3.9. Spectral Analysis: Amide I/Amide II Band Ratio

Intensities of characteristic peaks within the Amide I region corresponding to specific secondary structures were normalized and compared between samples of native HEWL in H₂O, and HEWL incubated for one, four, and 60 days in 90% C₂H₅OH. Bands corresponding to α-helix (1644 cm⁻¹), random coil (1654 cm⁻¹), and aggregation specific β-sheets (1620 and 1698 cm⁻¹) were identified, and their intensities for each sample were normalized by dividing with the intensity of the Amide II maximum (at 1542 cm⁻¹ in H₂O and 1534 cm⁻¹ in the case of 90% C₂H₅OH in H₂O) intensity. Normalized values are thus Amide I/Amide II ratios for characteristic peaks and they are presented as dimensionless values suggesting overall trends in the secondary structure change upon condition switch from water solution to high-ethanol solution and prolonged incubation. Normalized values of two aggregation-specific β-sheet bands were summed.