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 anti body 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 wil d-type sFv and a mutant sFv D31A (Asp(31) 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-t ype sFv is almost the same as that of D31A in the presence of more tha n 1 M NaCl. These results demonstrate that Asp(31) of the heavy chain interacts electrostatically with a positively charged amino acid resid ue of RNase A. (C) 1998 Elsevier Science Ltd. All rights reserved.