Stability parameters for one-step mechanism of irreversible protein denaturation: a method based on nonlinear regression of calorimetric peaks with nonzero deltaCp.
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 deltaCp 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 deltaCp. This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (Tm), (2) calorimetric enthalpy at Tm (deltaHm), (3) deltaCp, 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 deltaCp 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.
Arroyo-Reyna
Alfonso
A
Area de Biofisicoquímica, Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Apartado Postal 55-534, 09340 Iztapalapa, D.F., México. aar@xanum.uam.mx
Tello-Solís
Salvador R
SR
Rojo-Domínguez
Arturo
A
D001412:Bacillus subtilis / Q000201:enzymology; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017711:Nonlinear Dynamics; D011489:Protein Denaturation; D012044:Regression Analysis; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D013381:Subtilisins