Immunochemical studies of the a allotypes of rabbit heavy chain variable regions—II

Immunological properties of peptides from two different heavy chains of a3 allotype and restricted heterogeneity were studied using radioimmunoassays that involved inhibition by these peptides of a reaction between¹²⁵I labeled anti-a3 antibodies and Sepharose-bound a3 IgG. A purified fraction of a2-anti-a3 which cross reacts with at IgG was used for the assays of peptides from one of the heavy chains (AH80-5). The picomoles of various inhibitors required for 50% inhibition of the a3-anti-a3 reactions were determined and the corresponding ΔG⁰ values were calculated. Citraconylation of the lysine side chains (CH) markedly affected the inhibitory activity of one of the two heavy chains (AH80-5) and had a lesser effect [Δ(ΔG⁰) 1.4 kcal/mole] on the other. In both instances when the lysines of the tryptic peptides were deblocked by exposure to low pH, we observed gains in activity (1.3–1.7 kcal/mole). Although some of the primary structure which contributes to some a3-antigenic determinants may have been removed by tryptic digestion, the immunopeptides which we recovered inhibited at least 60% of the maximal binding of purified, labeled a 2 anti-a3 antibodies to a3 IgG. Thus major antigenic determmant(s) recognized by these particular anti-a3 antibodies, were retained on the portions of V_H region recovered from the tryptic digests. It is probable that losses in conformation contributed to the changes in ΔG⁰