Insight into the stability of protein in confined environment through analyzing the structure of water by temperature-dependent near-infrared spectroscopy.

To understand the stability of protein in confined environment, the near-infrared (NIR) spectra of aqueous solutions and reverse micelles (RMs) containing bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OVA) were measured at different temperature. With the resolution enhanced spectra calculated by continuous wavelet transform (CWT), the intensity change of the α-helix band at 4617 cm with temperature shows a clear denaturation of the protein in aqueous solution but not in RMs. The effect of the confined environment on the stability of the proteins is indicated. More importantly, the intensity change of the spectral bands of water around 6956 and 6842 cm provide an evidence for the denaturation, suggesting that water can be a probe exhibiting the structural change of proteins. Furthermore, comparing the spectral features of different water structures obtained by principal component analysis (PCA) from the spectra of RM with and without BSA, it is demonstrated that the bridging water connecting NH in protein and SO in the inner surface of RM may be the reason for the stabilization.