Abstract
A highly sensitive and rapid radiometric assay for the determination of specific epoxide hydratase activity with a steroid epoxide (16α, 17α‐epoxy‐1,3,5(10)‐estratrien‐3‐ol, ‘estroxide’) has been developed. The unreacted substrate was separated from the product 1,3,5(10)‐estratrien‐3,16β,17α‐triol by extraction into light petroleum. The product was then extracted into ethyl acetate and measured by scintillation spectrometry. Radiochromatography established that after subtraction of the blank the entire radioactivity measured in the ethyl acetate phase resulted from the product 1,3,5(10)‐estratrien‐3,16,17‐triol, whilst high performance liquid chromatography with the four possible isomers of 1,3,5(10)‐estratrien‐3,16,17‐triols as standards showed that the product was the 3,16β,17α‐triol. Limits of linearity with time and protein were established with microsomal fractions from liver, testes and ovaries.The apparent Km and V values were determined with liver and testes microsomes. The apparent Km for estroxide was very similar in both organs (liver 10 μM; testes 6.3 μM) which was similar to the value obtained for the series of epoxides derived from polycyclic hydrocarbons but substantially lower than that for styrene oxide. The specific activity of epoxide hydratase with estroxide as substrate was about twice as high as with benzo(a)pyrene 4,5‐oxide. The ratio of the specific epoxide hydratase activity with the two substrates was similar (1.6–2.2) in all investigated organs [liver, kidney, lung, testes (ovaries) and adrenals] of either sex of rats. Immunoprecipitation of solubilized liver microsomes by antibodies raised against epoxide hydratase purified to homogeneity was measured with estroxide, benzo(a)pyrene 4,5‐oxide and styrene oxide as substrates. The extent of precipitation of activity towards all three substrates with increasing amounts of added antibody was the same which suggests that all three reactions are catalysed by the same microsomal enzyme.
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