Fluorescence-based assay of estrogen receptor using 12-Oxo-9(11)-dehydroestradiol-17β

Abstract

12-Oxo-9(11)-dehydroestradiol-17β (12-oxo-E 2) was used to assay estrogen receptor binding in uterine cytosol preparations by an indirect fluorescence assay. In alkaline solution, 12-oxo-E 2 has a fluorescence excitation maximum at 402 nm (ϵ = 24,000) and an emission maximum at 480 nm ( φ f = 0.57), and its fluorescence can be observed down to 5 × 10 −11 m. The minimum detection limit of 12-oxo-E 2 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-E 2 with the receptor preparation and removal of excess free ligand, the receptor-12-oxo-E 2 complex is disrupted, and fluorescence measurements are made on the dissociated 12-oxo-E 2 in alkaline medium. This fluorometric assay was validated quantitatively by performing simultaneously, on the same receptor preparation, radiometric and fluorometric assays with [ 3H]E 2 and [ 3H]-12-oxo-E 2. 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]E 2 radiometric assay. The use of 12-oxo-E 2 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.