Kinetics of epidermal growth factor/receptor binding on cells measured by total internal reflection/fluorescence recovery after photobleaching

Total internal reflection fluorescence (TIRF) microscopy is used to measure the dissociation kinetic rate of fluorescein-labeled epidermal growth factor from its specific receptors on the surface of intact but mildly fixed A431 human epidermoid cells in culture. Prior applications of TIRF microscopy have been limited to nonreceptor binding or to model membrane systems. The evanescent field excites fluorescence selectively at the surface of the cell proximal to the coverslip. Prismless epiillumination TIR is employed to avoid space limitations and is achieved by passing the excitation laser beam through a high (1.4)-aperture objective so that the light is incident at the glass/water interface beyond the critical angle. Long-term focus is maintained by a special feedback system. Of the possible effects that can influence the time course of the postbleach fluorescence recoveries—the EGF/receptor dissociation ratek₂, the bulk solution diffusion rate of EGF, and the cell surface motion of the receptors—we infer that the dissociation ratek₂ dominates. Several fitting schemes are compared and indicate the presence of a multiplicity of values fork₂, ranging from about 0.05 to 0.004 s^–1, with an average value of about 0.012 s^–1. These results compare well with values previously obtained by radiolabel/washing techniques. The significance of the results in terms of kinetic models and the advantages of the TIRF technique for these sorts of measurements are discussed.