Kinetics and mechanism of the reaction of cysteine and hydrogen peroxide in aqueous solution

The oxidation of thiol-containing small molecules, peptides, and proteins in the presence of peroxides is of increasing biological and pharmaceutical interest. Although such reactions have been widely studied there does not appear to be a consensus in the literature as to the reaction products formed under various conditions, the reaction stoichiometry, and the reaction mechanisms that may be involved. This study examines the reaction kinetics of cysteine (CSH) with hydrogen peroxide (H2O2) in aqueous buffers (in the absence of metal ions) over a wide range of pH (pH 4-13) and at varying ratios of initial reactant concentrations to explore the range of conditions in which a two-step nucleophilic model describes the kinetics. The disappearance of CSH and H2O2 and appearance of cystine (CSSC) versus time were monitored by reverse-phase high-performance liquid chromatography (HPLC). The effects of oxygen, metal ions (Cu2+), pH (4-13), ionic strength, buffer concentration, and temperature were evaluated. Data obtained at [H2O2](0)/[CSH](0) ratios from 0.01-2.3 demonstrate that the reaction of CSH with H2O2 in the absence of metal ions is quantitatively consistent with a two-step nucleophilic reaction mechanism involving rate-determining nucleophilic attack of thiolate anion on the unionized H2O2 to generate cysteine sulfenic acid (CSOH) as an intermediate. Second-order rate constants for both reaction steps were generated through model fitting. At [H2O2](0)/[CSH](0) > 10, the % CSSC formed as a product of the reaction declines due to the increased importance of alternative competing pathways for consumption of CSOH. A thorough understanding of the mechanism in aqueous solution will provide valuable background information for current studies aimed at elucidating the influence of such factors on thiol oxidation in solid-state formulations. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.