Immunological Activity of Cytochrome c

Formationof antibodies to human cytochrome c was observed in nearly all rabbits immunized with the antigen incorporated in complete Freund's adjuvant.About 30 to 40% of the antibodies failed to react with Macaca mulatta cytochrome c, which differs from the human protein only at Residue 58, where M. mulatta cytochrome c has a threonyl residue and the human protein has isoleucine.Only human and kangaroo cytochromes c, which have isoleucyl residues at this position, reacted with those antibodies which failed to combine with M. mulatta cytochrome c.In contrast, antibodies elicited in rabbits by M. mulatta cytochrome c reacted identically with M. mulatta and human cytochromes c.Thus, the presence of the hydrophobic isoleucine correlates with the occurrence of an immunogenic determinant, whereas in the absence of isoleucine, this region is immunogenitally inert.Those antibodies to human cytochrome c which do combine with the M. mulatta protein reacted equally well with both proteins, indicating that the identity in amino acid sequence, outside the "isoleucine site," correlates with identity in antigenic structure.In contrast, some of the antigenic sites of kangaroo cytochrome c which interacted with antibody to human cytochrome c displayed a lower affinity for the antibody than corresponding sites on the human protein.The "isoleucine site," however, was found to be antigenically identical in the human and kangaroo proteins.The appearance of this isoleucine in human and kangaroo cytochromes c, and its importance in immunogenicity, emphasize the difficulties inherent in attempting to analyze evolutionary pathways through antigenic analysis.In the preceding paper (1) we described some of the properties and cross-reactions of antibodies to human, horse, and ~l~acaca mulatta cytochromes c.Evidence was presented that suggested the presence of an important antigenic determinant in human