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 IgG2b 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 protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties 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 noncooperatively. The charge on IgG2b at pH 8.3 is estimated to be -8.0 +/- 0.2; this value is obtained by analysis of the electrophoretic mobilities of complexes IgG2bL2, where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).
Mammen
M
M
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Gomez
F A
FA
Whitesides
G M
GM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004136:Dinitrobenzenes / Q000737:chemistry* / Q000276:immunology; D019075:Electrophoresis, Capillary; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D051381:Rats
D000911:Antibodies, Monoclonal; D004136:Dinitrobenzenes; D007074:Immunoglobulin G; D008024:Ligands