Two-dimensional attenuated total reflection/infrared correlation spectroscopy of adsorption-induced and concentration-dependent spectral variations of β-lactoglobulin in aqueous solutions

This paper demonstrates the potential of generalized two-dimensional (2D) attenuated total reflection/infrared (ATR/IR) spectroscopy in studies of spectral variations in the amide I region of aqueous solutions of protein. Two examples of the 2D correlation analysis are discussed in this paper. The first is concerned with adsorption-dependent spectral changes of beta-lactoglobulin (BLG) in solution. The second approach is dedicated to the concentration-dependent spectral changes. To generate the 2D correlation spectra, the original spectra have been subjected to pretreatment procedures consisting of ATR correction, subtraction of the spectrum of the buffer solution, smoothing, and normalization over the concentration. The adsorption-dependent 2D study shows that the interaction between the crystal surface and the protein molecules can be monitored successfully by the synchronous and asynchronous correlation spectra. This interaction is characterized by pronounced intensity changes at frequencies assigned to beta-sheet elements buried in the hydrophobic core of the molecule. The concentration-dependent 2D correlation maps, which develop 10 times more intense features than the adsorption-dependent 2D maps, are concerned with changes in various secondary structure elements located in the hydrophilic parts facing the solvent. The present study has also aimed at expanding generalized 2D correlation spectroscopy to quantitative utilization. The quantitative analysis of the 2D maps reveals that the intensity changes observed for the series of aqueous solutions of BLG with different concentrations are predominantly composed of concentration-induced secondary structure changes even in the presence of the adsorption of protein molecules to the ATR crystal.