Epoxide hydrase in human liver biopsy specimens: Assay and properties

Abstract

A sensitive assay for the determination of epoxide hydrase activity in needle biopsy specimens of human liver has been developed with [ 3H]styrene oxide as substrate and was used for the study of some properties of human epoxide hydrase. Levels of epoxide hydrase in liver of man are 4.71 ± 0.41 nmoles styrene glycol/mg protein per min, comparable to guinea-pig (5·00 ± 0·38) rather than to Rhesus monkey (13·16 ± 0·88). Human hepatic epoxide hydrase which was found exclusively in the microsomal fraction, was solubilized with Cutscum, a neutral detergent, and purified. The enzyme was remarkably stable as long as it was particle-bound. Neither dialysable cofactors nor endogenous activators or inhibitors appeared to exist in the homogenate. The optimum pH for the purified enzyme was 9. The apparent K m was 0.38 mM and the apparent V max was 62.1 nmoles product/mg protein per min with respect to styrene oxide as the substrate. The product, styrene glycol, had no inhibitory effects. High (5–17 mM) concentrations of substrate, styrene oxide, markedly inhibited epoxide hydrase activity at low (0·2 mg/ml) but not at high (2 mg/ml) concentrations of protein, indicating that this inhibition may be due to the alkylating properties of the subsirate, styrene oxide. Sulfhydryl reagents slightly but significantly inhibited the enzyme suggesting that no sulfhydryl group is essentially involved in the catalytic mechanism at the active site, but that sulfhydryl group(s) may be of importance for holding the enzyme molecule in the optimal conformation for maximal activity. Chelating agents, a carbonyl reagent and β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) had no effect.