Fluorimetric determination of d-amino acid oxidase

Abstract

The method depends on the formation of a fluorescent derivative, 2-hydroxy-3-methyl-quinoxaline, on condensation of pyruvate with o-phenylenediamine in acid medium. 2-Hydroxy-3-methylquinoxaline fluoresces strongly in 50% (v/v) sulfuric acid after excitation at 375 m[mu]. A single emission peak is observed at 480 m[mu]. Formation of the quinoxaline is dependent on time, temperature, acidity and relative concentration of reactants. A particulate preparation from a mouse kidney required FAD for optimum activity at pH 8.5; Km was 3.8 x 10-3 [image]; KFAD was 1.4 x 10-7 [image] and the reaction was strongly inhibited by p-chloro-mercuribenzoate and phenylmercuric acetate. Subcellular fractionation on a sucrose density gradient confirmed that the D-amino acid oxi-dase was localized on small granules.