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Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A
Y Katakura1, T Kumamoto, Y Iwai
1Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan. katakura@bio.eng.osaka-u.ac.jp
Abstract:
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. Whereas 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.