MONOPHENOLASE ACTIVITY OF POLYPHENOL OXIDASE FROM VERDEDONCELLA APPLE

Apple polyphenol oxidase (PPO) has been isolated and partially purified by using two sequential phase partitionings with Triton X-114 reported here for the first time. The enzyme showed monophenolase activity when assayed on (p-hydroxyphenyl)propionic acid (PHPPA) with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in a new and reliable continuous spectrophotometric method, with high sensitivity, accuracy, and precision. The initial monophenolase activity showed a lag period (tau) prior to the attainment of the steady state rate (V-ss). Both kinetic parameters, tau and V-ss, depended on the pH, the enzyme and substrate concentrations, and the presence of catalytic amounts of o-diphenol. These dependencies can be explained in terms of a reaction mechanism involving one single active site, two enzyme forms, E(met) and E(oxy), and a set of nonenzymatic reactions from o-quinones to chromophoric MBTH-quinone adduct, with regeneration of one molecule of o-diphenol. The effect of pH was related with two significant pK(a) values of the free enzyme forms but not of enzyme-substrate complexes. This kinetic characterization was essential for understanding and choosing optimal assay conditions for determining enzyme activity and concentration when using PHPPA as a monophenolic substrate of apple PPO.