Analysis of Monophenol Oxidase Activity Using High Pressure Liquid Chromatography with Electrochemical Detection

Abstract

A very sensitive, specific and reliable quantitative assay was developed for measuring the rate of hydroxylation of tyrosine by mushroom tyrosinase using high pressure liquid chromatography with electrochemical detection (HPLC-ED). The assay employs N-acetyldopamine (NADA) as cofactor and ascorbate as a reducing agent. The product of the reaction, L-dopa (3,4-dihydroxyphenylalanine), was readily separated by HPLC-ED from the remaining interacting components. The reaction was linear with time and proportional to the amount of enzyme present. The amount of ascorbate gradually decreased during the hydroxylation of tyrosine, but as long as ascorbate was present in the reaction mixture the levels of L-dopa and NADA were not altered. The data