Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits--IV. Amino-terminal light chain sequence analyses of restricted homozygous b4 rabbit anti-SIII and SVIII antibodies from partially inbred rabbits.

The structural homogeneity of the light polypeptide chains, derived from rabbit antibodies directed against types III and VIII pneumococcal polysaccharides from 6 individual b⁴ homozygous highly restricted responding rabbits from two partially inbred strains (ACEP and type III), was investigated. Ten antibody fractions were obtained by affinity chromatography using specific immunoadsorbents. The light chains, obtained by complete reduction and alkylation, were examined both by alkaline polyacrylamide disc gel electrophoresis in 10 M urea by polyacrylamide gel isoelectric focusing in 8 M urea. Each antibody fraction showed restricted electrophoretic mobility demonstrating 1 or 2 major light chain zones. Each pair of anti-SIII antibody fractions demonstrated different electrophoretic mobilities for all rabbits studied. The electrophoretic mobility of the isolated antibody light chains was directly related to the electrophoretic mobility of the whole antibody fractions. The amino-terminal sequences, obtained by automatic Edman degradation, of the kappa light chains, following complete reduction and alkylation and gel filtration, were determined for the 10 antibody fractions. Amino acid sequence analyses extended for 19–40 positions (a few included the first hypervariable region). Sequences obtained for all antibody light chain fractions, from restricted responders, were as homogeneous, for the first 28 positions, as a human kappa light chain, from an IgG(γ1) myeloma protein, sequenced by the same methods. In contrast, sequences obtained for antibody light chain fractions from partially restricted responders yielded two or three residues at a number of different positions. Two antibody kappa light chains, obtained from rabbit 723569, were both blocked at their animo-terminal residue. When the rabbit 723569 antibody light chain was reacted with CNBr, two sequences were obtained. Presumably, the CNBr cleaved the methionyl residue at position 4 yielding a peptide resembling the rabbit 722369 antibody light chain sequence from positions 5–19. Also themic acid used for the CNBr cleavage presumably hydrolyzed the Asp-Pro peptide bond in the constant part of the switch region yielding a second sequence which corresponded to the first 24 residues of the constant domain of b₄ allotypic light chains (positrons 110–133). The two antibody light chains obtained from rabbit 711319 and 724812 each gave different amino-terminal sequences, while the two antibody light chains obtained from rabbit 722714 both gave the same amino-terminal sequence for the first 20 residues (except for a Glu/Gln interchange at position 12). The light chains from the different antibody fractions had sequences whied in the V_kI, V_kII, and V_kIV subgroups. No strong primary sequence correlations could be shown to exist within the two strains of rabbits studied nor within the various rabbits shown to have a common lineage.