Direct imaging of lysozyme adsorption onto mica by atomic force microscopy

The adsorption dynamics of hen-egg-white lysozyme onto muscovite mica is followed using in situ tapping mode atomic force microscopy (AFM) in aqueous solution. Under stagnant conditions and at a bulk concentration of 2 mug/mL, lysozyme clusters were imaged on mica several minutes after exposure to the protein solution. Force-volume imaging confirmed the existence of different tip-surface interaction forces corresponding to areas of bare mica and areas covered with a single layer of adsorbed protein. C-14-labeled lysozyme was used to determine the mass of protein adsorbed. This information, in conjunction with the AFM images of the submonolayers, reveals that lysozyme, once adsorbed, diffuses on a time scale of minutes or less on the surface and forms clusters consisting of about five molecules. In contrast, at a protein concentration of 5 mug/mL in the AFM cell, surface coverage increases uniformly until a complete monolayer is established after 2 h. Once the initial protein monolayer is established, additional protein molecules adsorb onto the first layer, but at a slower rate. Washout of the protein solution at both short and long times and under both submonolayer and multilayer conditions has no influence on surface coverage, indicating irreversible adsorption. Our AFM data are consistent with a model in which the protein, once adsorbed onto the surface, undergoes conformation change due to protein-surface interactions. Previously buried hydrophobic residues are exposed and available for interaction with the exposed hydrophobic residues of other adsorbed proteins, resulting in surface aggregation. By the study of surface coverages below that of a monolayer, protein clustering can be observed.