Monoclonal antibody recognizes a conformational epitope in a random coil protein.
The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not recognize intact native cytochrome c, but they crossreact with the heme-containing peptides 1-38 and 1-65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2-4 is conformational and discontiguous, it is composed of residues close to the N-terminus and around position 25. The other monoclonal antibody, Cyt-1-59, seems to recognize a contiguous epitope close to the N-terminus. The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme-catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2-4 is particularly interesting because this antibody inhibits the proliferation of a T-cell clone specific for apo-cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547-548].
Saad
B
B
Biochemisches Institut, Universität Zürich, Switzerland.
Corradin
G
G
Bosshard
H R
HR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001059:Apoproteins / Q000276:immunology*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000276:immunology*; D045304:Cytochromes c; D000939:Epitopes / Q000276:immunology*; D006418:Heme / Q000276:immunology; D006736:Horses; D007124:Immunoenzyme Techniques; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000276:immunology; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D001059:Apoproteins; D003574:Cytochrome c Group; D000939:Epitopes; D010455:Peptides; D006418:Heme; D045304:Cytochromes c