beta-Casein adsorption at the silicon oxide--aqueous solution interface

Neutron reflectometry was used to investigate the time-dependent beta-casein adsorption at the silica-aqueous solution interface. The transient and steady-state structural characteristics of the adsorbed layer were determined from reflectivity curves, fitted to three-layer and two-layer models. The results show that the beta-casein adsorption to silica is very slow. The adsorption process involves the formation of an inner dense protein layer with a mean thickness of about 30 A onto which a more hydrated outer layer is self-associated. The surface excess and the total layer thickness of the asymmetric bilayer were, after 5 h adsorption time, estimated to be about 6.5 mg/m2 and 105 A, respectively. The adsorption behavior observed on silica contrasts with that previously reported for hydrophobic substrates, where beta-casein adsorption is much more rapid and the final surface excess is less than half of that observed for silica. Rinsing the silica surface with protein-free buffer resulted in a substantial desorption; much more pronounced than observed for hydrophobic substrates. This behavior suggests a weak adsorption affinity for a fraction of the adsorbed casein molecules; most likely the outer self-associated casein molecules in the adsorbed bilayer. The comparative desorption from hydrophobic surfaces was shown to be marginal. The difference between the layer structures adopted on hydrophobic and hydrophilic surfaces is also mirrored in the effects that the addition of a specific proteolytic enzyme (endoproteinase Asp-N) has on the adsorbed layer properties. The rinsing and endoproteinase cleavage processes result together in more than 80% reduction of the originally adsorbed mass at the silica surface. Only a thin but dense adsorbed layer remains after these treatments. The corresponding reduction reported for the hydrophobic adsorbent system was only about 20%. It is concluded that beta-casein adsorption on silica results in the formation of an asymmetric surface bound bilayer that stands in strong contrast to the monolayer structure formed at hydrophobic surfaces. This finding support the previous results obtained by using ellipsometry. The study also shows that neutron reflection, despite its limitations in time resolution, can be used for studying dynamic interfacial phenomena in protein systems.