Immunochemistry of cytochrome c. Identification of antigenic determinants through the study of hybrid molecules.

P.W Berman,H.A Harbury

doi:10.1016/s0021-9258(18)43711-8

Abstract

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.

Highlights

Antibodies elicited in rabbits against horse cytochrome c cross-react in varying degree with the cytochromes c of other species
It has been shown that other cytochromesc with a methionine residue in position 65 can be split and reconstituted in a comparablemanner,andthat hybrid molecules can be formedbyjoining the heme peptide of the protein of one species to the non-heme peptide of another (11)
Reconstituted Horse cytochrome c-The antibody-binding properties of reconstituted horse cytochrome c were compared with those of the parent protein through quantitative precipitation and radioimmunoassay experiments. Both studies yielded results that showed the two preparations to be antigenically very similar(Figs.4 and 5).it was apparent from the radioimmunoassay experiments that, to a small but significant extent, the reconstituted molecule competed less effectively with'"I-cytochrome c for the binding of RcHo antibodies than did the naturally occurring protein

Summary

IDENTIFICATION OF ANTIGENIC DETERMINANTS THROUGH THE STUDY OF HYBRID MOLECULES*

Studies of hybrid cytochromes, prepared bya reconstitution procedure involving the cleavage and re-formation of peptide bond 65-66,show that these differences result from single amino acid replacements. The immune response to protein antigens involves recognition processes keyed to a limited number of residues Attempts to identify these groups are at an early stage in the case of events at thecellular level, but have long constituted animportanttheme in studies of antibody binding. It has been demonstrated that thereconstitution process affords a suitable basis for the preparation of semisynthetic analogs of the cytochromesc (12) Both procedures provide opportunities for the generation of structures required for the immunochemical studies indicated.

ADnteitgeernmicinants of Cytcochrome

RESULTS

Rabbit Ala

DISCUSSION