Evaluation of a Highly Efficient Aryl Azide Photoaffinity Labeling Reagent for the Progesterone Receptor

Philip R. Kym, Kathryn E. Carlson, John A. Katzenellenbogen

Research output: Contribution to journalArticlepeer-review

Abstract

16α, 17α-[(R)-l'-(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.

Original languageEnglish (US)
Pages (from-to)115-122
Number of pages8
JournalBioconjugate Chemistry
Volume6
Issue number1
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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