The pH effects on thermal amyloid fibrillation kinetics of hen egg‐white lysozyme using new normalization factor for Raman spectroscopy

Abstract Amyloid fibrillation kinetics of proteins associated with neurodegenerative diseases has been extensively studied using Raman spectroscopy. The normalization factor for the spectra is crucial for obtaining correct kinetics of Raman indicators, especially vibrational band intensities. Here, we compared the concentration dependences between the absorption at 280 nm in UV–vis spectroscopy and the phenylalanine (Phe) Raman band intensity at 1003 cm −1 in amyloid fibrillation kinetics of lysozyme. The former exhibits better performance as normalization factor. Using this new normalization factor, the effect of p H value on the transformation of hen egg‐white lysozyme (HEWL) tertiary and secondary structures was studied subsequently. With increasing acidity, the unfolding of tertiary structures and the transformation of secondary structures are significantly accelerated. Notably, the populations of various secondary structures in the final state remain in the p H < 2.0 solutions, indicating that the branching ratios of “on‐pathway” to amyloid fibrils and “off‐pathway” to gel‐like aggregates are independent on the p H value in the range of 1.1–1.9.