AN ALTERNATIVE 7-ETHOXYRESORUFIN O-DEETHYLASE ACTIVITY ASSAY - A CONTINUOUS VISIBLE SPECTROPHOTOMETRIC METHOD FOR MEASUREMENT OF CYTOCHROME-P-450 MONOOXYGENASE ACTIVITY
AN ALTERNATIVE 7-ETHOXYRESORUFIN O-DEETHYLASE ACTIVITY ASSAY - A CONTINUOUS VISIBLE SPECTROPHOTOMETRIC METHOD FOR MEASUREMENT OF CYTOCHROME-P-450 MONOOXYGENASE ACTIVITY
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DOI：
10.1016/0003-2697(84)90144-1
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发表时间：
1984-01-01
影响因子：
2.9
通讯作者：
WALSH, C
中科院分区：
文献类型：
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作者：
KLOTZ, AV;STEGEMAN, JJ;WALSH, C
描述了用可见分光光度计直接测量细胞色素 P-450 依赖性 7-乙氧基试卤灵 O-脱乙基酶活性的程序，并与标准荧光方法进行了比较。用β-萘黄酮处理的哺乳动物（大鼠）和鱼类（scup，Stenotomus chrysops）肝微粒体进行的两种测定均产生相同的结果。该测定利用了高度纯化的 7-乙氧基试卤灵（底物）和试卤灵（酶产物）获得的可见吸收光谱的明显区别。详细介绍了酶产物试卤灵的纯化和表征，其消光系数 (ε572 = 73 mM-1 cm-1) 提供了酶活性的准确定量。产品发色团的大可见光消光系数为低活性样品提供了高灵敏度。该酶测定在可见分光光度计中的应用，以及大量证据表明单一芳烃诱导细胞色素 P-450 同工酶负责催化，增强了该底物在微粒体单加氧酶测定中的效用。通过简单测定 scup 肝微粒体中 7-乙氧基试卤灵氧化的偶联比以及 7,8-苯并黄酮和苯基咪唑对酶促反应抑制的 Ki，进一步证明了可见测定的实用性。
A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The 2 assays yielded identical results with both .beta.-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (.epsilon.572 = 73 mM-1 cm-1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the Ki for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.