6533b7dbfe1ef96bd1270829

RESEARCH PRODUCT

ISOLATION OF RAT LIVER EPOXIDE HYDRATASE: PROPERTIES AND SUBSTRATE SPECIFICITY OF THE PURE ENZYME

subject

description

ABSTRACT The microsomal enzyme epoxide hydratase has been purified to homogeneity as judged by electrophoretical, ultracentrifugal and immunological criteria and by C- and N-terminal analysis. The preparation procedure consisted of solubilisation using the non-ionic detergent cutscum, (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-cellulose and cellulose phosphate and hydrophobic chromatography on butyl-sepharose. The product was detergent-free, had a relatively high content of hydrophobic amino acids and tended to aggregate in aqueous solutions. The protein had a minimum molecular weight of 49,000 ± 500 with a sedimentation coefficient of S20w≃ 3. Antibodies raised against the homogeneous preparation precipitated the entire hydratase activity in solubilised microsomes towards benzo(a)pyrene 4,5-(K-region-)oxide and styrene oxide as substrates. Moreover, the pure enzyme had a very broad substrate specificity, hydrating both arene and alkene oxides. The same general relationship between the hydration velocities of 6 K-region epoxides of polycyclic hydrocarbons was obtained with rat liver microsomal fractions and pure epoxide hydratase. These findings suggest that the enzyme isolated is the only one present in the rat liver microsomal fractions which is responsible for the hydration of such substrates.