Abstract
Dihydrodiol dehydrogenase (DD) oxidizes naphthalene dihydrodiol to 1, 2-dihydroxynaphthalene, which is immediately autoxidized to 1, 2-naphthoquinone. Here we established a fluorometric assay for the enzyme, which is based on the conversion of 1, 2-naphthoquinone to fluorescent compounds by reacting with ethylenediamine. The formed fluorescent compounds were synthetically identified as 6-(2-aminoethylamino)benzo[ƒ]quinoxaline and 2, 6- or 3, 6-bis(2-aminoethylamino)benzo[ƒ]quinoxaline, which showed the same fluorescence at 550 nm at an excitation wavelength of 420 nm. This method provides a 9000-fold increase in sensitivity over a currently available assay which measures the change in the absorbance of a cofactor, NADPH. Since this simple and sensitive method allowed many samples to be assayed simultaneously, we applied it to the analysis of multiple forms of DD, separated by an anion-exchange chromatography, from six human liver specimens.
