Effect of surface properties and added electrolyte on the structure of beta-casein layers adsorbed at the solid/aqueous interface

The adsorption of beta-casein at hydrophobic and hydrophilic silica surfaces has been studied by time-resolved ellipsometry. Marked differences in, e.g., adsorption kinetics and plateau adsorption coverage, were observed on the two types of surfaces. The miscellaneous adsorption mechanisms at the two surfaces resulted in different structures of the adsorbed layers as evident from the thicknesses and protein densities measured on the two substrates as well as the effect on the adsorbed layer properties of a subsequently added specific proteolytic enzyme, endoproteinase Asp-N. At the hydrophobic surface, the adsorption is fast and the surface is saturated within a relatively short period. The addition of endoproteinase Asp-N reduces the surface excess and the thickness by 24 and 45%, respectively. This corresponds to cleavage at amino acid residues 43 and/or 47 in the hydrophilic portion of the protein. Adsorption from solutions containing added electrolyte leads to significant increase of the surface excess. However, no significant change was observed in the ellipsometric layer thickness. At constant ionic strength, the surface excess increased in the order NaCl < MgCl2 < CaCl2. From the experimental evidence, it was concluded that the adsorbed layer structure at the hydrophobic surface can be described as a monolayer with an inner dense region comprising the relatively large hydrophobic portions of the protein molecules and an outer region of the highly charged N-terminal portions protruding into the aqueous phase. The adsorption kinetics at the hydrophilic silica surface, although initially the same as on the hydrophobic surface, was found to be much slower and plateau surface excess values were not reached even after 2 h of adsorption. This suggests that substantial rearrangements of the protein molecules take place within the adsorbed layer during the adsorption process. Although the maximum surface excess at the hydrophilic surface of 4.3 mg m(-2) is higher than the value of 2.8 mg m(-2) measured at the hydrophobic surface, the thickness is slightly smaller, i.e., 60 Angstrom and 66 Angstrom respectively. Hence, the protein adopt a more compact structure at the hydrophilic surface, at least in the inner part of the adsorbed layer. The different structure at the hydrophilic surface was confirmed by the larger reduction of the surface excess and layer thickness associated with the addition of endoproteinase Asp-N, leaving a very thin compact layer at the surface.