An alternative 7-ethoxyresorufin O-deethylase activity assay: a continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase activity.
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 two 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 K1 for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.
D000818:Animals; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System; D005399:Fishes; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000032:analysis*; D010105:Oxygenases / Q000032:analysis*; D051381:Rats; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D010105:Oxygenases; D019363:Cytochrome P-450 CYP1A1