The His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values.
We examined the effects of 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 for 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 values, 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.
Ito
W
W
Institute for Comprehensive Medical Science, Fujita Health University, Aichi, Japan.
Sakato
N
N
Fujio
H
H
Yutani
K
K
Arata
Y
Y
Kurosawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D005260:Female; D005803:Genes, Immunoglobulin; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D006639:Histidine; D009113:Muramidase