Cytosol-mediated reduction of resorufin: a method for measuring quinone oxidoreductase.
The reduction of resorufin (7-hydroxyphenoxazone) fluorescence was catalyzed by enzymes present in the hepatic cytosol of rats and hamsters. This reaction was mediated by either NADH or NADPH, was completely inhibited by 10 microM dicumarol, and was not affected by anaerobic conditions (purging the reaction cuvette with nitrogen). The enzyme-mediated decrease in resorufin fluorescence was also associated with the loss of the primary absorbance maximum at 570 nm as well as the shoulders at 530 and 600 nm. Similar spectral changes were observed after resorufin was nonenzymatically reduced by sodium dithionite. The enzymatic activity was induced 20- to 40-fold by animal pretreatment with Aroclor-1254 or methylcholanthrene, but only minimally by phenobarbital. A 2.5-fold increase in the rate of the reaction was noted when the pH of the reaction mixture was lowered from pH 7.5 to 6.0. This pH optimum was not a result of slower rates of reoxidation of the reduced resorufin at lower pH, but was due to increased rates of reduction of the compound. Several of the characteristics of the reaction were congruent with the involvement of DT-diaphorase (quinone oxidoreductase, EC 1.6.99.2), and this newly developed fluorimetric assay would appear to be a rapid, sensitive, and direct method for measurement of DT-diaphorase activity.
D000818:Animals; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008647:Mesocricetus; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism*; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D010078:Oxazines; D009243:NAD; D009249:NADP; C014180:resorufin; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases