Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide
Introduction
Thiol-containing compounds play an important role in protecting biologic systems against oxidative injury [1], [2]. There is also increasing evidence for thiol involvement in metabolic regulation [3], signal transduction and regulation of gene expression [4], [5], [6], [7], [8]. Oxidants and antioxidants are proposed to participate in this redox regulation by shifting the balance between reduced and oxidized cellular thiols [4], [6], [9]. Reduced glutathione (GSH) is the most abundant intracellular low molecular weight thiol, but other thiols can also protect against oxidative injury or inhibit signal transduction. Not all these effects can be explained by the thiols acting as precursors of GSH [10].
This suggests that they may interact directly with oxidants rather than by an enzymatic mechanism specific for GSH. Such a mechanism could involve either radical scavenging or direct reaction with hydrogen peroxide. Thiols have been shown to react with superoxide [11], [12], [13], [14], [15] and hydrogen peroxide [14], [16], [17], but a systematic comparison of the reactivities of different thiols with each oxidant has not been carried out. There is also no consensus view on thiol reactivity with superoxide. Some authors report no reaction [18], [19] and estimates of rate constants range from 15 to 1000 M−1s−1 [14], [15], [20], [21] to greater than 105 M−1s−1 [22], [23], [24]. In this paper we have compared GSH with dithiothreitol (DTT), cysteine, cysteamine, penicillamine, N-acetylcysteine, and the angiotensin converting enzyme inhibitor captopril, in their reactivity with superoxide generated by xanthine oxidase. We have also measured rate constants for reaction with hydrogen peroxide and related them and superoxide reactivity to thiol group pK.
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Materials and methods
Thiols, Cu/Zn superoxide dismutase, xanthine oxidase, bovine liver catalase and other biochemicals were obtained from Sigma Chemical Co. (St, Louis, MO, USA), and hydrogen peroxide was obtained from BDH (Poole, UK).
Reactions were all performed at 37°C, in 50 mM phosphate buffer containing 10 μM diethylenetriaminepenta-acetic acid (DTPA), at pH 7.4 unless stated otherwise, as previously described for GSH [15]. Superoxide was generated from hypoxanthine (50 μM) and xanthine oxidase (usually about
Reaction rates of thiols with hydrogen peroxide
Hydrogen peroxide was added to varying concentrations of each thiol and rates of exponential decay were measured. These values of kobs were plotted against thiol concentration to obtain the second order rate constants (Fig. 1). As expected for the thiolate being the reactive species [16], these were highest for the thiols with the lowest pK (Table 1). Rate constants calculated for the reaction of the thiolate ions were similar for the different compounds.
Thiol consumption with superoxide
To establish whether the thiols react
Reactivity with superoxide
All the thiol compounds that we examined, with the exception of captopril, reacted with superoxide as demonstrated by a loss of thiol groups and enhanced oxygen uptake in a xanthine oxidase system. Their relative reactivity at pH 7.4, determined either as thiol loss or oxygen uptake, was inversely related to the pK of the thiol group. Thus, N-acetylcysteine had a low reactivity, and the lack of reaction with captopril is likely to be a consequence of its high pK. The reactivity of most of the
Acknowledgements
This work was supported by a grant from the Health Research Council of New Zealand. We are grateful To Dr. M. J. Davies for helpful comments.
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The present address for D. Metodiewa is Institute of Applied Radiation Chemistry, Technical University of Lodz, 93-590 Lodz, Poland.