Fluorescence-based assay of estrogen receptor using 12-oxo-9(11)-dehydroestradiol-17 beta.
12-Oxo-9(11)-dehydroestradiol-17 beta (12-oxo-E2) was used to assay estrogen receptor binding in uterine cytosol preparations by an indirect fluorescence assay. In alkaline solution, 12-oxo-E2 has a fluorescence excitation maximum at 402 nm (epsilon = 24,000) and an emission maximum at 480 nm (phi f = 0.57), and its fluorescence can be observed down to 5 X 10(-11) M. The minimum detection limit of 12-oxo-E2 is 25 fmol by spectrofluorometry and 5 fmol by HPLC-fluorometry. Although this compound is not appreciably fluorescent at neutral pH (i.e., at conditions under which it binds to the estrogen receptor), receptor binding by fluorometry can be measured indirectly: After equilibration of 12-oxo-E2 with the receptor preparation and removal of excess free ligand, the receptor-12-oxo-E2 complex is disrupted, and fluorescence measurements are made on the dissociated 12-oxo-E2 in alkaline medium. This fluorometric assay was validated quantitatively by performing simultaneously, on the same receptor preparation, radiometric and fluorometric assays with [3H]E2 and [3H]-12-oxo-E2. The radiometric determinations with both compounds gave nearly equivalent estimates of receptor site concentrations, but the fluorometric estimate of binding site concentration was somewhat less (70-85%) than that expected on the basis of the [3H]E2 radiometric assay. The use of 12-oxo-E2 in an indirect spectro- or HPLC-fluorometric assay provides a means for assaying estrogen receptor concentrations by fluorescence with a sensitivity approaching that of radiometric techniques.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives*; D005260:Female; D005456:Fluorescent Dyes; D011869:Radioligand Assay; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis*; D012756:Sheep; D013050:Spectrometry, Fluorescence; D014599:Uterus / Q000378:metabolism
D005456:Fluorescent Dyes; D011960:Receptors, Estrogen; C051866:12-oxo-9(11)-dehydroestradiol; D004958:Estradiol