Dissociation kinetics of bivalent ligand-immunoglobulin E aggregates in solution.
We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented.
Posner
R G
RG
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853.
Erickson
J W
JW
Holowka
D
D
Baird
B
B
Goldstein
B
B
D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D004058:Diffusion; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007073:Immunoglobulin E / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D051379:Mice; D008962:Models, Theoretical; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D012996:Solutions; D013861:Thiocyanates; D007073:Immunoglobulin E; D016650:Fluorescein-5-isothiocyanate