DETERMINATION OF THE BINDING CONSTANTS FOR 3 HPR-SPECIFIC MONOCLONAL-ANTIBODIES AND THEIR FAB FRAGMENTS

Jel42, Jel44 and Jel323 are mouse monoclonal antibodies specific for H Pr, the histidine-containing phosphocarrier protein, of the Escherichi a coli phosphoenolpyruvate:sugar phosphotransferase system. The bindin g constants, K-d, of the three antibodies and their Fab fragments have been determined: Jel42, 2.8 +/- 1.6 nM; Jel42Fab, 3.7 +/- 0.3 nM; Jel 44, 5.1 +/- 0.4 nM; Jel44Fab, 6.3 +/- 1.1 nM; Jel323, 5.7 +/- 0.5 nM; Jel323Fab, 5.1 +/- 0.9 nM. The binding constants were determined by a fluorescence polarization assay that used the mutants Arg17Cys HPr and Phe2Cys HPr specifically labeled with fluorescein-5-maleimide. The la tter was used for Jel323 as interaction with fluorescein-5-maleimide-l abeled Arg17Cys HPr gave quenching of the fluorescence intensity. The specificity of each antibody and the Fab fragments for binding to many HPr mutants was,determined by this solution assay. The Fab fragments had the same specificity or cross-reactivity as the antibodies. Compar ison of relative binding specificity determined by a solid phase assay showed that the results from both types of assay are comparable. Neit her Jel42 nor Jel323 binding was affected by ionic strength (similar t o 45 to 245 mM salt), but Jel44 varied about two- to threefold. Charge d residues are prominent in the Jel44 epitope and paratope. Initial th ermodynamic characterization was investigated by temperature-dependent determinations of the K-d. The binding of Jel42 and Jel323 to HPr was entropic at low temperatures and enthalpic at physiological temperatu res. Jel44 showed no change in the contributions of entropy and enthal py over the temperature range 3 to 37 degrees C. The 2.5 Angstrom reso lution structure of the complex of Jel42 Fab fragment bound to HPr des cribed in the accompanying paper provides some structural intepretatio n for the mutational effects. (C) 1998 Academic Press.