On the adsorption of β-lactoglobulin on hydrophilic gold surfaces: Studies by infrared reflection-absorption spectroscopy and ellipsometry

Abstract

A systematic analysis of the adsorption of β-Lactoglobulin onto hydrophilic gold surfaces is reported. Films formed by adsorption from aqueous solution at pH 4.5–13.0 and dried in air were analyzed by infrared reflection-absorption spectroscopy (IRAS), and compared with standard transmission spectra of the protein in the aqueous and solid phases. Ellipsometric measurements were performed in solution at pH 4.5–10.0. Both the infrared and ellipsometric results indicated a decreasing surface concentration for increasing pH (increasing net charge of the protein). The lineshape and frequency of the Amide I bands in the reflection-absorption spectra were significantly changed in relation to the same bands in the aqueous and solid phases. Blue shifts of about 10 to 40 cm −1 were observed upon adsorption over the investigated pH region, while only a weak pH dependence was observed in the lineshape and exact frequency of the Amide I bands. These findings strongly suggested that a conformational transition of the protein molecules occurred upon adsorption. From a comparison with spectra in the aqueous and solid phases it was concluded that the proteins existed in an unfolded conformation on the gold surface. The refractive indices and thicknesses of the optically equivalent films, obtained from the ellipsometric measurements, were modeled to obtain an approximate picture of the molecular extension and packing of the proteins on gold. These results indicated that β-Lactoglobulin was adsorbed in a native conformation at pH 4.5, while a transition toward a flat unfolded conformation occurred at pH 6.0 and 10.0. The fact that the IRAS experiments were performed on dried films may be one of the reasons why the conformation change with pH was not observed as a major change in the IR spectra. Only small variations in the parameters used for describing the secondary structure of the protein were observed with pH. Changes in certain parts of the spectra could, however, be used to gain detailed information about specific protein-surface interactions.