Journal of Biochemistry Advance Access originally published online on March 3, 2008
Journal of Biochemistry 2008 143(6):781-792; 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 
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-Diaminoalkanes as a Tracer for Estrogen Receptor
1Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581; 2Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Saitama 345-0043; and 3Department of Applied Molecular Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 812-8581, Japan
To whom correspondence should be addressed. 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
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Abstract |
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Steroidal nuclear receptors (NRs) 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 NRs. We designed and synthesized a series of conjugates of 17
-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with ,
-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 (ER) in the radiolabel binding assay using [3H]17β-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. 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 ER, 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, fluorescence intensity, fluorescent tracer, receptor-binding assay
Abbreviations:
Cbz, carbobenzoxy; DMF, N,N-dimethylformamide; DMSO, N,N-dimethyl sulfoxide; E2(n)cF, the conjugates between 17-ethinylcarboxyestradiol (E2) and caryboxyfluorescein (cF) via ,-diaminoalkanes -NH-(CH2)n-NH-; EDC·HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ER, estrogen receptor; ERR, estrogen-related receptor; ESI, electrospray ionization; EtOAc, ethyl acetate; FAB, fast atom bombardment; GST, glutathione-S-transferase; HOBt, 1-hydroxybenzotriazole; HP-TLC, high-performance thin-layer chromatography; LBD, ligand binding domain; MS, mass spectrometry; PBS, phosphate buffer saline; RP-HPLC, reverse-phase high-performance liquid chromatography; TFA, trifluoroacetic acid and THF, tetrahydrofuran
*Present address: Department of Microbiology, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, 216-8511, Japan.