Refinement of noncalorimetric determination of the change in heat capacity, ΔCp, of protein unfolding and validation across a wide temperature range

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Abstract
The change in heat capacity, ΔCp, on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs‐Helmholtz relationship to determine ΔCp has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which ΔG is zero, Tm, and change in enthalpy at Tm are determined by thermal denaturation and ΔCp is then calculated using the Gibbs‐Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of ΔCp consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, Tm, and the van't Hoff enthalpy, ΔHvH, from a thermal denaturation, gives a reasonable estimate of ΔCp, albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs‐Helmholtz equation as a function of stability and temperature while simultaneously fitting ΔCp, Tm, and ΔHvH gives values for the last two parameters that are in excellent agreement with experimental values. Proteins 2008.