4-HYDROXYANISOLE - THE MOST SUITABLE MONOPHENOLIC SUBSTRATE FOR DETERMINING SPECTROPHOTOMETRICALLY THE MONOPHENOLASE ACTIVITY OF POLYPHENOLOXIDASE FROM FRUITS AND VEGETABLES

A continuous spectrophotometric method for determining the monophenola se activity of polyphenol oxidase from several plant sources is descri bed. This assay method is based on the coupling reaction between 3-met hyl-2-benzothiazolinone hydrazone and the quinone product of the oxida tion of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydr oxyanisole proved to be the best monophenol assayed to measure the mon ophenolase activity of polyphenol oxidase from apple, artichoke, avoca do, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisol e were compared to those of p-hydroxyphenyl propionic acid, a very sen sitive monophenol previously reported to assay the monophenolase activ ity of polyphenol oxidase from apple, pear, and mushroom. The high val ues of the maximum steady state rate obtained for 4-hydroxyanisole sug gest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assa ys which predicted the highest reactivity of 4-hydroxyanisole. Therefo re nuclear magnetic resonance assays proved to be a valuable and usefu l tool to predict the best monophenolic substrate for plant polyphenol oxidases, The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyaniso le was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydr oxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone a s coupling reagent the most sensitive and precise assay method up to d ate reported in the literature to determine the monophenolase activity of polyphenol oxidase from fruits and vegetables. (C) 1998 Academic P ress.