Changes in protein hydration during thermal denaturation of rnase A

We have used the osmotic stress technique to measure the difference in the number of water molecules thermodynamically associated with ribonuciease A in its native and heat-denatured states. For reversible denaturation in 30 mM glycine, pH 2.2, five osmolytes (betaine, glycerol, sarcosine, sorbitol and triethylene glycol) gave apparent water stoichiometry differences (δn_w) in the range 37 < δn_w < 153. If each bound water molecule occupies 8-10Ų of solvent accessible protein surface, these stoichiometries indicate that the change in solvent accessible surface area (δ5ASA) is < 1530 Å2, Denaturation in the presence of lOmM 2-mercaptoethanol gave only slightly greater values, indicating that disulfide crosslinking has little effect on the change of solvent exposure accompanying denaturation. This experimental estimate of δUASA is considerably smaller than theoretical values reported in the literature. We suggest that current models of the denatured state of ribonuciease A overestimate its soivent exposure.