A colorimetric method for the determination of phenol oxidase in plant material.

Enzymes catalyzing the oxidation of phenols occur widely in plant tissues and have been extensively studied (l-7), but the diversity of their properties and the complexities of the reactions catalyzed have made assays and their interpretations uncertain. In only a few instances have essentially pure enzyme preparations been studied (1, 6, S), and even in these cases the mechanism of oxidation is not well understood. The two principal types of phenol oxidase or tyrosinase can best be defmed in terms of the substrates used for their detection; for example, catechol for catecholase or polyphenolase, and p-cresol or phenol for cresolase or monophenolase. In each case the first reaction product is believed to be a highly reactive o-quinone (1). Early methods of determination (2, 5) were based on measuring the colored oxidation products of phenols or aromatic amines, but the conditions controlling color formation were complex with generally inaccurate results. More recently, manometric methods in which oxygen uptake is measured with catechol or p-cresol as substrates have been preferred. In the former case, in which the enzyme tends to be rapidly inactivated during the oxidation, a secondary substrate system has often been employed to keep the o-quinone reduced. Kubowitz (9) used the hexose monophosphate dehydrogenase-triphosphopyridine nucleotide system, while Nelson and Damson (1) employed hydroquinone or ascorbic acid. This device, however, does not eliminate enzyme inactivation, since the latter appears to depend on the actual amount of oxygen reduced (1). This sets definite limits on the accuracy of manometric methods. The goal of the present work was to provide a phenol oxidase method suitable for routine assay of large numbers of samples of plant material, as well as for the estimation of the enzyme in small amounts of tissue.