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

John A. Katzenellenbogen, Kathryn E. Carlson, Rajeshward D. Bindal, Richard L. Neeley, Pierre M. Martin, Henri P. Magdelenat

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)336-348
Number of pages13
JournalAnalytical Biochemistry
Issue number2
StatePublished - Dec 1986


  • fluorescence
  • HPLC techniques
  • organic synthesis
  • radioactivity measurement
  • receptors-hormonal
  • steroids

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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