N-bromosuccinimide oxidation and amino acid content of isoelectrofocused fractions from a DNP antibody population.

The reagent -bromosuccinimide (NBS) has been employed to investigate the role of antibody tryptophan in the binding of 2,4-dinitrophenyl (DNP) ligands by isoelectric anti-DNP antibody components. NBS titration in 0.1 M acetate—8 M urea (pH 4) buffer established a direct relationship between the extinction coefficients, fluorescence yields, maximum quench values, and tryptophan contents of the isoelectric antibodies. However, there was no consistent correlation between these properties and the relative affinities of these antibodies. Treatment with NBS in 0.05 M acetate (pH 4) buffer resulted in the modification of approximately 50–62 per cent of the tryptophans which were modified in the 0.1 M acetate—8 M urea (pH 4) buffer. This was accompanied by an approximate 15 to 20-fold reduction in the hapten binding affinity of the isoelectric antibodies. The presence of hapten protected from approximately five to nine tryptophans against NBS modification, and prevented to a great extent the reduction in binding activity caused by NBS treatment. Though NBS treatment in the 0.05 M acetate (pH 4) buffer eliminated 80–90 per cent of antibody fluorescence, the residual fluorescence was to some degree quenchable by hapten. This finding prohibits definitive conclusions with regard to the functional role of antibody tryptophan in antigen binding. It is suggested, however, that while tryptophan is instrumental in the maintenance of full hapten binding affinity, its primary influence is environmental (possibly by establishing a hydrophobic region conducive to the binding of DNP ligands) rather than as a contact residue. The amino acid contents of the isoelectric components were found to differ significantly in a number of amino acids. This supports the conclusion that anti-DNP antibodies exhibit a great degree of degeneracy with respect to properties characteristic of the whole antibody molecule.