Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent

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Abstract
2-Amino-4-nitrophenol was tritiated in an acid-catalyzed H 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 [beef heart] mitochondria. Based on produce 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 .mu.M) 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 bore 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.