Local Diffusion and Concentration of IgG near Planar Membranes:  Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy

Abstract

The local diffusion coefficients and concentrations of a fluorescently labeled, monoclonal IgG in the solution adjacent to substrate-supported phospholipid bilayers have been measured by using total internal reflection with fluorescence correlation spectroscopy (TIR−FCS). In these measurements, the evanescent depth was ≈1000 Å. TIR−FCS data were obtained as a function of membrane composition, pH, and ionic strength. Best-fits to previously determined theoretical forms provided measures of the average local IgG concentrations and diffusion coefficients. These values were compared to the bulk concentrations and diffusion coefficients, measured far from the membrane, by using FCS with a focused beam. The bulk diffusion coefficient was also measured with dynamic light scattering. The IgG concentrations close to the membrane surface were not significantly different from those in the bulk. However, the local diffusion coefficients, as measured by TIR−FCS, were faster at low ionic strength and slower at high ionic strength. The observed ionic strength dependence of the local diffusion coefficient was not observed in the bulk. This work describes the first measurement of protein concentration and diffusion close to model membrane surfaces by using TIR−FCS. Future investigations with smaller evanescent wave depths, obtained by using high refractive index substrates, are expected to provide much more information about the physical dynamics of proteins in close proximity to membranes. This information should contribute significantly to the current picture of the physical factors that govern interactions between soluble ligands and membrane-associated receptors.