TY - JOUR
T1 - Fluorescence-based assay of estrogen receptor using 12-Oxo-9(11)-dehydroestradiol-17β
AU - Katzenellenbogen, John A.
AU - Carlson, Kathryn E.
AU - Bindal, Rajeshward D.
AU - Neeley, Richard L.
AU - Martin, Pierre M.
AU - Magdelenat, Henri P.
N1 - Funding Information:
Estradiol was a generous gilt of Dr. John Baran of Searle Laboratories. Support of this work by grants from the National Institutes ofhealth (PHS 5 ROl AM15556 to J.A.K.), the Institut de la Sante et la Recherche Medicale, the Association de la Reserche Contre de la Cancer (Villejuif) and the Faculty de Medicine, Universite Aix-Marseille (to P.M.M.), and from the Comite de Coordination Institut Curie, Paris and the Comite de Paris de la Ligne Nationale Francaise Contre le Cancer (to H.P.M.) are gratefully acknowledged.
PY - 1986/12
Y1 - 1986/12
N2 - 12-Oxo-9(11)-dehydroestradiol-17β (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 (ε{lunate} = 24,000) and an emission maximum at 480 nm (φf = 0.57), and its fluorescence can be observed down to 5 × 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.
AB - 12-Oxo-9(11)-dehydroestradiol-17β (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 (ε{lunate} = 24,000) and an emission maximum at 480 nm (φf = 0.57), and its fluorescence can be observed down to 5 × 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.
KW - fluorescence
KW - HPLC techniques
KW - organic synthesis
KW - radioactivity measurement
KW - receptors-hormonal
KW - steroids
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U2 - 10.1016/0003-2697(86)90351-9
DO - 10.1016/0003-2697(86)90351-9
M3 - Article
C2 - 3826620
AN - SCOPUS:0022898286
SN - 0003-2697
VL - 159
SP - 336
EP - 348
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -