Use of high-performance liquid chromatography in the evaluation of the synthesis and binding of fluorescein linked steroids to estrogen receptors.
A fluorescein-linked estrogen was synthesized as a non-invasive, non-radiochemical means of detecting the levels and distribution of estrogen receptors in histological preparations of breast and endometrium. 17 alpha-Ethynylestradiol-21-carboxylic acid was coupled via octane-1,8-diamine to fluorescein-isothiocyanate yielding a promising ligand, N-fluoresceinyl-5,N"-[8-(3,17 beta-dihydroxy-19-nor-17 alpha-pregna-1,3,5 (10)-triene-20-yne-21-carboxylic acid amide)]octylthiourea (F8DE) for an estrogen receptor. High-performance liquid chromatography on preparative reversed-phase C18 columns was used to purify the final product. Using cytosolic receptor preparations from bovine uterus and human uterus and breast cancer, the binding of F8DE was determined by competition analyses to have a Kd value of 10(-8) M. High- and low-molecular-weight forms of estrogen receptors were separated on TSK 3000SW and 4000SW columns by high-performance size-exclusion chromatography. Specific binding of radio labeled estradiol-17 beta to these forms was inhibited in the presence of F8DE, indicating association with the fluorescein-linked steroid.
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D005260:Female; D019793:Fluorescein; D005452:Fluoresceins; D006801:Humans; D011960:Receptors, Estrogen / Q000302:isolation & purification / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism
D005452:Fluoresceins; D011960:Receptors, Estrogen; D013256:Steroids; D019793:Fluorescein