in 0.5 mol dmm3 H,SO, at (a) platinum and (b) gold electrodes [44,45]. Other details as in Fig. 13.

T.R Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part 1

~~-26

13

-*e*

RSSR

(ads.)

-2e*

Hz0 I ‘: RSSR

(ads.) -2e-

Hz0 I I: RSSR + 0

x2

(ads.)

Hz0

-eI

RSSOd

(ads.) + RCOOH (ads.) * l/2 R2 + l/2 cq

slow hydrolysis

I RSH

/

(ads.)

+

SO$-

RSOi

+

+

CR

H’

.t

UNKNOWNPRODUCTS

Fig. 15. Proposed reaction pathway for the oxidation of L-cystine and L-cysteine at pH 1.7 on a gold electrode [39].

rates. This suggested the L-cystine surface reactions (21)-(23) were kinetically controlled, whilst the thiol surface reaction (24) was under mixed kinetic-diffusion control. It was concluded that much less than a monolayer of L-cysteine was adsorbed on the electrodes. As shown in Figs. 13 and 14, surface oxide formation complicated the study at the noble metals. Pradac and co-workers proposed possible involvement of the oxide in the mechanism of L-cystine oxidation at both metals. They cited the lack of separation of the two processes, even though oxidation commenced at electrode potentials some 150 mV more positive on gold (Fig. 13). In contrast, oxidation of r_-cysteine was inhibited by surface oxide formation at the electrodes. During the reverse negative potential scan, oxidation continued as the surface oxide was reduced (Fig. 14). From measurements at a platinum RDE, Reddy and Krishnan 146,471 supported mechanism (24) for L-cysteine oxidation. Tafel slopes of 120 mV per decade and an electrochemical reaction order of - 1 for protons were recorded.

At gold electrodes, Safranov et al. [48,49] reported that mechanism (24) for L-cysteine oxidation was applicable in neutral solution. From pH 3 to 12, Tafel slopes were 120 mV per decade and dE/dpH, at constant current, was 60 mV. Fractional electrochemical reaction orders, for L-cysteine and protons, were ascribed to a change in the amino acid adsorption with coverage. From studies at a gold RDE, with ex situ X-ray photoelectron spectroscopic (XPS) analysis of the electrode surface, Reynaud et al. [39] proposed a complex reaction pathway (Fig. 15) for the oxidation of L-cystine and L-cysteine in a buffer solution of pH 1.7. Much additional evidence is required, however, to support this pathway as XPS is inherently an ex situ technique. Reynaud et al. [39] confirmed that adsorption of L-cystine and L-cysteine ocurred by ac voltammetric experiments. Steady-state voltammograms were consistent with the cyclic voltammograms (Figs. 13 and 14) reported by Pradac and co-workers [44,45]. Constantpotential electrolysis of L-cystine solutions negative of + 1.20 V (vs. SCE), followed by XPS analysis, identi-

14

T.R. Ralph et al. / Electrochemistry of L-cystine and r-cysteine: Part 1

fied RSSO; and RCOOH as adsorbed products. At potentials more positive than + 1.40 V (vs. SCE), sulphur intermediates were not detected on the electrode surface. Dissolved RSO, was proposed as the oxidation product. Adsorbed RSSO; and RCOOH were also detected after constant potential electrolysis of L-cysteine solutions, negative of + 1.30 V (vs. SCE). Reynaud et al. concluded that the first step in the oxidation of L-cysteine was to r_-cystine. 5. Conclusion The fundamental electrochemistry of r_-cystine, L-cysteine and related compounds has been widely studied from both thermodynamic and kinetic perspectives owing to the biological and industrial importance of these species. Measurements are complicated by the tendency for slow equilibration and adsorption of the amino acids on solid electrode surfaces and the formation of cysteinate complexes on mercury. In the case of precious metals, interaction with surface oxides is also important. Voltammetry indicates the reduction of dissolved L-cystine to r_-cysteine is electrochemically irreversible. At lower overpotentials the kinetics of electron transfer are sluggish and the reduction is kinetically controlled. At higher overpotentials the electrochemical kinetics are significantly enhanced and the rate of mass transport of L-cystine now determines the rate of reduction. At intermediate overpotentials the reduction is under mixed kinetic-mass transport control. Consequently, for the electrosynthesis selection of an electrocatalytic electrode material which promotes the sluggish electrochemical kinetics of L-cystine reduction is key. There is some evidence that cathode materials capable of adsorbing the disulphide molecule neither very strongly nor very weakly (cf the volcano plot effect) may be required. L-cystine is reportedly adsorbed at all the electrode metals apart from vitreous carbon and a WISGE. Significantly, L-cystine is not reduced at vitreous carbon or the WISGE at electrode potentials positive of hydrogen evolution (e.g. approximately - 1.2 V (vs. SCE) in buffer solution of pH 1.7 at vitreous carbon). Conversely, at mercury and transition metal macrocycles, rates of L-cystine reduction are high at much less negative electrode potentials at a similarly low pH (see refs. 5 and 34, respectively). Adsorption of L-cystine does not, however, guarantee efficient electrosynthesis of L-cysteine. For example, at platinum, gold and silver, although a significant amount of the disulphide is absorbed at the electrode surface, reduction of the amino acid is masked by hydrogen evolution at these low hydrogen overpotential metals.

Clearly, it is required that the overpotential for disulphide reduction should be lower than that for hydrogen evolution and low hydrogen overpotential metals are not favoured. This has been well demonstrated by the studies of Arai et al. [38]. At platinum, palladium and silver electrodes, current efficiencies for the disulphide reduction were 10% or less. At the corresponding metal thiolate film surfaces, however, promotion of the disulphide reduction kinetics and retardation of hydrogen evolution combine to produce near 100% current efficiencies for thiol formation. The literature also provides some selection criteria for an appropriate electrochemical reactor. The kinetic facility of L-cysteine oxidation at all the electrode materials examined and L-cystine oxidation at platinum, gold and carbon suggests that the electrosynthesis requires a divided reactor. Further, mass transport control of r_-cystine reduction at high over-potentials implies that a cell design offering high rates of mass transport may be favourable. Acknowledgements

Part of this work was supported by an SERC CASE studentship (to T.R.R.) in collaboration with ECRC (now EA Technology) at Capenhurst. The authors are grateful to a referee who drew their attention to early work on the polarography of cysteine and cystine. References 1 R.M.C. Dawson, D.C. Elliot, W.H. Elliot and K.M. Jones, Data for Biochemical Research, Clarendon Press, Oxford, 1986, pp. 12 and 13. 2 T.-Y. Liu, in H. Neurath, R.L. Hill and C.-L. Boeder (Eds.), The Proteins, Academic Press, New York, 1977, Vol. 3, Chapter 3. 3 T.R. Ralph, M.L. Hitchman, F.C. Walsh and J.P. Millington, to be published. 4 H. Borsook, E.L. Ellis and H.M. Huffman, J. Biol. Chem. 117 (1937) 281, 5 J.C. Ghosh, S.N. Raychaudhurri and S.C. Ganguli, J. Indian Chem. Sot., 9 (1932) 43, 53. 6 D.E. Green, Biochem. J., 27 (1933) 678. 7 M. Dixon and H.J. Quastel, J. Chem. Sot., 123 (1923) 2943. 8 J.W. Williams and E.M. Drissen, J. Biol. Chem., 87 (1930) 441. 9 L.K. Ryklan and C.L.A. Schmidt, Univ. Calif. Publ. Physiol., 8 (1944) 257. 10 G.S. Fruton and H.T. Clarke, J. Biol. Chem., 106 (1934) 667. 11 N. Tanaka, I.M. Kolthoff and W. Stricks, J. Am. Chem. Sot., 77 (1955) 2004. 12 I.M. Kolthoff and C. Barnum, J. Am. Chem. Sot., 62 (1940) 3061. 13 I.M. Kolthoff and C. Barnum, J. Am. Chem. Sot., 63 (1941) 520. 14 0. Grubner, Collect. Czech. Chem. Commun., 19 (1954) 444. 15 I.M. Kolthoff, W. Stricks and N. Tanaka, J. Am. Chem. Sot., 77 (1955) 5211. 16 B. Nygard, Acta Chem. Stand., 15 (1961) 1039. 17 B. Nygard, Ark. Kemi, 27 (1967) 341, 405, 425; 28 (1967) 75, 89. 18 B. Nygard, Acta Univ. Ups., (19671 104. 19 B. Nygard, J. Olefsson and G. Bergson, Ark. Kemi, 28 (1967) 41.

T.R. Ralph et al. / Electrochemistry of L-cystine and L-cysteine: Part I 20 M. Kalousek,

21 22 23 24 25 26 27 28 29 30 31 32 33 34

0. Grubner and A. Tockstein, Collect. Czech. Chem. Commun., 19 (1954) 1111. I.M. Kolthoff, W. Stricks and N. Tanaka, J. Am. Chem. Sot., 77 (1955) 4739. I.M. Issa, A. El Samahy, R.M. Issa and Y.M. Temerik, Electrochim. Acta, 17 (1972) 1615. CA. Ducarmois-Mairesse, G.J. Patriarche and J.L. Vandenbalck, J. Pharm. Belg., 28 (1973) 300. CA. Ducarmois-Mairesse, G.J. Patriarche and J.L. Vandenbalck, Anal. Chim. Acta, 71 (1974) 165. I.R. Miller and J. Teva, J. Electroanal. Chem., 36 (1972) 157. M.T. Stankovich and A.J. Bard, J. Electroanal. Chem., 75 (1977) 487. M.J. Pena and I. Alarcon, An. Quim., 86 (1990) 594. P. Bianco and J. Haladjian, J. Electrochim. Acta, 2.5 (1980) 1317. J. Zagal, C. Fierro and R. Rozas, J. Electroanal. Chem., 119 (1981) 403. J. Zagal, P. Herrera, K. Brinck and S. Zanartu-Ureta, Proc. Electrochem. Sot., 84-12 (1984) 602. J. Zagal and P. Herrera, Electrochim. Acta, 30 (1985) 449. J. Zagal, C. Paez and S. Barbato, Proc. Electrochem. Sot., 87-12 (1987) 211. B.Z. Nikolic, R.R. Adzic and E.B. Yeager, J. Electroanal. Chem. 103 (1979) 281. Z. Wang, C. Li, Z. Deng and Q. Zha, Huaxue Xuebao, 44 (1986) 863; Chem. Abstr., 105 (1986) 1990302.

15

35 Z. Wang, C. Li, Z. Deng and Q. Zha, Huaxue Xuebao, 45 (1987) 931; Chem. Abstr. 108 (1988) 64491s. 36 Z. Wang, C. Li, Z. Deng and Q. Zha, Gaodeng Xuexiao Huaxue Xuebao, 8 (1987) 453; Chem. Abstr. 108 (1988) 12940t. 37 Z. Wang and D. Pang, J. Electroanal. Chem., 283 (1990) 349. 38 G. Arai, T. Sugaya, H. Sakamoto and Y. Iwao, Bull. Chem. Sot. Jpn., 65 (1992) 594. 39 J.A. Reynaud, B. Maltoy and P. Canessan, J. Electroanal. Chem., 114 (1986) 195. 40 R. Eggli and R. Asper, Anal. Chim. Acta, 101 (1978) 253. 41 V. Brabec and V. Mornstein, Biophys. Chem., 12 (1980) 159. 42 T. Watanabe and H. Maeda, J. Phys. Chem., 93 (1989) 3258. 43 D.G. Davis and E. Bianco, J. Electroanal. Chem., 12 (1966) 254. 44 J. Pradac and J. Koryta, J. Electroanal. Chem., 17 (1968) 167, 177, 185. 45 J. Pradac, N. Ossendorfova and J. Koryta, Croat. Chem. Acta, 45 (1973) 97. 46 S.J. Reddy and V.R. Krishnan, Anal. Chem. Symp. Ser., 2 (1980) 155. 47 S.J. Reddy and V.R. Krishnan, Trans. SAEST, 15 (1980) 99. 48 A.Yu. Safranov, M. Tarasevich, V.A. Bogdanovskaya and A.S. Chernyak, Sov. Electrochem., 19 (1983) 378. 49 A.Yu. Safranov, M. Tarasevich, V.A. Bogdanovskaya and A.S. Chernyak, J. Electroanal. Chem., 202 (1986) 147. 50 H.D. Jakobke and H. Jeschkeit, Amino Acids, Peptides and Proteins: an Introduction, Akademie Verlag, Berlin, 1977, p. 25.