Ligand binding by murine IgM antibodies: Intramolecular heterogeneity exists in certain, but not all, cases

Abstract

The ligand binding properties of eight hybridoma-derived murine anti-DNP IgM(ϰ) antibodies were analysed by equilibrium dialysis. Four of these proteins exhibited the expected valances of ~ 10 and relatively low affinities (⩽2.2 × 10 5 M −1). The remaining four proteins exhibited valences of considerably less than 10 (⩽8) and relatively high affinities (> 10 6 M −1). When these proteins were subjected to two cycles of lyophilization, those of the former group were observed to still exhibit ~10 sites per molecule with homogeneous affinities similar to those of the respective untreated molecules. However, molecules in the latter group (valences of (⩽ 8) were observed to exhibit only five to six binding sites subsequent to lyophilization with no changes in affinities. When the reductive subunits from each of the IgM(κ) proteins were subjected to trypsinization, two different patterns were observed in terms of the yields of Fabμ fragments. Each of the proteins originally exhibiting ~10 binding sites yielded >90% of the expected Fabμ fragments. In contrast each of the proteins exhibiting ⩽8 binding sites yielded only ~50% of the expected Fabμ fragments. Collectively these results indicate the existence of at least two different forms of murine IgM molecules, those with ~ 10 homogeneous, relatively stable sites and those with only approx. five stable sites. It is suggested that these intramolecular functional differences may be attributable to intramolecular conformational differences.