Fluorometric microassay to quantify microsomal epoxide hydrolase in 96-well plates

Analytical Biochemistry
M E Herrero, J V Castell

Abstract

The microfluorometric assay, suitable for measuring microsomal epoxide hydrolase activity in cultured cells, is based on the conversion of benzo[a]pyrene-7,8-epoxide to the corresponding trans-benzo[a]pyrene-7,8-dihydrodiol, a compound that fluoresces at 403 nm when excited at 365 nm. Activity is determined by incubating S9 fractions obtained from microcultures with this epoxide and monitoring the fluorescence with a microplate reader. Under the assay conditions selected, the photodecomposition of the reaction product was minimized and the linearity of the reaction was extended. The major advantages of this method are: (1) high sensitivity with a detection limit of 5 pmol/well of trans-benzo[a]pyrene-7,8-dihydrodiol formed, which is comparable to the most sensitive radioactive methods; (2) minimal sample requirement (1-5 micrograms liver microsomes; 10-50 micrograms S9 fraction from cultured cells); (3) reduced consumption of hazardous reagents; and (4) a considerable reduction in assay time and facility for simultaneous determination of enzyme activity in multiple samples.

Citations

Sep 29, 2004·Journal of Biochemical and Molecular Toxicology·Mary Beth Genter
Nov 24, 1999·Journal of Biochemical and Molecular Toxicology·P J KorytkoJ G Scott

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