Synthesis and characterization of a heterobifunctional photoaffinity reagent for modification of tryptophan residues and its application to the preparation of a photoreactive glucagon derivative

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Abstract
The synthesis of the heterobifunctional crosslinking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSCl) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan-containing polypeptides and proteins lacking SH groups. The sulfenyl Cl group of NAPSCl reacts with the indole ring of tryptophan following 2nd-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSCl gave 1 major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon activated the adenylate cyclase of [rat] hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.