Lysozyme solvation in monohydric alcohols with low water content: a calorimetric and preferential solvation study.

The main focus of this work is to clarify how protein macromolecules become solvated in monohydric alcohols with low water content. Alcohols with varying hydrophobicity (1-propanol, ethanol, and methanol) were used as model organic liquids. Two independent experimental techniques (isothermal immersion calorimetry and water/alcohol adsorption) were employed to provide complementary thermodynamic functions of the preferential solvation of hen egg-white lysozyme in alcohols with low and high water contents at 298.15 K. The advantage of this approach is that the enthalpies and Gibbs energies of water and alcohols can be simultaneously studied during the deep dehydration of lysozyme. Three distinct composition regimes are operative in water-alcohol mixtures. Protein stabilization, as a result of the preferential hydration of lysozyme, was observed in the water-rich region, which was driven by a positive change in entropy, with the residual enzyme activity values close to 100%. Alcohol-induced inactivation of lysozyme was detected in the middle region, with the residual catalytic activity close to zero. In the water-poor region, the bulky ethanol and 1-propanol molecules, unlike methanol, were preferentially excluded from the protein surface. In the water-rich region, protein stabilization was driven by an exothermic (favorable) enthalpy change. The pairwise (adsorbate-adsorbate) interaction coefficients demonstrated that alcohols with longer alkyl chains stabilize water-water association at low water contents to a larger extent than methanol with a shorter alkyl chain. The results obtained may be highly relevant for elucidating the distinct roles of water bound to the amphiphilic surfaces of biomacromolecules.