A rapid and sensitive radiometric assay for cytosolic epoxide hydrolase is described. The assay is based on the highly efficient partitioning of unreacted 1-phenyl-1,2-epoxybutane (β-ethylstyrene oxide) from an aqueous mix into isooctane, with the product diol being retained in the aqueous phase. The trans-epoxide is an excellent substrate for both the glutathione transferase and epoxide hydrolase present in mouse liver cytosol, and both enzymes may be monitored simultaneously after appropriate modifications of the assay are made. Also, trans-β-ethylstyrene oxide is hydrated much faster than the cis-isomer in the hepatic cytosol of three different mammalian species, and both isomers are poor substrates for the microsomal epoxide hydrolase. Mouse liver cytosol hydrates the trans-epoxide at 69 nmol/min-mg protein, and a Km of 7.2 × 10−5m is observed. Reduction of 1-phenyl-1-bromo-2-butanone with sodium borotritide and subsequent ring closure of the bromohydrin mix with base was a highly efficient route for radiolabeling β-ethylstyrene oxide. The resulting cis- and trans-epoxides were separable by semipreparative high-performance liquid chromatography.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology