Comparison of Two Hapten-specific Rabbit Antibodies

From the ¹ From the Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Individual rabbits received injections of bovine serum albumin (BSA) coupled with equivalent amounts of p-azophenylarsonate (As) and p-azophenyl-N-trimethylammonium (R₄N) ions. Injection of the doubly labeled azoprotein ensured initial equal distribution of the two haptens among the cells involved in antibody formation.

The immune sera of the injected animals contained hapten-specific antibodies of the types anti-As and anti-R₄N, and a mixture of protein-specific antibodies directed against the various determinant groups of BSA. The hapten-specific antibodies were separated from each other and from anti-BSA by means of immunosorbents containing As or R₄N residues bound to hydroxyphenylazobenzyl cellulose.

Anti-As and anti-R₄N were digested with trypsin and chymotrypsin. Peptide maps of the digests of the two antibodies were extremely similar. Usually, small differences in one or two peptide spots were observed. However, small differences were also found between antibodies produced by an individual rabbit at different times and between antibodies produced in various rabbits of the same injection series.

Column chromatograms of the enzymatic digests of the two hapten-specific antibodies, anti-As and anti-R₄N, showed only 20 to 25 peaks, most of them identical in both types of antibodies. Although differences in one or two peaks were observed, they were not the same when the two hapten-specific antibodies were prepared from an immune serum taken from the same animal at another time, or from the immune serum of another rabbit.

Starch gel electrophoresis of the reduced and alkylated peptide chains of the purified hapten-specific antibodies at pH 8.2 revealed the presence of two cathodic bands produced by the A chains, and 6 to 8 narrow anodic bands of B chains. The A chains of anti-As migrated consistently faster cathodically than those of anti-R₄N. The patterns of bands formed by the B chains of anti-As and anti-R₄N were very similar; although differences in the intensity of some of the bands were observed, they were not found consistently, and they varied in the antibodies isolated from an individual rabbit at different times. Larger differences were produced by disk electrophoresis in polyacrylamide gel.

Our results show that each of the two purified hapten-specific antibodies consists of a mixture of antibody molecules differing from each other by 6 to 8 types of B chains, possibly also by heterogeneity of the A chains. The absence of any consistent difference between the peptide maps or column chromatograms of the digests of anti-As and anti-R₄N raises the possibility that heterogeneity involves not only the non-specific parts of the antibody molecules but also the combining sites, and that the specific combining site of a hapten-specific antibody such as anti-As may be formed by various amino acid sequences, each of these resulting in a conformation which fits closely to the homologous hapten.

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