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Journal of Biochemistry Advance Access published online on March 3, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn031
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© 2008 The Japanese Biochemical Society

Direct Measure of Fluorescence Intensity for Efficient Receptor-binding Assay: Conjugates of Ethinylcarboxyestradiol and 5(and 6)-Carboxyfluorescein via {alpha}({omega}-Diaminoalkanes as a Tracer for Estrogen Receptor

Daisuke Asai1,*, Takatoshi Tokunaga1, Kaoru Kondo2, Tsutomu Kawaguchi1, Sayaka Takayanagi1, Teruo Shinmyozu3, Makoto Nakai2, Yoshikuni Yakabe2 and Yasuyuki Shimohigashi1,* *

1Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan, 2Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Saitama 345-0043, Japan, and 3Department of Applied Molecular Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 812-8581, Japan

**To whom correspondence should be addressed: Prof. Yasuyuki Shimohigashi: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan. Tel: +81-92-642-2584, Fax: +81-92-642-2584, E-mail: shimoscc{at}mbox.nc.kyushu-u.ac.jp

Received October 17, 2007; Accepted February 19, 2008


  Abstract

Steroidal nuclear receptors have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of nuclear receptors. We designed and synthesized a series of conjugates of 17{alpha}-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with {alpha},{omega}-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n = 2, 4, 6, 8, 10, and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor in the radiolabel binding assay using [3H]17β-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without estrogen receptor. They exhibited sufficiently large specific binding characteristics with adequate Kd and Bmax values. When these fluorescent ligands were used as a tracer for the binding assay against the estrogen receptor, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [3H]17β-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.

Key Words: endocrine disruptors, estrogen receptor, fluorescent tracer, fluorescence intensity, receptor-binding assay

* Present Address: Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, 216-8511, Japan.


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