Phylogeny of immunoglobulin structure and function. IX. Intramolecular heterogeneity of shark 19S IgM antibodies to the dinitrophenyl hapten.

IgM antibodies to DNP were isolated by affinity chromatography from the sera of nurse sharks immunized with dinitrophenylated streptococcal cells. The isolated antibodies from some animals were of the 19S variety, although in other cases both 19S and 7S forms were seen; there was no evidence of a temporal sequence when both species were present. Equilibrium dialysis studies of the isolated antibodies demonstrated several important points: 1) the 19S antibodies exhibited heterogeneity of ligand binding with an average of five high and five low affinity sites per molecule; 2) the 7S antibodies, when isolated, appeared to have one high and one low affinity site of similar affinity as those of the 19S molecules from the same bleeding; and 3) no evidence of increased affinity of either antibody population was seen for up to 21 months of immunization. To study the basis for the ligand binding heterogeneity of the 19S antibodies, several different approaches were employed. The results of studies with proteolytic fragments showed that neither steric hindrance nor allosteric effects could account for the observed heterogeneity. Preparative liquid isoelectric focusing was used in attempting to separate structurally homogeneous 19S antibodies. The focused 19S anti-DNP preparations contained an average of five high and five low affinity sites per molecule. Recombination studies with mildly reduced heavy and light chains from focused 19S antibodies resulted in the recovery of active ~7S recombinants containing high and low affinity sites indistinguishable from those of the original molecules. These findings suggest that the isoelectrically focused preparations were homogeneous and the heterogeneity of ligand binding was an intramolecular phenomenon. Furthermore, it was observed that treatment of shark 19S anti-DNP antibodies with guanidine HCl resulted in the conversion of the five high affinity sites to low affinity ones, i.e., the resultant 19S molecules contained 10 low affinity sites. The conclusion of this study is that the heterogeneity of ligand binding by nurse shark 19S antibodies to the DNP moiety cannot be attributed solely to intermolecular structural heterogeneity but rather likely involves intramolecular heterogeneity at the conformational level.