AN OSMOLYTE EFFECT ON THE HEAT-CAPACITY CHANGE FOR PROTEIN-FOLDING

We have carried out a differential scanning calorimetry study into the pH effect on the thermal denaturation of ribonuclease A at several co ncentrations of the osmolyte sarcosine. In order to properly analyze t hese data, we have elaborated the thermodynamic theory of the linkage between temperature, cosolvent, and pH effects. The denaturation heat capacity increases with sarcosine concentration. The effects of temper ature and sarcosine concentration on the denaturation enthalpy and ent ropy values are well described by convergence equations, with converge nce temperatures of around 100 degrees C for the enthalpy and around 1 12 degrees C for the entropy; we suggest that these effects might be r elated to a solvent-induced alteration of the apolar-group-hydration c ontribution to the folding thermodynamics. From our data, we estimate that about 70 extra molecules of water are thermodynamically bound upo n ribonuclease denaturation in diluted aqueous solutions of sarcosine; this number is 6-9 times smaller than that predicted on the basis of the following two premises: (a) the osmolyte is strongly excluded from the surface of both the native and the denatured protein and (b) the denatured state is a fully solvated chain. We suggest that at least on e of these two premises does not hold. We briefly comment on the poten tial use of cosolvent effects on thermal denaturation to evaluate the degree of hydration of denatured proteins (thus providing an independe nt measure of the consequence of their possible residual structure) an d, also, on the possibility of finding substances that are more effici ent protein stabilizers than known osmolytes are.