DETERMINATION OF THE BINDING OF LIGANDS CONTAINING THE N-2,4-DINITROPHENYL GROUP TO BIVALENT MONOCLONAL RAT ANTI-DNP ANTIBODY USING AFFINITY CAPILLARY ELECTROPHORESIS

Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG(2b) antibody and charged ligands that contained a N-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobility of the complex between Ig and its ligand(s). Zwitterionic buffer additives lessened adsorption of pr otein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certa in multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about elec trophoretic mobilities of protein and protein-ligand complexes. The un certainties and assumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monovalent ligands , the dissociation of the ligands from the antibody occurred noncooper atively. The charge on IgG(2b) at pH 8.3 is estimated to be -8.0 +/- 0 .2; this value is obtained by analysis of the electrophoretic mobiliti es of complexes IgG(2b)L(2), where the ligands L are structurally simi lar but have different charges (the charges on the ligands were also d etermined by CE).