title: Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.
abstract: 2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D012710:Serum Albumin, Bovine; D013367:Submitochondrial Particles / Q000378:metabolism; D014475:Uncoupling Agents
chemical_list: D000345:Affinity Labels; D009596:Nitrophenols; D010743:Phospholipids; D014475:Uncoupling Agents; D012710:Serum Albumin, Bovine