Different functional boundaries for the major antigenic region of two cytochromes c

The antigenic sites of horse and rat cytochromes c in the major antigenic region were compared using a panel of variant cytochromes c and a large number of BALB/c mouse monoclonal antibodies (mAbs) in competitive ELISAs. The major antigenic region of cytochrome c is located on the surface opposite of that containing the exposed heme crevice. mAbs specific for this region on rat cytochrome c were affected in binding by amino acid substitutions at positions 62 and at one or more of positions 3, 100, 103, and 104 but not by a substitution at position 89. In contrast, mAbs specific for the same region on horse cytochrome c were affected by amino acid substitutions at positions 62, (probably) 60, and 89. Some, but not all, of the anti-horse cytochrome c mAbs were affected by amino acid substitutions at one or more of positions 3, 100, 103, and 104. Thus, the functional boundaries of this antigenic region are different for these two cytochromes c, as shown primarily by the differential effects of residue 89. This distinction is most likely due to one or more of the four amino acid differences between horse and rat cytochromes c on the antigenic surface that result in different physicochemical properties for the horse and rat proteins. The results suggest that as yet unidentified physicochemical parameters, possibly surface topography and/or charge distribution, influence the focusing of antibodies onto the surface of a protein antigen.