Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations.
Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal antibodies specific for this protein. Thus, we have N-formylated the single tryptophan found in horse cytochrome c at position 59 and N-carbethoxylated one of the histidyl residues, which was determined to be at position 26 by the analysis of proteolytic cleavage fragments of the modified protein using liquid secondary ion-mass spectrometry on triple quadropole or tandem quadropole Fourier transform instruments. We discuss the impact of these modifications on the antigenicity of horse cytochrome c with regard to the conformational perturbations introduced by such modifications and with reference to our previous studies on the binding sites of these antibodies using other methodologies (Jemmerson, R., and Paterson, Y. (1986) BioTechniques 4, 18-31).
D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate; D000939:Epitopes / Q000378:metabolism*; D006736:Horses; D007834:Lasers; D013058:Mass Spectrometry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013237:Stereoisomerism; D014364:Tryptophan / Q000378:metabolism
D003574:Cytochrome c Group; D000939:Epitopes; D010446:Peptide Fragments; D014364:Tryptophan; D004047:Diethyl Pyrocarbonate