Determination of estradiol 2- and 16 alpha-hydroxylase activities in rat liver microsomes using high-performance liquid chromatography.
We have developed a sensitive and nonradiometric assay of estradiol 2- and 16 alpha-hydroxylase activities using reverse-phase high-performance liquid chromatography with voltametric detector. The 2- and 16 alpha-hydroxylated estrogens produced by the incubation of estradiol with rat liver microsomes were initially separated into the catechol and phenolic fractions using a QAE-Sephadex A-25 borate column. The metabolites were detected in quantities as low as 0.5-1 ng using 3-methoxy-1,3,5(10)-estratriene-2,16 alpha,17 beta-triol or 4-hydroxyestrone 17-oxime as an internal standard. Apparent Km and Vmax of the 2- and 16 alpha-hydroxylases were 41.9 microM and 1.3 nmol/mg protein/min, and 82 microM and 480 pmol/mg protein/min, respectively.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000302:isolation & purification*
D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D039181:Steroid 16-alpha-Hydroxylase; C020120:estrogen 2-hydroxylase