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 and practical consequences. In the present study we provide strong evidence by dielectric relaxation studies on RNase A–glycerol–water solutions that the protein creates for itself an aqueous environment in its immediate vicinity by attracting water molecules from the bulk solvent, thereby increasing the glycerol concentration of the bulk solution. This finding provides direct spectroscopic support for the thermodynamic results of Gekko and Timasheff (K. Gekko and S. N. Timasheff, Biochemistry, 1981, 20, 4668; K. Gekko and S. N. Timasheff, Biochemistry, 1981, 20, 4677) who reported preferential exclusion of glycerol from the protein domain.