Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.
The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Timasheff
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D004795:Enzyme Stability; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism*; D013012:Sorbitol / Q000494:pharmacology*; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D014867:Water; D013012:Sorbitol; D012259:Ribonuclease, Pancreatic