title: Spectrofluorometric analysis of CYP2A6-catalyzed coumarin 7-hydroxylation.
abstract: A spectrofluorometric method is described for the determination of CYP2A6-catalyzed coumarin 7-hydroxylation. Following acidification of the reaction mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and assayed using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This assay is applicable to enzymatic studies of cDNA-expressed CYP2A6 and can be used to monitor coumarin 7-hydroxylation activity in microsomes prepared from liver and other tissues and in isolated hepatocytes and cultured cells that express this cytochrome P450 activity.
authors.affiliation: Department of Biology, Boston University, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: Thomas K H
authors.initials: TK
authors.lastname: Chang
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D065637:Cytochrome P-450 CYP2A6; D006900:Hydroxylation; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014468:Umbelliferones / Q000378:metabolism*
chemical_list: D014468:Umbelliferones; C031477:7-hydroxycoumarin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6