Arginyl residues in the active sites of antibody against the 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP) group

Abstract

Anti-NIP antibody preparations from individual rabbits lost 40–80 per cent of their sites when 70 per cent of their arginyl residues were modified by glyoxal. Antibodies whose sites were protected by hapten and then similarly modified retained more than 90 per cent of their sites. Anti-NIP preparations whose amino groups were completely maleylated lost only 5–13 per cent of their sites. When such maleylated antibodies were treated with glyoxal, there was extensive loss of anti-NIP sites. In view of the known reactivity of glyoxal, it is very likely that only arginyl residues were modified in those antibodies since ammonium, the only other group reactive with glyoxal, was already blocked. These observations establish the presence of a glyoxal-reactive amino acid, most probably arginine, in the affected anti-NIP sites and indicate that few, if any, anti-NIP sites contain an amino group. The absence of amino groups near a significant proportion of anti-NIP sites was also indicated by the failure of anti-NIP antibodies to be affinity labeled with NIP-azide. Thus, guanidinium groups of arginine side chains seem to account for the positive charge involved in binding the phenolate form of the NIP determinant to the active site of its antibodies, while ammonium groups appear to be of minor importance.