Delineation of the antigenic sites of oxidized ribonuclease in the mouse by passive cutaneous anaphylaxis and hemagglutination inhibition

Antigenic regions of oxidized bovine pancreatic ribonuclease (O-RNase) in the mouse were analyzed by passive cutaneous anaphylaxis (PCA) and hemagglutination, using peptide segments encompassing nearly the entire molecule, Purified peptides. 1–20, 11–31, 40–61, 67–85, 67–98, and 105–124 were obtained by enzymatic digestion. Two smaller car☐y (C)-terminal peptides, ala¹¹⁴-124 and 118–124, were synthesized by solid phase procedures. Only the C-terminal half (residues 67 on) inhibited the hemagglutination of O-RNase with mouse antibodies. Studies on the antigenicity of the eicosapeptide 105–124 were particularly interesting. This peptide elicited PCA and was an efficient inhibitor of PCA and hemagglutination produced with the intact antigen. The two synthetic derivatives, ala¹¹⁴-124 and 118–124, reacted with antibody in the hemagglutination of sheep erythrocytes coated with peptide-protein conjugate. These peptides did not inhibit the hemagglutination of peptide 105–124-conjugate, suggesting that peptide 105–124 has an antigenic site not found on them. In addition, the 11-member peptide ala¹¹⁴-124 elicited PC A, demonstrating a valence of two or more. Thus, peptide 105–124 appears to be at least trivalent. The above observations further substantiate the multivalence of this eicosapeptide previously observed in the goat. This C-terminal peptide with its multiple antigenic sites contributes a major share of the antigenicity of O-RNase for the mouse. Species differences were observed in the antigenic map of O-RNase. Unlike rabbits and goats, mice responded to the 67–98 region but not to the 40–61 region which is a major determinant in the other species.