Spectroscopic evidence for the preferential hydration of RNase A in glycerol-water mixtures: Dielectric relaxation studies

The elucidation of the molecular mechanism of the interaction of denaturing and stabilizing agents with proteins is a problem of eminent fundamental a nd practical consequences. In the present study we provide strong evidence by dielectric relaxation studies on RNase A-glycerol-water solutions that t he protein creates for itself an aqueous environment in its immediate vicin ity by attracting water molecules from the bulk solvent, thereby increasing the glycerol concentration of the bulk solution. This finding provides dir ect spectroscopic support for the thermodynamic results of Gekko and Timash eff (K. Gekko and S. N. Timasheff, Biochemistry, 1981, 20, 4668;(1) K. Gekk o and S. N. Timasheff, Biochemistry, 1981, 20, 4677(2)) who reported prefer ential exclusion of glycerol from the protein domain.