Thermal Stability of Proteins in Aqueous Polyol Solutions: Role of the Surface Tension of Water in the Stabilizing Effect of Polyols

Abstract

Polyols and sugars are known to increase the thermal stability of proteins in aqueous solutions and lead to their preferential hydration. While the mechanism of action of sugars has been proposed to be essentially due to the increase in the surface tension of water in their presence, polyols including glycerol have been reported to lower the surface tension of water and act via the solvophobic effect. Surface tension measurements of polyols in water carried out by us, on the contrary, indicate a substantial increase in the surface free energy of water. These increments are comparable to those found in the case of sugars. To investigate the role of the surface tension of the solvent medium on the thermal stability of proteins, we have carried out a comprehensive study on the effects of a series of polyols with varying numbers of hydroxyl groups and stereochemistry, such as mannitol, inositol, sorbitol, xylitol, and adonitol, on the thermal stability of five proteins varying in their physicochemical characteristics, viz., ribonuclease A, α-chymotrypsinogen, lysozyme, cytochrome c, and trypsin inhibitor. The studies have been carried out at pH 2.5, 4.0, and 7.0 to elucidate the contribution of the surface charges toward the polyol-mediated thermal stability of proteins. A very good correlation between an increase in the surface tension of water in the presence of a polyol and an increase in the themal stability of proteins has been observed. Correlation of the thermal stability of proteins with physicochemical properties such as net charge and net hydrophobic surface exposed on unfolding indicates that none of these properties is a dominant factor in governing protein thermal stability. The results indicate that polyol-induced water-mediated effects such as an increase in the surface tension of water play a major role in the stabilization of proteins and that the preferential hydration of proteins observed in their presence is a consequence of the increase in the surface free energy of water.