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 β-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 β-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.
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