Specific photoaffinity labeling of Tyr-49 on the light chain in the steroid-combining site of a mouse monoclonal anti-estradiol antibody using two epimeric 6 alpha- and 6 beta-(5-azido-2-nitrobenzoyl)amidoestradiol photoreagents

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6 alpha- and 6 beta-(5-azido-2-nitrobenzoyl)amido[17 alpha-H-3]estradiol photoreagents (6 alpha- and 6 beta-ANBA-[17 alpha-H-3]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents, In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([H-3]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue, Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6 beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6 alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled, This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain, Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6 alpha- or 6 beta-ANBA-estradiol nitrene derivatives to the peptide.