title: 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.
abstract: 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 17alpha-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 (ER) in the radiolabel binding assay using [(3)H]17beta-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate K(d)- and B(max)-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 [(3)H]17beta-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asai
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Tokunaga
authors.forename: Kaoru
authors.initials: K
authors.lastname: Kondo
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: Sayaka
authors.initials: S
authors.lastname: Takayanagi
authors.forename: Teruo
authors.initials: T
authors.lastname: Shinmyozu
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakai
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Yakabe
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Shimohigashi
mesh_terms: D000473:Alkanes / Q000737:chemistry*; D001667:Binding, Competitive; D001681:Biological Assay; D004958:Estradiol / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D011869:Radioligand Assay
chemical_list: D000473:Alkanes; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D005452:Fluoresceins; C024098:6-carboxyfluorescein; D004958:Estradiol