First derivative synchronous fluorometric method to continuously measure monophenolase activity

Abstract

Tyrosinase plays an essential role in melanin biosynthesis and inherently exhibits both monophenolase and diphenolase activity. A first derivative synchronous fluorometric assay was established for directly monitoring monophenolase activity. The zero-crossing point at 322 nm for the first-derivative under synchronous fluorescence with Δλ = 67 nm was utilized to selectively quantify tyrosine in the presence of the reaction product dihydroxyphenylalanine (DOPA). The limit of detection (LOD) for tyrosine was 0.54 μM. The fluorescence intensity of tyrosine was monitored at intervals of 30 s to establish the time course of tyrosine consumption. The LOD for the monophenolase activity was 0.0706 U⋅ mL^-1. The Michaelis-Menten e constant and maximum speed were 21.83 μM and 1.12 μM min^-1, respectively. Zinc ions competitively inhibited the monophenolase activity, with an IC₅₀ value of 14.36 μM. This assay is easily and rapidly executed and is of great significance for analyzing the kinetics of enzymatic reactions and in fundamental research on monophenolase. This approach has potential applications in the discovery of tyrosinase inhibitors for medicine and cosmetics, as well as in the industrial synthesis of substituted o-diphenol intermediates.

Keywords: Borate; First derivative synchronous fluorescence; Inhibitor; Monophenolase; Tyrosine.