The thermal denaturation of ribonuclease A in aqueous solutions of 2-methyl-2,4-pentanediol(MPD) was investigated by differential scanning calorimetry at pH 5.8, i.e., conditions similar to those used to crystallize the protein, and also at pH 3. A two-state reversible denaturation occurred in aqueous MPD up to 50%(v/v) MPD. Under both conditions the denaturation temperature, Td, decreased almost linearly with increasing MPD concentration; there was a slightly greater effect at pH 5.8. The calorimetric enthalpy, HD, first increased with increasing MPD concentration and then decreased slightly at MPD concentrations above 20-30%. This behavior is analogous to that observed for organic additives, such as ethanol and dimethyl sulfoxide, with denaturing agents. The plot of δHD against δSd, the denaturation entropy at Td, gave a linear correlation between these parameters, indicating a close relationship between the MPDinduced unstabilization of the protein and the water structure around the protein molecule. The standard thermodynamic parameters for denaturation, δG, δH, and δS, were calculated from Td and HD, assuming a constant heat capacity change. The unstabilizing effect of MPD on the thermal stability of the protein results from that the increase in δzH, induced by MPD, is compensated by a large increase in δS, with a resulting decrease in δG; it is in accord with an effect on solvent ordering around the exposed nonpolar groups of the protein. It also indicates a temperature dependence of the MPD effect on the protein stability. © 1984 The Chemical Society of Japan.
