Fluorescence polarization study of a salt bridge between a single-chain Fv and its antigen ribonuclease A.
The interaction between a single-chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M-1 in PBS at pH 7.2 and 37 degrees C, respectively. While the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.
Katakura
Y
Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan. katakura@bce.bio.eng.osaka-u.ac.jp
Kumamoto
T
T
Iwai
Y
Y
Kurokawa
Y
Y
Omasa
T
T
Suga
K
K
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001216:Asparagine / Q000235:genetics; D016231:Fluorescence Polarization Immunoassay; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000276:immunology; D012259:Ribonuclease, Pancreatic / Q000276:immunology*; D012492:Salts / Q000276:immunology; D012965:Sodium Chloride / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D012492:Salts; C055966:immunoglobulin Fv; D012965:Sodium Chloride; D001216:Asparagine; D012259:Ribonuclease, Pancreatic; D000409:Alanine