4-Hydroxyanisole: the most suitable monophenolic substrate for determining spectrophotometrically the monophenolase activity of polyphenol oxidase from fruits and vegetables.
A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those of p-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolas activity of polyphenol oxidase from fruits and vegetables.
Espín
J C
JC
Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Spain.
Tudela
J
J
García-Cánovas
F
F
D000840:Anisoles; D004156:Catechol Oxidase / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D013045:Species Specificity; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology*
D000840:Anisoles; C009760:mequinol; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase