Ohno Ken-ichi, Fukushima Takeshi, Santa Tomofumi, Waizumi Nobuaki, Tokuyama Hidetoshi, Maeda Masako, Imai Kazuhiro
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Anal Chem. 2002 Sep 1;74(17):4391-6. doi: 10.1021/ac020088u.
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 17alpha-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 17beta-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.
利用雌激素受体（ER）和荧光偏振（FP）开发了一种快速、简单且无危害的内分泌干扰物检测方法。在荧光化合物中，17α-荧光素标记的雌二醇衍生物被选为ER结合测定中最合适的配体，因为它对ER表现出最高的亲和力。在Scatchard图分析中，其凸曲线呈现正协同结合，表明随着配体结合形成二聚体，ER发生构象变化，从而增加了对额外配体的亲和力。根据Hill图分析，其解离常数和Hill系数分别为10.4 nM和1.63。用未标记的17β-雌二醇（E2）进行的竞争性结合测定产生的IC50值为2.82 nM，Hill系数为1.67，因此得到的Ki值为0.65 nM。同样，雌酮、雌三醇、己烯雌酚和他莫昔芬的Hill系数分别测定为0.99、1.17、1.59和2.44。
