Studies on the Structural Localization of Rabbit H Chain Allotypic Determinants Controlled by the a Locus PURIFICATION AND IMMUNOLOGICAL PROPERTIES OF AN IMMUNOPEPTIDE BEARING

An immunopeptide bearing a3 allotypic determinant(s) was isolated from the y chain of an a3 homozygous rabbit (G222-2) immunized with type III pneumococcal vaccine. Immunological properties of peptides were studied using a radioimmunoassay that involved inhibition by these peptides of a reaction between ““I-labeled anti-a3 antibody and Sepharose-bound a3 immunoglobulin G (IgG). The y chain was isolated from IgG of restricted heterogeneity and then citraconylated and digested with trypsin. The tryptic digest (TDl) was passed through an anti-a3 immunoabsorbent column either directly or after an intermediate step of Sephadex G-75 chromatography. The bound peptides (Tl) were eluted with 0.1 M acetic acid and further digested with tryps@. The digest (TD2) was again run on the anti-a3 immunoabsorbent column to purify the bound immunopeptide T2. In the radioimmunoassay this immunopeptide was found to have major a3 det,erminant(s). Its molecular weight was found to be approximately 6,000, which decreased to about 3,000 after reduction and alkylation. These data, together with NH,and COOH-terminal analyses and cysteine peptide mapping, demonstrated that T2 is composed of two polypeptide chains linked by a disulfide bond, one from the cysteine 22 region having lysine at the COOH terminus and the other from the cysteine 92 region having arginine at the COOH terminus. The lysine peptide was separated from the arginine peptide and its NH,-terminal sequence was found to be Gly-Asx-Glx-Ser-Thr-Cys. Since the cysteine is at position 22, the lysine peptide starts at position 17. It has approximately 22 residues. The framework sequence from 17 to 20 is different from those reported so far. In addition, the heavy chain used in these studies has some other unusual features including a histidine, probably in the first hypervariable region. The presence of histidine in the first hypervariable region of rabbit heavy chain has not been reported previously. The other peptide which is about 30 amino acids in length and ends with arginine 94, probably includes positions 67, 70, 71, 84, and 85 that are believed to have substitutions correlating with a allotypes. In a hypothetical three-dimensional model of the F, portion of rabbit anti-S111 antibody BS-5, residues 17 to 33 of the lysine peptide and 67 to 79 and 84 to 85 which may be present in the arginine peptide are fully exposed on the surface and are far removed from the antibody combining site.