MONOPHENOLASE ACTIVITY OF POLYPHENOL OXIDASE FROM VERDEDONCELLA APPLE

Apple polyphenol oxidase (PPO) has been isolated and partially purifie d by using two sequential phase partitionings with Triton X-114 report ed here for the first time. The enzyme showed monophenolase activity w hen assayed on (p-hydroxyphenyl)propionic acid (PHPPA) with 3-methyl-2 -benzothiazolinone hydrazone (MBTH) in a new and reliable continuous s pectrophotometric method, with high sensitivity, accuracy, and precisi on. The initial monophenolase activity showed a lag period (tau) prior to the attainment of the steady state rate (V-ss). Both kinetic param eters, tau and V-ss, depended on the pH, the enzyme and substrate conc entrations, and the presence of catalytic amounts of o-diphenol. These dependencies can be explained in terms of a reaction mechanism involv ing one single active site, two enzyme forms, E(met) and E(oxy), and a set of nonenzymatic reactions from o-quinones to chromophoric MBTH-qu inone adduct, with regeneration of one molecule of o-diphenol. The eff ect of pH was related with two significant pK(a) values of the free en zyme forms but not of enzyme-substrate complexes. This kinetic charact erization was essential for understanding and choosing optimal assay c onditions for determining enzyme activity and concentration when using PHPPA as a monophenolic substrate of apple PPO.