Thermodynamic analysis of osmolyte effect on thermal stability of ribonuclease A in terms of water activity.

Thermal unfolding of ribonuclease A (RNase) was analyzed in various osmolyte solutions of glycine, proline, sarcosine, N,N-dimethylglycine, betaine, myo-inositol, taurine, and trimethylamine-N-oxide (TMAO). All the osmolytes tested stabilized the protein. The thermal unfolding curve was described well by the van't Hoff equation and the melting temperature and the enthalpy of protein unfolding were obtained. The Wyman-Tanford equation, which describes the unfolded-to-folded protein ratio as a function of water activity, was successfully applied to obtain a linear plot. In consideration of this experimentally obtained linearity, the Wyman-Tanford plot could be integrated to calculate the stabilization free energy of the protein (∆∆G) in the solution. The ∆∆G was proved to be described by the property of the microstructure around the protein surface, which is composed of the protein hydration, the cosolute-binding, and the preferential exclusion, and the property of the bulk solution; water activity. The m-values of osmolytes for protein unfolding were obtained from ∆∆G calculated. Among the osmolytes tested, myo-inositol showed the highest m-value.