Effect of hydration on the secondary structure of lyophilized proteins as measured by fourier transform infrared (FTIR) spectroscopy

The impact of hydration on the secondary structure of proteins using FTIR spectroscopy was investigated. Alternative sampling techniques were investigated since KBr pelletization of hydrated proteins is not recommended. Spectra of lyophilized dry proteins were collected in transmission mode by palletizing, mulling, and in ATR mode. Spectra for hydrated proteins were collected in mulls and in ATR mode. Spectra for reconstituted solutions were collected in transmission mode. Spectra of Protein–sucrose colyophilized mixtures were collected in KBr pellets and in ATR mode. Pure proteins underwent significant change in structure upon lyophilization, reforming upon reconstitution. ATR spectra differed from transmission spectra in peak intensity and position, suggesting a more nativelike structure even after correction for refractive index dispersion. No significant differences were found between KBr pellet and mull spectra. Colyophilization with sucrose led to protection of structure. The effect of hydration on the structure was protein dependent, ranging from loss of native structure (IgG) to partial reformation of native structure (BSA). It is concluded that spectra collected in different modes are not directly comparable and caution must be exercised in interpreting the data. Contrary to general view, the secondary structure of proteins in a hydrated state was not equivalent to that in solution. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2910–2921, 2007