[A sensitive fluorometric assay for dihydrodiol dehydrogenase].
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[f]quinoxaline and 2,6- or 3,6-bis(2-aminoethylamino)benzo[f]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.
Miyabe
Y
Y
Gifu Prefectural Tajimi Hospital, Japan.
Matsuura
K
K
Nakayama
T
T
Deyashiki
Y
Y
Ohya
I
I
Hara
A
A
D005029:Ethylenediamines; D005260:Female; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D009285:Naphthoquinones; D010088:Oxidoreductases / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D005029:Ethylenediamines; D009285:Naphthoquinones; C031234:ethylenediamine; C040756:1,2-naphthoquinone; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase