Evidence for Ammonium and Guanidinium Groups in the Combining Sites of Anti-p-azobenzenearsonate Antibodies— Separation of Two Different Populations of Antibody Molecules

Abstract The effect of modification of amino and guanidinium groups of rabbit antibodies directed against the negatively charged p-azobenzenearsonate hapten (anti-Rp antibodies) was studied. Purified antibodies from either a single rabbit or from serum pooled from several rabbits lost at least 85 to 90% of their combining sites when 100% of their amino groups were selectively modified with maleic anhydride and 75 to 80% of their guanidinium groups were modified by subsequent treatment with glyoxal. However, they lost 50% or less of their combining sites when only the amino groups were modified. The loss of combining sites could be prevented by the presence of specific hapten (p-nitrobenzenearsonate) during the modification reactions. These results suggest that some anti-Rp antibodies have combining sites which contain guanidinium groups, while others have combining sites which contain amino groups. It was possible to separate these antibody molecules into two such populations. Maleylated anti-Rp antibody molecules were separated into two fractions with a specific solid immunoadsorbent. Approximately half of the maleylated antibodies remained in the solution because they had lost their combining sites as a result of maleylation of an amino group in the combining sites. It was possible to recover completely the antibody activity by removing most of the maleyl groups by acid hydrolysis. These antibodies did not contain glyoxal-reactive groups in their combining sites because treatment with glyoxal either before or after hydrolysis of the maleyl groups resulted in no loss of antibody combining sites. The second fraction of the maleylated antibodies, those which were adsorbed on the solid immunoadsorbent, was eluted and treated again with maleic anhydride, and their combining sites were found to be 100% effective. This showed that there were no amino groups in the combining sites of these antibodies. When these antibodies were treated subsequently with glyoxal, 75% of their combining sites were lost, showing the presence of glyoxal-reactive groups in these sites. Modification with both maleic anhydride and glyoxal of antibodies directed against a positively charged (p-azophenyltrimethylammonium) hapten and a neutral (3-azopyridine) hapten did not result in any losses of combining sites, showing that these antibodies did not contain amino- or glyoxalreactive groups in their combining sites. This investigation showed the presence and separation of at least two distinct populations of anti-Rp antibody molecules —those with combining sites containing guanidinium or other glyoxal-reactive groups and no amino groups, and those with combining sites containing amino groups and no other glyoxal-reactive groups. Amino acid analyses of the glyoxal-modified antibodies showed that arginyl residues had been irreversibly modified. No evidence was found that any other residues had been modified. However, the possibility still exists that other residues were modified, but that the modification was reversed during hydrolysis of the antibodies. Nevertheless, the immunochemical evidence is that the important group altered was a guanidinium group, since only antibodies directed against negatively charged haptens are affected while antibodies directed against neutral and positively charged haptens are not affected.