Immunological activity of cytochrome c. I. Precipitating antibodies to monomeric vertebrate cytochromes c.

The production of antibodies by rabbits in response to monomeric preparations of human, Macaca mulatta, and horse cytochromes c, incorporated in complete Freund’s adjuvant, is described. These antibodies precipitate with the monomeric homologous antigens, whereas no such precipitates occurred with antisera elicited by monomeric preparations of the kangaroo (Macropus canguru), turkey, and tuna proteins, or by preparations of these last two cytochromes c conjugated to acetylated bovine y-globulin. The most potent immunogen was the human protein and the antibodies elicited were shown to be associated with the yG-globulin fraction of serum. The cross-reactions of the three precipitating antisera with the cytochromes c from 25 different eukaryotic species were studied by quantitative complement fixation and by competition of unlabeled heterologous antigen with 1251labeled homologous antigen for its combination with antibody. Cytochromes c from different species which have identical amino acid sequences cannot be distinguished immunologically. With some antisera there is a rough correlation between the number of differences in primary structure among various cytochromes c and the homologous antigen and the extent of their immunological cross-reactivity. Some antisera do not distinguish between cytochromes c of difIerent amino acid sequences. For those cytochromes c which differ by single residues, the immunological distinction may vary from very minor to major. In a small globular protein such as cytochrome c, the effects of amino acid substitutions on antigenic properties can serve to identify and characterize antigenic determinants.