Basic studies on spectrophotometric and chemiluminescent determination of D-aspartate using D-aspartate oxidase from Cryptococcus humicolus UJ1

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A spectrophotometric method and a chemiluminescent method were studied for determination of minute amounts of D-aspartate using D-aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that catalyzes very specifically the oxidative deamination of D-aspartate. The spectrophometric method was based on the measurement of 2,4-dinitrophenylhydrazones derived from keto acids produced from D-aspartate by the action of the enzyme. Addition of catalase to the enzyme reaction mixture was required to degrade hydrogen peroxide, another reaction product from D-aspartate and oxygen, so that the keto acids would not be decreased by the reaction with the hydrogen peroxide. The detection limit for D-aspartate was 1nmol, and the presence of 100nmol D-glutamate did not affect the detection at all. The chemiluminescent method depended on the measurement of the hydrogen peroxide as a product by the chemiluminescent reaction of hydrogen peroxide with bis (2,4,6-trichlorophenyl) oxalate in the presence of a fluorescent dye, 8-anilinonaphthalene-1-sulfonic acid. The detection limit appeared to be around 20 pmol of D-aspartate,
長岡技術科学大学の論文
1999-02-10