Chromatographic Analyses of the Effects of Glutathione, Cysteine and Ascorbic Acid on the Monophenol and Diphenol Oxidase Activities of Tyrosinase

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Abstract
The effects of glutathione, cysteine, and ascorbic acid on the monophenol and diphenol oxidase functions of tyrosinase were assessed by high performance liquid chromatography with electrochemical detection (HPLC-ED) at both oxidative and reductive potentials. The enzyme-catalyzed hydroxylations of tyrosine to dopa and tyramine to dopamine were inhibited completely by glutathione and cysteine, but not by ascorbic acid. However, the rates of oxidation of dopa and dopamine were enhanced approximately 5 % by cysteine and 75 % by glutathione. There was no chromatographic evidence to indicate that either thiol reduced o-quinones back to their respective o-diphenols, a reaction that was documented for ascorbic acid. Glutathione and cysteine each formed sulfnydryl conjugates with the oxidation products of both dopa and dopamine. The thiol-mediated alterations in tyrosinase activity are likely due to the direct interactions of these sulfhydryl compounds with the enzyme, suggesting that the availability and relative quantities of glutathione and cysteine at the sites of o-quinone formation may have a profound effect on quinone cytotoxicity. Under certain conditions the nucleophilic addition of glutathione and cysteine to o-quinones may represent a mechanism for regulating quinone cytotoxicity. However, glutathione-enhanced diphenol oxidase activity can potentiate cytotoxic damage by generating oxyradicals, depleting cells of o-diphenols, and lowering the level of glutathione available for antioxidant activity.