To test the validity of thermodynamic parameters from the equilibrium method, we have studied the reversible heat-induced denaturations of lysozyme, ribonuclease A, cytochrome c and myoglobin at various pH values, using absorption spectral measurements. For each protein, if a linear temperature-dependence of the pre- and post-transition baselines is assumed for the analysis of the conformational-transition curve, the estimate of ∆H (the enthalpy change on denaturation at Tm, the midpoint of denaturation) is significantly less than ∆H, the value obtained by the calorimetric measurements. If the analysis of thermal-denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function, there exists an excellent agreement between ∆Hm values of all proteins obtained from equilibrium and calorimetric methods. The latter analysis is supported by the studies on model compounds, for measurements of absorption properties of tyrosine, tryptophan and haem as a function of temperature suggested that the temperature-dependencies of the optical properties are indeed non-linear. We have observed that for each protein the constant-pressure heat-capacity change on denaturation (∆Cp) determined from the plots of ∆H versus Tm is not only independent of the method of analysis of the transition curve, but it is also in excellent agreement with calorimetric ∆Cp. An important conclusion of this study is that for these proteins that exhibit two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.

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