Calorimetric observation of lysozyme degradation at elevated temperature in water and DMSO-water mixtures

Abstract

The reversibility of protein denaturation is an essential factor for biotechnology. Previous differential scanning calorimetry (DSC) studies demonstrated the development of the low-temperature shoulder on the calorimetric denaturation peak of lysozyme in successive heating-cooling cycles, which implies irreversible denaturation. However, this effect was not thoroughly investigated. In the present work, we have quantitatively studied the effect of incubation at the elevated temperature on the state of lysozyme in water and mixtures of water with dimethyl sulfoxide (DMSO) using DSC. The changes to the state of the lysozyme molecule indicated by DSC thermograms were also evaluated by circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) techniques. It is noted that with the increase of duration or temperature of incubation, the low-temperature peak on DSC thermograms grows, while the height of the unfolding peak of native structure decreases. The increase in the height of the low-temperature peak in DSC scans correlates with the development of the sideband associated with the absorption of the carboxyl group in the infrared spectra. This result suggests that the low-temperature endothermic peak corresponds to the unfolding of the deamidated protein. At the same time, DLS measurements indicate absence of aggregation, while FTIR and CD data demonstrate that deamidated protein maintains a native-like structure. The evaluation of the DSC thermograms allowed to determine the rates and activation energy of the degradation of protein molecules at the elevated temperatures. The addition of DMSO slows down the protein degradation but has little effect on the apparent activation energy of the process.