The His-probe method: Effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values

AbstractWe examined the effects or histidine residues that were artificially introduced into complementarity-determining regions of antibodies on antigen-antibody Interactions at different pH values. Using a monoclonal antibody specific ror hen egg-white lysozyme and three mutant antibodies that contained a histidine residue. we measured binding constants for antibodies and lysozyme at different pH values (PH 5–8). No gross conformational changes were evident over this range of pH vulues, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH-dependent antigen-binding patterns observed between the wild-type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH-dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues