The stability behavior of four globular proteins (glucose oxidase, ribonuclease, lysozyme, and carbonic anhydrase) in pure buffer and in the presence of water-miscible hydroxylic additives (alcohols, polyols, and sugars) was analyzed. Attention was focused on the influence of these compounds on the melting temperature of the proteins. For all of the proteins examined, this latter quantity was found to be linearly related to the bulk surface tension of the mixed solvent. To provide a quantitative interpretation to the above observation, a molecular thermodynamic model, based on the additive-induced perturbation of the equilibrium between the folded and the unfolded protein forms, was developed. It is shown that, under some limiting conditions, the Gibbs equilibrium criterion applied to the two-state unfolding process yields a linear dependence of the melting temperature on the bulk surface tension, as observed for the proteins considered. The results obtained appear to indicate that the conformational stability of heat-stressed proteins in water-hydroxylic cosolvent mixtures does not rely on any special property of these substances but rather on their ability to affect the interfacial free energy between the protein and the solvent through perturbations of the surface tension of water. The model proposed can be used for interpretation and correlation of thermal unfolding data and, as a diagnostic tool, to assess whether the surface tension mechanism provides the overwhelming contribution to protein unfolding.
IntroductionThe elaborate molecular mechanisms causing the transformation of the random-coil polypeptide chain into the compact, highly ordered, structure of proteins are mainly mediated by hydrophobic interactions 1 and by nonlocal entropic effects due to steric constraints in the folded state. 2 Their contributions to the free energy of stabilization are nearly equal in magnitude, 3 but whereas hydrophobic interactions favor the native conformation, nonlocal entropic effects destabilize this state. The interplay of these two major and opposing driving forces manifests itself in the small free energies of stabilization commonly observed: 4-6 typical values for globular proteins are in the range 5-15 kcal/mol. Native proteins, therefore, are only marginally stable, and this intrinsic lability makes them easily susceptible to denaturation.Thermal inactivation is one of the most important forms of protein denaturation. 7,8 Although there is still considerable uncertainty about the mechanisms by which the polypeptide chain loses its compactness and biological activity, it now seems to be ascertained that the first and, perhaps, the only universal step in thermal inactivation is represented by partial unfolding. [9][10][11] Valuable information about the physical bases of this process can be gained by perturbing the protein environment, for instance by addition of a water-miscible component, and analyzing the resulting changes in stability.The stability behavior of several proteins in the presence of stabilizers ...
