Effect of polyol osmolytes on ΔGD, the Gibbs energy of stabilisation of proteins at different pH values

Thermal denaturation curves of lysozyme and ribonuclease-A were determined by measuring their far-UV circular dichroism (CD) spectra in the presence of different concentrations of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at various pH values in the range 7.0-1.9. The denaturation curve at each polyol concentration and pH was analysed to obtain values of Tm (midpoint of denaturation) and ΔHm (enthalpy change at Tm), and these ΔHm and Tm values obtained at different pH values were used to obtain ΔCp (constant-pressure heat capacity change) at each polyol concentration. Using values of ΔHm, Tm and ΔCp in the Gibbs-Helmholtz equation, ΔGD° (Gibbs energy change at 25 °C) was determined at a given pH and polyol concentration. Main conclusions of this study are that polyols have no significant effect on ΔGD° at pH 7.0, and they stabilise proteins in terms of ΔGD° against heat denaturation at lower pH values. Other conclusions of this study are: (i) Tm at each pH increases with increasing polyol concentration, (ii) ΔHm remains, within experimental error, unperturbed in the presence of polyols, and (iii) ΔCp depends on polyol concentration. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of both proteins in their native and denatured states are not perturbed on the addition of polyols.