Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation
Language English Country Netherlands Media print
Document type Comparative Study, Journal Article, Research Support, Non-U.S. Gov't
PubMed
10574186
Knihovny.cz E-resources
- MeSH
- Aryl Hydrocarbon Hydroxylases * MeSH
- Cytochrome P-450 CYP2A6 MeSH
- Escherichia coli genetics MeSH
- Spectrometry, Fluorescence MeSH
- Hydroxylation MeSH
- Microsomes, Liver enzymology MeSH
- Kinetics MeSH
- Rats MeSH
- Coumarins metabolism MeSH
- Humans MeSH
- NADPH-Ferrihemoprotein Reductase metabolism MeSH
- Mixed Function Oxygenases analysis metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Sensitivity and Specificity MeSH
- Cytochrome P-450 Enzyme System analysis metabolism MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- Aryl Hydrocarbon Hydroxylases * MeSH
- coumarin MeSH Browser
- CYP2A6 protein, human MeSH Browser
- Cytochrome P-450 CYP2A6 MeSH
- Coumarins MeSH
- NADPH-Ferrihemoprotein Reductase MeSH
- Mixed Function Oxygenases MeSH
- Recombinant Proteins MeSH
- Cytochrome P-450 Enzyme System MeSH
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.
In Vitro Interaction of Binuclear Copper Complexes with Liver Drug-Metabolizing Cytochromes P450
Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs
