Electrochemical determination of diphenol oxidase activity using high-pressure liquid chromatography

A quantitative assay for the diphenol oxidase activity of tyrosinase (EC 1.14.18.1) using high-pressure liquid chromatography with electrochemical detection is described. The assay is based on the observation (M. Sugumaran, 1986, Biochemistry 25, 4489-4492) that tyrosinase catalyzes the oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde. The substrate and product were readily separated on a reverse-phase column equilibrated with 0.1 M citrate buffer, pH 3.2, containing 0.5 mM Na2 EDTA, and 5% (v/v) acetonitrile. The reaction of DHMA with mushroom tyrosinase was linear with time and proportional to the amount of enzyme present. The specific activity of mushroom tyrosinase using the method was about fourfold greater than that obtained using a spectrophotometric assay for diphenol oxidase following dopachrome formation from L-3,4-dihydroxyphenylalanine. The applicability of the high-pressure liquid chromatographic assay to determination of diphenol oxidase activity in small biological sample sizes was demonstrated by using microgram quantities of crude, cell-free hemolymph from Aedes aegypti mosquitoes.