Thermodynamics of the denaturation of lysozyme in alcohol-water mixtures

Abstract

The thermal denaturation of [hen egg white] lysozyme (EC 3.2.1.17) was studied at pH 2 in aqueous mixtures of methanol, ethanol and 1-propanol by high sensitivity differential scanning calorimetry (DSC). The most obvious effect of alcohols was the lowering of Td, the temperature of denaturation, increasigly with higher alcohol concentration and longer alkyl chain. The calorimetric and van''t Hoff enthalpies of denaturation initially increased and then decreased with increasing alcohol concentration, the ratio of the 2 enthalpies being nearly unity, 1.007 .+-. 0.011, indicating the validity of the 2-state approximation for the unfolding of lysozyme in these solvent systems. The reversibility of the denaturation was demonstrated by the reversibility of the DSC curves and the complete recovery of enzymic activity on cooling. The changes in heat capacity on unfolding decreased with increasing alcohol concentration for each alcohol. Experimentally determined values of denaturation temperature and of entropy and heat capacity changes were used to derive the additional thermodynamic parameters .DELTA.G.degree. and .DELTA.S.degree. for denaturation as a function of temperature for each alcohol-water mixture. Comparison of the thermodynamic parameters with those reported in aqueous solution at various values of pH and guanidine hydrochloride concentration showed that these latter changes have no effect on the heat capacity change, the addition of alcohols causes a sharp decrease.