Oxidation of catechol in plants. IV. Purification and properties of the 3,4,3',4'-tetrahydroxydiphenyl-forming enzyme system from Tecoma leaves

Abstract

Acetone powders prepared from leaf extracts of Tecoma stans L. were found to catalyze the oxidation of catechol to 3,4,3',4'-tetrahydroxydiphenyl. Fractionation of the acetone powders obtained from Tecoma leaves with acetone, negative adsorption of the acetone fraction with tricalcium phosphate gel, and chromatography of the gel supernatant on DEAE-Sephadex yielded a 68-fold purified enzyme with 66% recovery. The enzyme had an optimum pH around 7.2. It showed a temperature optimum of 30° and the K_m for catechol was determined as 2 × 10^-4 M. The purified enzyme moved as a single band on polyacrylamide gel electrophoresis. Its activity was found to be partially stimulated by Mg²⁺. The reaction was not inhibited by ο-phenanthroline and α,α'-dipyridyl. The purified enzyme was highly insensitive to a range of copper-chelating agents. It was not affected appreciably by thiol inhibitors. The reaction was found to be suppressed to a considerable extent by reducing agents like GSH, cysteine, cysteamine, and ascorbic acid. The purified enzyme was remarkably specific for catechol. Catalase affected neither the enzyme activity nor the time course of the reaction. Hydrogen peroxide was not formed as a product of the reaction.