A SENSITIVE FLUOROMETRIC ASSAY FOR DIHYDR ODIOL DEHYDROGENASE

Dihydrodiol dehydrogenase (DD) oxidizes naphthalene dihydrodiol to 1,2 -dihydroxynaphthalene, which is immediately autoxidized to 1,2-naphtho quinone. Here we established a fluorometric assay for the enzyme, whic h is based on the conversion of 1,2-naphthoquinone to fluorescent comp ounds by reacting with ethylenediamine. The formed fluorescent compoun ds were synthetically identified as 6-(2-aminoethylamino)benzo[f]quino xaline and 2,6- or 3,6-bis(2-aminoethylamino)benzo[f]quinoxaline, whic h showed the same fluorescence at 550 nm at an excitation wavelength o f 420 nm. This method provides a 9000-fold increase in sensitivity ove r a currently available assay which measures the change in the absorba nce of a cofactor, NADPH. Since this simple and sensitive method allow ed many samples to be assayed simultaneously, we applied it to the ana lysis of multiple forms of DD, separated by an anion-exchange chromato graphy, from six human liver specimens.