Structural Conformation of Bovine Serum Albumin Layers at the Air–Water Interface Studied by Neutron Reflection

Abstract

The adsorption of bovine serum albumin (BSA) at the air–water interface has been studied by specular neutron reflection. The variation of the adsorbed amount and the total thickness of the BSA layer with respect to bulk BSA concentration was determined at pH 5, close to its isoelectric point (IP). While the surface excess showed a steady increase with bulk concentration the thickness of the protein layer was found to be close to the short axial length of 40 Å of the globular solution structure of BSA at concentrations below 0.1 g dm−3, suggesting that BSA molecules adsorb with their long axes parallel to the surface of water. At 1 g dm−3the adsorbed layer can be modeled as an upper layer of 40 Å with a volume fraction of 0.4 and a sublayer of 30 Å underneath the top main layer with a volume fraction of 0.12. The results suggest that, although there is some structural deformation accompanying adsorption, there is no denaturation. The extent of immersion of the BSA in water was determined by performing the measurements in D2O and in a mixture of H2O and D2O whose contrast matches that of BSA. The signal is then only from the part of the layer out of water. At pH 5 this layer was about 10 ± 5 Å at a bulk concentration of 5 × 10−4g dm−3and decreased to 5 ± 3 Å at 1 g dm−3. The fraction of the BSA layer immersed in water therefore varies from about 70 to over 90%. The effect of pH on the adsorption was examined at two BSA concentrations. While pH had little effect on the adsorption at a low BSA concentration of 5 × 10−3g dm−3, both surface excess and layer thickness showed pronounced peaks at pH 5 at the higher concentration of 1 g dm−3. The increased adsorption at pH 5 is attributed to the reduced lateral electrostatic repulsion around the IP. This adsorption pattern became less pronounced when the total ionic strength was increased from 0.02 to 1 M, indicating that the electrolyte screens the electrostatic repulsions within the adsorbed layer.