6533b86dfe1ef96bd12caa78

RESEARCH PRODUCT

Epoxides derived from various polycyclic hydrocarbons as substrates of homogeneous and microsome-bound epoxide hydratase. A general assay and kinetic properties.

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A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the incubation mixture after incubation allowed unreacted epoxide and its phenolic by-product to be extracted into light petroleum whilst the product dihydrodiol remained in the aqueous phase. The product was then extracted into ethyl acetate and estimated radiochemically. This assay gave low extraction blanks (0.8-3.8%) when six K-region epoxides of polycyclic hydrocarbons were used, with high recoveries of the corresponding dihydrodiol in the ethyl acetate phase (65-89%). Radiochromatograms demonstrated that all the radioactivity in the ethyl acetate extracts of active incubations above that of boiled enzyme blanks was confined to a single band that always cochromatographed with the authentic trans-dihydrodiol. Using this assay, the kinetic parameters of six K-region epoxides were estimated. In all cases the apparent Km was low (2-5.9 muM). This is about 100-fold lower than the known apparent Km of epoxide hydratase for styrene oxide, an alkene oxide that is widely used as a substrate for epoxide hydratase. The rate of hydration varied with the substrate. Thus the maximum velocity for hydration of phenanthrene 9,10-oxide greater than 7-methylbenz[a]anthracene 5,6-oxide approximately benz[a]anthracene 5,6-oxide approximately benzo[a]pyrene 4,5-oxide greater than 3-methylcholanthrene 11,12-oxide greater than dibenz[a,h] anthracene 5,6-oxide. This relationship between the individual epoxides was found in microsomal fractions from both rat skin and rat liver, although the activity was always much lower in skin microsomes. All six arene oxides derived from polycyclic hydrocarbons were substrates for the homogeneous epoxide hydratase that was isolated from rat liver microsomal fractions using styrene oxide, an alkene oxide, as substrate to follow the purification.