Suzuki Sachiko, Ohno Ken-ichi, Santa Tomofumi, Imai Kazuhiro
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Anal Sci. 2003 Aug;19(8):1103-8. doi: 10.2116/analsci.19.1103.
In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.
在本研究中，我们通过应用荧光配体荧光素标记的雌二醇（F-E2），开发了一种利用荧光偏振（FP）的快速、简单且均相的人重组雌激素受体β（hrER-β）结合测定方法。研究了使用F-E2和hrER-β进行饱和结合测定的Scatchard图和Hill图分析。F-E2对hrER-β显示出高亲和力，解离常数为5.53 nM，表明F-E2适用于hrER-β结合测定。进行了使用F-E2的竞争性结合测定，其中加入化合物（竞争者）后FP值降低，以评估具有和不具有生物活性的化合物与hrER-β的结合特性。检测了21种化合物，如激素、药物、工业化学品和植物雌激素。将获得的S形抑制曲线转换为伪Hill图，并从回归方程中获得50%抑制浓度（IC50）和Hill系数（ER-配体结合中的协同程度）。拮抗剂的Hill系数大于激动剂，表明Hill系数与雌激素/抗雌激素活性之间存在相关性。
