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.

Semisynthetic cytochrome c.

Horse heart cytochrome c can be split with cyanogen bromide into a heme peptide (residues 1-65) and a nonheme peptide (residues 66-104). In a process involving (i) complex formation between the two fragments and (ii) restoration of the severed peptide linkage, a fully active cytochrome c preparation can be re-formed. Use has been made of this process to couple the heme peptide to peptide 66-104 synthesized by the Merrifield solid-phase procedure. The semisynthetic product formed in this manner is indistinguishable from reconstituted cytochrome c prepared with nonsynthetic peptide 66-104.

Reconstitution of horse heart cytochrome c: interaction of the components obtained upon cleavage of the peptide bond following methionine residue 65.

Horse heart cytochrome c can be divided into a heme peptide of 65 residues (H65CNBr) and a nonheme peptide of 39 residues (N39CNBr) by treatment of the molecule with cyanogen bromide. Upon mixture of the two peptides in aqueous solution, a 1: 1 complex with properties closely resembling those of the parent heme protein can be formed. The reaction can conveniently be effected in sodium acetate buffer of pH 4.7, with the H65CNBr in the reduced form. The heme peptide is predominantly in the high-spin state under these conditions, and, upon the addition of N39CNBr, is converted to a complex with absorption and circular dichroism spectra which correspond closely to those of ferrocytochrome c. If N39CNBr is added to the oxidized form of H65CNBr, the spectral properties of the product differ appreciably from those of the parent protein. A complex with absorption and circular dichroism spectra comparable to those of ferricytochrome c can readily be obtained, however, through oxidation of the product of the reaction of N39CNBr with H65CNBr in the reduced state. The complex formed in this manner has an absorption band at 695 nm, exhibits no tryptophan or tyrosine fluorescence, and is active in the succinate oxidase system.