Characterization of the partial binding sites present on the isolated light and heavy chains of anti-naphthalenesulfonate antibodies

Abstract

Specifically purified monomeric heavy chains from anti-6-azonaphthalene-2-sulfonate (anti-6,2-aNS) and anti-4-azonaphthalene-1-sulfonate (anti-4,1-aNS) antibodies have been prepared from maleylated, reduced and alkylated antibodies by gel filtration in low ionic strength buffer which allows the high charge density of the maleylated proteins to cause effective repulsion of the individual peptide chains. The light chains from such antibodies or from non-maleylated antibodies have also been prepared. The values of the binding constants of the partial binding sites for specific hapten on the separated H chains have been determined by fluorescence enhancement measurements and by equilibrium dialysis and compared to the binding constant values of the sites in the intact antibodies from which the chains were obtained. The values for H chains are about one-tenth those for intact antibodies, indicating that a large proportion of the original site structure of the antibody is retained in the separated H chains. The presence of a partial binding site on isolated L chains from anti-6,2-aNS antibody has been demonstrated by fluorescence enhancement measurements, extending the previous demonstration of such partial sites on L chains of anti-4,1-aNS antibody by Yoo et al. (1967). The enhanced fluorescence observed is shown to be due to specific binding by L chain alone and not due to the presence of a small amount of H chain in the L chain preparation, through the use of a specific antibody adsorbent for H chain. The microspecificity of the partial binding sites on the isolated H and L chains of anti-4,1-aNS and anti-6,2-aNS antibodies was investigated by determining the relative binding constants of a series of related haptenic structures to these sites by the technique of displacement of fluorescence-enhancing ligand from the sites, as developed previously (Nakamura et al., 1970). The results indicated some differences in the specificity of sites on H chains as compared to sites on L chains, consistent with the view that the partial sites on the two chains from a given antibody are directed against different portions of the specific haptenic structure.