Rebinding of IgE Fabs at Haptenated Planar Membranes: Measurement by Total Internal Reflection with Fluorescence Photobleaching Recovery

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• 作者：B. Christoffer Lagerholm ; Tammy E. Starr ; Zoya N. Volovyk ; and Nancy L. Thompson
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：February 29, 2000
• 年：2000
• 卷：39
• 期：8
• 页码：2042 - 2051
• 全文大小：152K
• 年卷期：v.39,no.8(February 29, 2000)
• ISSN：1520-4995

文摘

In previous work, a general analytical theory for ligand rebinding at cell surfaces was developedfor a reversible bimolecular reaction between ligands in solution and receptors on a membrane surface[Lagerholm, B. C., and Thompson, N. L. (1998) Biophys. J. 74, 1215-1228]. This theory can be used topredict theoretical forms for data obtained by using total internal reflection with fluorescence photobleachingrecovery (TIR-FPR) [Thompson, N. L., Burghardt, T. P., and Axelrod, D. (1981) Biophys. J. 33, 435-454]. Thus, one method by which the rebinding theory can be tested is to use TIR-FPR. In the workdescribed herein, the reversible kinetics of mouse monoclonal anti-dinitrophenyl (DNP) IgE Fabs atsubstrate-supported planar membranes composed of 25 mol % DNP-conjugated phosphatidylethanolamineand 75 mol % dipalmitoylphosphatidylcholine have been examined by using TIR-FPR. Data were obtainedas a function of the Fab solution concentration. Higher Fab concentrations reduce rebinding (and increasethe fluorescence recovery rate) because different Fab molecules compete for the same surface-bindingsites. Data were also obtained for solutions containing different volume fractions of glycerol. In thesemeasurements, higher glycerol concentrations increase rebinding (and decrease the fluorescence recoveryrate) because the solution viscosity is increased and the Fab diffusion coefficient in solution is decreased.The TIR-FPR data were quantitatively compared with theoretical predictions which follow from the generaltheory for rebinding at the membrane surface. The data were consistent with the theoretical predictionsand, therefore, provide experimental verification of the previously developed theory.