Immunochemistry of cytochrome c. Identification of antigenic determinants through the study of hybrid molecules.
Antibodies elicited in rabbits against horse cytochrome c cross-react in varying degree with the cytochromes c of other species. Radioimmunoassay experiments are described in which 23% of the immunoglobulin shown to bind to horse cytochrome c fails to react with beef cytochrome c, and 14% of that bound by beef cytochrome c does not react with rabbit cytochrome c. Studies of hybrid cytochromes, prepared by a reconstitution procedure involving the cleavage and re-formation of peptide bond 65-66, show that these differences result from single amino acid replacements, Beef cytochrome c differs in binding capacity from horse cytochrome c as a result of the substitution of a glycine for a lysine residue in sequence position 60, and the difference between rabbit cytochrome c and beef cytochrome c reflects the substitution of a valine for a proline residue in position 44. Reconstituted horse cytochrome c and reconstituted beef cytochrome c have binding capacities indistinguishable from those of the parent proteins. The presence of a homoserine rather than a methionine residue in position 65 results, in the case of the reconstituted horse molecule, in a slightly lower affinity for the antibody population directed against the lysine-60 region. A corresponding difference is not observed in the case of beef cytochrome c and its reconstitution product, which do not bind the population in question.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001666:Binding Sites, Antibody; D002417:Cattle / Q000276:immunology; D003574:Cytochrome c Group / Q000032:analysis / Q000276:immunology*; D000939:Epitopes; D006321:Heart; D006736:Horses / Q000276:immunology; D007106:Immune Sera; D010527:Perissodactyla / Q000276:immunology; D011487:Protein Conformation; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay; D013045:Species Specificity
D000596:Amino Acids; D000906:Antibodies; D003574:Cytochrome c Group; D000939:Epitopes; D007106:Immune Sera