A CONTINUOUS SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF MONOPHENOLASE ACTIVITY OF TYROSINASE USING 3-METHYL-2-BENZOTHIAZOLINONE HYDRAZONE

A continuous spectrophotometric method for the rapid determination of monophenolase activity of tyrosinase is described. This method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone products of the oxidation of various monophenols in the presence of tyrosinase. The chemical reaction between MBTH and o-quinone has been kinetically characterized, the λ(max) and the molar absorptivity coefficients of the adducts have been calculated, and the stoichiometry of the reaction has been determined. The method is illustrated by measuring the enzymatic activity of mushroom tyrosinase during the hydroxylation of phenol and tyramine. The presence of MBTH in the reaction medium decreases the lag period present during the expression of monophenolase activity and the high ε values at 500-505 nm of the adducts make this method more sensitive than other continuous methods. The MBTH reaction in the presence of monophenols or o-diphenols has been optimized to stain tyrosinase obtained from different biological sources in electrophoresis gels. © 1994 Academic Press, Inc.