Method for the determination of molar absorptivities of thiol adducts formed from diphenolic substrates of polyphenol oxidase.

Metabolic thiols such as cysteine and glutathione are involved in the biosynthetic pathway of phaeomelanins. They attack the o-quinones generated by polyphenol oxidase in its action on mono and o-diphenols and thus generate adducts. Determination of the molar absorptivities of these adducts is useful for spectrophotometric studies of phaeomelanin biosynthesis, antibrowning reagents in plants, and polyphenol oxidase assay methods. For their calculation, a method based on the depletion of o-diphenol by the action of polyphenol oxidase in the presence of thiol has been proposed. However, the method is slow and presents certain problems, for which reason we propose a new and faster method based on the action of polyphenol oxidase on o-diphenols which are in excess with respect to oxygen. Under these assay conditions there is rapid enzymatic formation of o-quinones, which react stoichiometrically with a thiol giving rise to the corresponding thiol-diphenol adduct. The method has been successfully applied to adducts of cysteine and glutathione with several o-diphenolic substrates of polyphenol oxidase involved in phaeomelanin biosynthesis in skin, neurones, and plants.