Testing protein adsorption models by off-null ellipsometry: Determination of binding constants from a single adsorption curve

Abstract

The kinetics of prothrombin adsorption on planar bilayers, prepared from 40% dioleoyl phosphatidylserine and 60% dioleoyl phosphatidylcholine, are studied by off-null ellipsometry. A simple expression for the adsorption rate is derived directly from measured adsorption rates. The interpretation of this expression in terms of transport processes shows that up to a surface coverage Γ = 1.8 pmol cm −2, i.e. 50% of the maximal surface coverage Γ max, the adsorption rate appears to be regulated by diffusion-limited transport of prothrombin from the bulk solution to the adsorbing surface. The adsorption rate is proportional to the concentration difference between the bulk and the solution immediately adjacent to the surface. The latter concentration appears to be instantaneously in equilibrium with the surface excess, conforming to the classical Langmuir isotherm. For low surface coverages, below 50% of maximal adsorption, the kinetics are thus described by a simple model containing the mass transfer coefficient, the dissociation constant K d and the maximal surface coverage Γ max, With known Γ max this model allows the estimation of K d from a single adsorption curve. However, for surface coverages exceeding 60% of maximal adsorption, drastic deviations from this model are found. The intrinsic adsorption rate is substantially reduced, presumably by surface exclusion.