Thermodynamics of Unfolding of β-Trypsin at pH 2.8

The unfolding equilibrium of β-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the vanʹt Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of β-trypsin estimated from spectroscopic data (ΔGu = 6.0 ± 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (ΔGuH2O = 5.5 ± 0.3 kcal/mol) at pH 2.8 and 300°K.