Molecular studies of subspecificity differences among phosphorylcholine-binding mouse myeloma antibodies using ^(31)P nuclear magnetic resonance

Abstract

We have determined the pH dependencies of the binding affinities of the mouse myeloma immunoglobulins M603, W3207, and M167 for the haptens phosphorylcholine (PC) and L-α-glycerophosphorylcholine (GPC). These affinities are generally maximal near neutral pH with the exception of the binding of PC by M167 which is strongest at pH 5.5. These data have helped to clarify the nature and relative importance of the ionic interactions between hapten and antibody. ^(31)P nuclear magnetic resonance (NMR) techniques were used to probe the influence of pH on the microenvironment of the phosphate group of several haptens when these were bound to M603, W3207, and M167. The phosphate subsites of M603 and W3207 are both electropositive and also show other similarities; that of M167 has a net electronegative character. The two hydrogen bonds known to be formed between M603 and the phosphate oxygens of PC are also involved in binding GPC and are essentially unaffected by pH in the region 3-9. Studies with the hapten 3-trimethylamino-l-propanol phosphate (TMAPP) show that the binding cavity of M 167 is substantially wider than those of M603 and W3207. These results lead to a detailed molecular model of the pH dependent binding of PC and related haptens to these three antibodies; they further indicate the roles of various amino acid residues in defining the differing ligand specificities of these antibodies.