Stability parameters for one-step mechanism of irreversible protein denaturation:: a method based on nonlinear regression of calorimetric peaks with nonzero ΔCp

Thermal transitions of many proteins have been found to be calorimetrically irreversible and scan-rate dependent. Calorimetric determinations of stability parameters of proteins which unfold irreversibly according to a first-order kinetic scheme have been reported. These methods require the approximation that the increase in heat capacity upon denaturation DeltaC(p) is zero. A method to obtain thermodynamic parameters and activation energy for the two-state irreversible process N --> D from nonlinear fitting to calorimetric traces is proposed here. It is based on a molar excess heat capacity function which considers irreversibility and a nonzero constant DeltaC(p). This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (T-m), (2) calorimetric enthalpy at T-m (DeltaH(m)), (3) DeltaC(p) and (4) activation energy (E). The thermal irreversible denaturation of subtilisin BPN' from Bacillus amyloliquefaciens was studied by differential scanning calorimetry at pH 7.5 to test our model. Transitions were found to be strongly scanning-rate dependent with a mean DeltaC(p) value of 5.7 kcal K-1 mol(-1), in agreement with values estimated by accessible surface area and significantly higher than a previously reported value. (C) 2004 Elsevier Inc. All rights reserved.