Determination of estradiol 2- and 4-hydroxylase activities in mouse liver microsomes by liquid...

A sensitive, selective, and nonradiometric assay for estradiol 2- and 4-hydroxylase activities using high-performance liquid chromatography with an electrochemical detector (LCEC) is developed. The assay relies on a liquid-solid extraction of the catechol estrogens onto alumina, followed by their elution with dilute acid. Chromatographic separation is accomplished on a C18 reversed-phase column with an isocratic system consisting of 0.05M sodium acetate in methanol/water/acetic acid 34:56:10 (V/V). Using this assay, the estrogen 2- and 4-hydroxylase activities contained in the microsomal fraction of liver from adult female mice is studied. The assay is optimized with regard to pH, substrate or enzyme concentrations, and time. The apparent km and Vmax of the 4- and 2-hydroxylase are 22.5 microM and 35.9 pmol/mg protein/min and 64.7 microM and 206.2 pmol/mg protein/min, respectively.