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A Direct, Highly Sensitive Fluorometric Assay for a Microsomal Cytochrome P450-mediated O-demethylation Using a Novel Coumarin Analog as Substrate

A Direct, Highly Sensitive Fluorometric Assay for a Microsomal Cytochrome P450-mediated O-demethylation Using a Novel Coumarin Analog as Substrate

R Roser, H Thomas

Z Naturforsch C J Biosci. Nov-Dec 2000;55(11-12):915-22.

PMID: 11204196

Abstract:

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP6174863	3-Chloro-7-hydroxy-4-methylcoumarin		6174-86-3	Price

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