[A method for determining theo-diphenoloxidase activity in the pyrocatechol oxidation reaction].

A new simple and sensitive spectrophotometric method is suggested for determining the catecholase activity of diphenoloxidase. The method is based on the enzymatic oxidation of pyrocatechol to 1,2-benzoquinone (BQ) in the presence of the excess of ethylenediamine sulphate (EDA). The condensation product (products) of BQ and EDA (P365) is stable in the solution and possesses strong absorption in the range of 365 nm. The molar absorption factor, E365 (under condition that the molar reaction ratio of catechol to P365 is 1:1) is 15500 M-1 cm-1 on the average. Optimal reaction conditions (pH 7.0, T=25-30 degrees C, [EDA]o = 5 mg/ml) are determined. The advantages and restrictions of the suggested technique in comparison with the methods described earlier are discussed.