Continuous Fluorometric Method for Determining the Monophenolase Activity of Tyrosinase on L-Tyrosine, through Quenching L-DOPA Fluorescence by Borate

Tyrosinase is the key enzyme in melanin biosynthesis and inherently involves both monophenolase activity and diphenolase activity. A continuous fluorometric assay method was developed for the first time to directly monitor the real monophenolase activity without the interference of diphenolase reactions through exclusively quenching the native fluorescence of DOPA by borate. Complexation with borate at pH 8.0 allowed for selective quantitation of tyrosine in a binary mixture of tyrosine and DOPA at 335 nm. The time course for consumption of tyrosine was established to measure the initial velocity by recording the tyrosine fluorescence intensity at discrete intervals. The assay worked in the monophenolase activity range from 0.13 to 2.01 U mL-1 with the limit of detection (LOD) of 0.10 U mL-1. The assay method exhibited a promising prospect in application in kinetics of monophenolase and high throughput screening for monophenolase inhibitors.