Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation.
In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790+/-690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48+/-0.37 microM and 3360+/-180 pmol product/min/nmol CYP, respectively.
Soucek
P
P
Biotransformations Group, Center of Occupational Diseases, National Institute of Public Health, Prague, Czech Republic. pavel.soucek@ecn.cz
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D009251:NADPH-Ferrihemoprotein Reductase