Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

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Abstract
We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wavelengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 + 0.4 °C and the enthalpy of unfolding at this temperature (ΔHm) is 40.8 + 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of ΔHm versus Tm yield a mean value of 590 + 120 cal/(mol·K) for the change in heat capacity upon unfolding (ΔCp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 + 110 cal/(mol·K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining ΔCp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520–2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (ΔGu) were plotted versus temperature, the resulting curve appeared linear; therefore, ΔCp could not be determined. However, the data for ΔGu versus denaturant concentration are linear over an extra ordinarily wide range of concentrations. Moreover, extrapolated values of ΔGu in urea are identical to values measured directly.