Study on Interactions of Endocrine Disruptors with Estrogen Receptor-β Using Fluorescence Polarization

抄録

In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-β(hrER-β) 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-β were studied. F-E2 showed a high affinity for hrER-β, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-β 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-β. 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.