The chemical nature of the combining site of rabbit anti- p-azophenylphosphorylcholine antibody

Abstract

Determinations were made of the amino acid residues involved as contact residues in the hapten combining site of rabbit antibodies raised against p-azophenylphosphorylcholine by reacting antibody with either diazoacetamide or maleic anhydride followed by glyoxal reagent. The effect of the reaction on ability of the antibody to bind hapten was followed. Reactions were carried out both in the absence and in the presence of the hapten p-nitrophenylphosphorylcholine. A loss of binding activity coupled with the demonstration that the presence of hapten during modification could reduce the loss of activity was taken as evidence that the type of amino acid modified was in the binding site. Glyoxalation of about 70% of the total arginine per molecule (under conditions in which lysyl residues were blocked by maleylation) reduced the number of active binding sites in all of the antibody preparations tested. As much as a 44% loss of sites was observed in one case. These losses could be partially prevented by carrying out the reaction in the presence of hapten. Arginine, therefore, appears to be a part of the combining site of a significant portion of the molecules of each antibody preparation studied. Esterification of about 20% the carboxyl groups with diazoacetamide resulted in a decrease in binding capacity (∼24%) which was protectable by hapten. Although extensive modification of carboxyl groups was not achieved, these results point to the probability that a glutamyl or asparyl residue is in the site. Maleylation of nearly all of the lysine present with maleic anyhdride was virtually ineffective in reducing the binding capacity of these antibodies, thus indicating the absence of lysine in the site. We conclude that a significant portion of the anti- p-azophenylphosphorylcholine antibody population has a positively charged arginyl residue, and a negatively charged carboxylate group, in the site. These residues are those which provide for electrostatic interaction of the site with the negatively charged phosphoryl group and the positively charged quaternary nitrogen of the p-azophenylphosphorylcholine hapten.