Nims R W, Prough R A, Lubet R A
Arch Biochem Biophys. 1984 Mar;229(2):459-65. doi: 10.1016/0003-9861(84)90176-0.
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.
大鼠和仓鼠肝细胞溶质中存在的酶催化了试卤灵（7-羟基吩恶嗪）荧光的降低。该反应由NADH或NADPH介导，被10 microM双香豆素完全抑制，且不受厌氧条件（用氮气吹扫反应比色皿）的影响。酶介导的试卤灵荧光降低还与570 nm处最大吸光度主峰以及530和600 nm处的肩峰的消失有关。用连二亚硫酸钠对试卤灵进行非酶还原后，也观察到了类似的光谱变化。用Aroclor - 1254或甲基胆蒽对动物进行预处理可使酶活性诱导20至40倍，但苯巴比妥的诱导作用极小。当反应混合物的pH从7.5降至6.0时，反应速率提高了2.5倍。这种最适pH不是较低pH下还原试卤灵再氧化速率较慢的结果，而是该化合物还原速率增加的结果。该反应的几个特征与DT-黄递酶（醌氧化还原酶，EC 1.6.99.2）的参与一致，这种新开发的荧光测定法似乎是一种测量DT-黄递酶活性的快速、灵敏且直接的方法。
