title: Evaluation of a highly efficient aryl azide photoaffinity labeling reagent for the progesterone receptor.
abstract: 16 alpha,17 alpha-[(R)-1'-(4-Azidophenyl)ethylidenedioxy]pregn-4-ene- 3,20-dione (7) was prepared in high specific activity tritium-labeled form (20 Ci/mmol) and shown to bind to the progesterone receptor with an affinity (Kd = 0.80 nM) that is 47% of that of [3H]-R 5020 (Kd = 0.38 nM). [3H]Progestin aryl azide 7 exhibits high photoattachment efficiency (60% at 1 h) compared to the commonly used progesterone receptor photoaffinity labeling reagent [3H]-R 5020 (2.2% at 1 h) and is the most efficient progesterone receptor photoaffinity labeling reagent prepared to date. The photoattachment observed with 7 proceeds in a time-dependent fashion, with most of the attachment occurring within the first 10 min of photolysis. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of two adducts of molecular weight 108,500 +/- 800 and 87,000 +/- 1,500 (n = 3), the same species as labeled by [3H]-R 5020. The ratio of progesterone receptor subunits A:B was determined to be 3.3:1 with both [3H]progestin azide 7 and [3H]-R 5020. Information on the specific amino acid(s) that attach to the ligand during photolysis awaits further analysis of the covalently bound ligand-protein adduct.
authors.affiliation: Department of Chemistry, University of Illinois, Urbana 61801, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Kym
authors.forename: K E
authors.initials: KE
authors.lastname: Carlson
authors.forename: J A
authors.initials: JA
authors.lastname: Katzenellenbogen
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001667:Binding, Competitive; D005260:Female; D010777:Photochemistry; D011282:Pregnenediones / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011980:Receptors, Progesterone / Q000737:chemistry / Q000378:metabolism*
chemical_list: C093050:16,17-(1'-(4-azidophenyl)ethylidenedioxy)pregn-4-ene-3,20-dione; D000345:Affinity Labels; D001386:Azides; D011282:Pregnenediones; D011980:Receptors, Progesterone