THERMODYNAMICS OF UNFOLDING FOR TURKEY OVOMUCOID 3RD DOMAIN - THERMAL AND CHEMICAL DENATURATION

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 (T(m)) occurs at 58.6 +/- 0.4-degrees-C and the enthalpy of unfolding at this temperature (DELTAH(m)) is 40.8 +/- 0.3 kcal/mol. When T(m) was perturbed by varying pH and denaturant concentration, the resulting plots of DELTAH(m) versus T(m) yield a mean value of 590 +/- 120 cal/(mol . K) for the change in heat capacity upon unfolding (DELTAC(p)). 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 DELTAC(p) (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 25202525). 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 (DELTAG(u)) were plotted versus temperature, the resulting curve appeared linear; therefore, DELTAC(p) could not be determined. However, the data for DELTAG(u) versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of DELTAG(u) in urea are identical to values measured directly.