CHROMATOGRAPHIC ANALYSES OF THE EFFECTS OF GLUTATHIONE, CYSTEINE AND ASCORBIC-ACID ON THE MONOPHENOL AND DIPHENOL OXIDASE ACTIVITIES OF TYROSINASE

The effects of glutathione, cysteine, and ascorbic acid on the monophe nol and diphenol oxidase functions of tyrosinase were assessed by high performance liquid chromatography with electrochemical detection (HPL C-ED) at both oxidative and reductive potentials. The enzyme-catalyzed hydroxylations of tyrosine to dopa and tyramine to dopamine were inhi bited completely by glutathione and cysteine, but not by ascorbic acid . However, the rates of oxidation of dopa and dopamine were enhanced a pproximately 5% by cysteine and 75% by glutathione. There was no chrom atographic evidence to indicate that either thiol reduced o-quinones b ack to their respective o-diphenols, a reaction that was documented fo r ascorbic acid. Glutathione and cysteine each formed sulfhydryl conju gates with the oxidation products of both dopa and dopamine. The thiol -mediated alterations in tyrosinase activity are likely due to the dir ect interactions of these sulfhydryl compounds with the enzyme, sugges ting that the availability and relative quantities of glutathione and cysteine at the sites of o-quinone formation may have a profound effec t on quinone cytotoxicity. Under certain conditions the nucleophilic a ddition of glutathione and cysteine to o-quinones may represent a mech anism for regulating quinone cytotoxicity. However, glutathione-enhanc ed diphenol oxidase activity can potentiate cytotoxic damage by genera ting oxyradicals, depleting cells of o-diphenols, and lowering the lev el of glutathione available for antioxidant activity.