Numazawa M, Satoh S, Ogura Y, Nagaoka M
Anal Biochem. 1985 Sep;149(2):409-14. doi: 10.1016/0003-2697(85)90591-3.
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.
我们利用带有伏安检测器的反相高效液相色谱法，开发了一种灵敏且非放射性的雌二醇2-羟化酶和16α-羟化酶活性检测方法。通过用大鼠肝脏微粒体孵育雌二醇产生的2-羟化和16α-羟化雌激素，最初使用QAE-葡聚糖A-25硼酸盐柱分离为儿茶酚和酚类组分。使用3-甲氧基-1,3,5(10)-雌三烯-2,16α,17β-三醇或4-羟基雌酮17-肟作为内标，可检测低至0.5-1 ng的代谢物。2-羟化酶和16α-羟化酶的表观Km和Vmax分别为41.9 μM和1.3 nmol/mg蛋白质/分钟，以及82 μM和480 pmol/mg蛋白质/分钟。
