Kinetics and mechanism of deuterium oxide-induced fluorescence enhancement of fluorescyl ligand bound to specific heterogeneous and homogeneous antibodies.
Comparative kinetics studies of ligand dissociation and D2O enhancement were performed with both heterogeneous and homogeneous anti-fluorescyl immunoglobulin G antibodies. Heterogeneous rabbit and homogeneous mouse (monoclonal) antibody preparations were purified by immunoadsorption and found to be pure IgG by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Relatively high affinities of all liganded antibody preparations were determined by dissociation rate studies, demonstrating comparatively long lifetimes for the dissociation of bound fluorescein. In addition, rabbit anti-fluorescyl preparations were found to display marked heterogeneity of off-rates while mouse monoclonal anti-fluorescyl preparations exhibited a single off-rate indicating homogeneity. D2O fluorescence enhancement studies showed that heterogeneous kinetics was observed with both heterogeneous and homogeneous antibody active sites. Temperature studies of ligand D2O enhancement and dissociation rates using homogeneous anti-fluorescyl antibodies revealed similar, yet different activation energies (22.7 +/- 0.8 cal and 20.2 +/- 0.3 cal, respectively) for both phenomena. The studies demonstrated that the anti-fluorescein antibody active site consists of both solvent accessible and relatively inaccessible components, and that the binding of ligand involves both exchangeable hydrogen atoms and other as yet unresolved interactions. The mechanism of D2O fluorescence enhancement is discussed in terms of its complexity involving heterogeneous rate mechanisms.
D000818:Animals; D002459:Cell Fusion; D005452:Fluoresceins; D006822:Hybrid Cells / Q000276:immunology; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits / Q000276:immunology; D013154:Spleen / Q000276:immunology; D013816:Thermodynamics
D005452:Fluoresceins; D007074:Immunoglobulin G; D008024:Ligands