Mapping antibody binding sites on cytochrome c with synthetic peptides: are results representative of the antigenic structure of proteins?
Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.
Schwab
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C
Biochemisches Institut der Universität Zürich, Switzerland.
Twardek
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A
Lo
T P
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Brayer
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GD
Bosshard
H R
HR
D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001059:Apoproteins / Q000276:immunology; D002846:Chromatography, Affinity; D003574:Cytochrome c Group / Q000276:immunology*; D045304:Cytochromes c; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology*; D011506:Proteins / Q000276:immunology; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D000906:Antibodies; D000941:Antigens; D001059:Apoproteins; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c