Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization

Ken ichi Ohno, Takeshi Fukushima, Tomofumi Santa, Nobuaki Waizumi, Hidetoshi Tokuyama, Masako Maeda, Kazuhiro Imai

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17α-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17β-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.

Original languageEnglish
Pages (from-to)4391-4396
Number of pages6
JournalAnalytical Chemistry
Volume74
Issue number17
DOIs
Publication statusPublished - 2002 Sep 1
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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