Analytical Sciences

Study on Interactions of EndocrineDisruptors with Estrogen Receptor-β Using FluorescencePolarization

Sachiko SUZUKI, Ken-ichi OHNO, TomofumiSANTA,  and Kazuhiro IMAI

Graduate School of PharmaceuticalSciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,Japan

In this study, we developed a rapid, simpleand homogeneous human recombinant estrogen receptor-β (hrER-β)binding assay method using fluorescence polarization (FP) by applying afluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plotand a Hill plot analysis of the saturation binding assay using F-E2 andhrER-β were studied. F-E2 showed a high affinity for hrER-β, thedissociation constant was 5.53 nM, indicating that F-E2 is applicable tothe hrER-β binding assay. Competitive binding assays using F-E2, inwhich the FP values decreased upon the addition of compounds (competitors)were carried out to evaluate the binding characteristics of compounds withand without biological activities to hrER-β. Twenty-one compounds,such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens,were examined. The obtained sigmoidal inhibition curves were transformedinto pseudo-Hill plots and the concentrations at 50% inhibition(IC₅₀) and Hill coefficients, the degree of cooperativity inER-ligand binding, were obtained from the regression equations.Antagonists exhibited larger Hill coefficients than agonists, showing thecorrelation between the Hill coefficients and the estrogenic/antiestrogenicactivities.

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