Changes of the secondary structure of β-lactoglobulin in water-ethanol solutions were studied using infrared spectroscopy. The efficiency of both principal component analysis and two-dimensional correlation spectroscopy in avoiding spectral subtraction of water was evaluated. The kinetic curves assessed from the scores of the first principal component indicated that changes were more pronounced in 50% ethanol solution than in 5% ethanol-water solution. The spectral patterns derived from the eigenvectors made it possible to characterize changes in the secondary structure of the protein. While aggregation was obtained in 50% ethanol solution, α-helices were formed at the detriment of turns in 5% ethanol solution. The two-dimensional correlation spectroscopy provided information on the sequence of events that occurred by adding ethanol to aqueous solutions of β-lactoglobulin. In 5% ethanol solution, the disappearance of turns preceded the formation of α-helices. Moreover, a small amount of aggregates were depicted at the end of the process. Aggregation of the protein was the main event observed in 50% ethanol-water solution. This event was preceded by the disappearance of α-helices. Application of both principal component analysis and two-dimensional correlation spectroscopy to spectra recorded in H<sub>2</sub>O made it possible to detect small changes not observed on second derivative spectra collected in D<sub>2</sub>O.
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