Theoretical and experimental investigations of adsorbed protein structure at a fluid interface

The structure of adsorbed layers of the disordered milk protein beta-casein at the air-water interface determined by specular neutron reflectivity measurements is presented and compared with results obtained from self-consistent-field modelling of a beta-casein look-alike molecule at a solid-water interface. Changes in the layer structure with substrate pH in the range pH 5.5-7.0 have been determined experimentally and have been estimated theoretically. At pH 7 the neutron reflectivity data are fitted using a two-layer model of the adsorbed protein film consisting of a thin (approximate to 10 Angstrom) protein-rich region (phi approximate to 0.95) at the interface and a thicker (approximate to 45 Angstrom), more diffuse (phi approximate to 0.15), layer extending into the bulk aqueous phase. On reducing the substrate pH towards the protein isoelectric point, a thickening of the adsorbed layer is observed which is attributed to secondary adsorption of protein parallel to the monolayer observed at pH 7. The thicknesses of both layers in the fitted model, and the volume fraction of protein in the diffuse layer, increase to accommodate the extra protein. A model, linear chain polyelectrolyte consisting of apolar, polar (uncharged) and polar (pH-dependent charge) residues is constructed in a sequence which mirrors the known primary structure of beta-casein. The adsorption behaviour of this casein look-alike at a solid-water interface is modelled using the self-consistent-field theories of Scheutjens and Fleer, under similar conditions of pH as for the specular neutron reflection experiments. Total segment density profiles are obtained which show very good qualitative agreement with the experimental results. At neutral pH, the monolayer profile phi(z) falls off in a smooth fashion from very high values at the interface (phi approximate to = 0.95) to values close to the bulk concentration over about 50 Angstrom. On reducing the pH, the adsorbed amount is seen to increase, with the additional material appearing as a shoulder on the phi(z) profile, as observed in the neutron experiments.