Effects of Dimethyl Sulfoxide, Glycerol, and Ethylene Glycol on Secondary Structures of Cytochrome c and Lysozyme As Observed by Infrared Spectroscopy

Effects of 10–30% (v/v) of dimethyl sulfoxide, glycerol, and ethylene glycol on the H–O–H bending vibration of water and the amide I bands of horse heart cytochrome c and chicken egg white lysozyme in 25mM sodium phosphate buffer (pH 7.4) were examined at 20°C by Fourier transform infrared spectroscopy. The H–O–H bending mode of water was strongly affected by these cryoprotectant solvents. Increasing the concentration of cryosolvents from 0 to 30% shifts the water bending band maximum from 1645 to about 1650cm−1. Second‐derivative analysis reveals significant changes in conformation‐sensitive amide I regions of lysozyme ascribed to α‐helix (1657cm−1), turn (1674cm−1), and unordered (1646cm−1) structures; each cryosolvent increases the intensity of the 1657cm−1 band at the expense of bands at 1674 and 1646cm−1. No changes in spectra deemed significant were observed for cytochrome c under the same conditions. There is no spectral evidence of structural randomization of proteins due to the presence of these cryosolvents. Cryosolvent‐induced changes in secondary structure of proteins may result from changes in water structure which, in turn, perturb the structure of the protein and/or from direct interactions between cryosolvent and protein.