Affinity Labelling of Antibodies with Aryl Nitrene as Reactive Group

THE covalent binding of a haptene to its specific antibody with a view to identifying the section of peptide chains which form the combining site has been pioneered by Singer et al.1,2, who named this approach affinity labelling. Antibodies were prepared in rabbits against several aromatic haptenes such as benzene arsonate, and affinity labelling was performed by reaction with p-(arsonic acid)-benzene diazonium fluoroborate through the reactive diazonium group. The reaction with specific antibody was considerably more rapid than that with inert IgG because of the concentration of reagent in the combining site. Subsequent separation of heavy and light chains followed by enzymic digestion led to the isolation of one predominant dipeptide (Val-Tyr) from the light chain and another (Thr-Tyr) from the heavy chain; both of these were substituted on the tyrosine residue. No sequence data were available on the peptide chains of rabbit IgG, but from comparison with the known sequences of the light chains of human and mouse IgG it was suggested that the tyrosine residue 86 in the light chain may have been the one labelled.