The immune response towards beta-adrenergic ligands and their receptors--VII. Equilibrium and kinetic binding studies of l-alprenolol to a monoclonal anti-alprenolol antibody.
Binding of the catecholamine beta-adrenergic antagonist, l-alprenolol, by the IgGl anti-alprenolol monoclonal antibody 37A4 was examined using the radioligand 3H-dihydroalprenolol as an extrinsic signal and the increase in antibody fluorescence upon l-alprenolol binding as intrinsic signal. Equilibrium binding studies based on both signals indicated that the binding process was exothermic with a positive entropy change. The difference in the affinity constants obtained by radioligand binding studies and by fluorescence analysis could be ascribed to the higher affinity of the hydrogenated tritiated l-dihydroalprenolol compared to the unsaturated l-alprenolol. The association rate constants determined by both signals were 10(4)-10(5)/M/sec and showed a high activation enthalpy (8-10 kcal/mol), thus excluding a diffusion controlled reaction. At low temp (7 degrees C), the fluorescence stopped-flow studies showed non-linear pseudo first order kinetics, indicating the existence of a fast pre-equilibrium of low affinity, followed by a conformational change leading to the tight binding of the ligand. The dissociation rate constants determined using both signals were very similar. Thus, the differences in affinity between the hydrogenated and non-saturated l-alprenolol could be ascribed to the association rate constants. Affinity constants and thermodynamic parameters calculated from the kinetic data were in close agreement with those determined by equilibrium binding. The mechanisms of ligand binding are discussed in terms of the interactions of idiotypes and anti-idiotypes in the anti-catecholamine immune response.
Hoebeke
J
J
Department de Biotechnologie, Institut Pasteur, Paris, France.
Engelborghs
Y
Y
Chamat
S
S
Strosberg
A D
AD
D000526:Alprenolol / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology; D000937:Antigen-Antibody Reactions; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004082:Dihydroalprenolol / Q000276:immunology; D006241:Haptens / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011863:Radioimmunoassay; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006241:Haptens; D007074:Immunoglobulin G; D004082:Dihydroalprenolol; D000526:Alprenolol