Dynamics of nonspecific adsorption of insulin to erythrocyte membranes

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Abstract
Molecules may arrive at targets (receptors, enzymes, etc.) localized on a membrane surface by first adsorbing onto the surface and then surface diffusing to the targets. The flux rate of molecules arriving at targets via this mechanism depends on the surface diffusion coefficient of the molecules and, in some circumstances, on the adsorption/desorption kinetics. The technique of total internal reflection with fluorescence recovery after photobleaching (TIR-FRAP) was used here to study these rate parameters of fluorescein-labeled insulin (f-insulin) interacting with erythrocyte ghosts. Ghosts were adhered to polylysine coated slides for TIR illumination. Some ghosts became flattened and unsealed on the polylysine so that both extracellular and cytoplasmic sides of the membrane were openly exposed to the solution. An aluminum thin film between the polylysine and the fused silica of a slide quenched ‘background’ fluorescence from f-insulin adsorbed directly onto the polylysine. An interference fringe pattern from two intersecting and totally internally reflecting laser beams provided surface-selective excitation with a spatial variation of illumination intensity across a ghost for surface diffusion measurements. Measured characteristic values of desorption rate constants ranged from 0.043 to 270 s−1. According to a preexisting theoretical model, the largest desorption rate constant in this range would result in some increase in the total flux rate to a perfect sink target due to capture from the surface, provided that the surface diffusion coefficient was ≥ about 10−8 cm2/s. However, based on TIR-FRAP measurements on our system, we estimate that the surface diffusion coefficient is less than about 5×10−10 cm2/s. The combination of novel techniques presented here may prove valuable to other workers seeking to make diffusive and chemical kinetic rate parameter measurements of biomolecules at biological cell membranes.