Coverage-dependent orientation of lysozyme adsorbed on silica

We use total internal reflection fluorescence (TIRF), streaming current, and optical reflectometry measurements, under a variety of solution conditions, to examine the mechanism of the chicken egg lysozyme reorientation on silica surfaces, originally proposed by Robeson and Tilton (Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104). The TIRF data suggest that a two-stage reorientation occurs in the lysozyme layer during adsorption. The first stage involves a reorientation to optimize lysozyme interaction with the charged surface and to reduce lateral repulsions between adsorbed protein molecules. This stage occurs when the adsorbed layer reaches a critical surface coverage and indicates the importance of lateral interactions between adsorbed proteins. Moreover, the reorientation rate depends on both the bulk protein concentration and the fluid wall shear rate, indicating that incoming protein molecules from the bulk solution also participate in the reorientation. The preferred orientation puts the active site face toward the solution and the most positively charged amino acid patch into contact with the negative silica surface. A second stage of slow restructuring occurs as the adsorbed layer coverage asymptotically approaches saturation and corresponds to reorientation of the protein molecules that fill surface vacancies made accessible by the first reorientation. Consistent with this proposed two-stage reorientation mechanism, streaming current measurements indicate that adsorbed lysozyme initially causes a significant interfacial potential reversal, followed by a slow relaxation to less positive zeta-potentials as the lysozyme optimizes its orientation. Accordingly, the desorbable fraction of the adsorbed layer after reorientation is significantly smaller than that before reorientation.