title: Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects.
abstract: Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.
authors.affiliation: Institute of Biochemical Physics, Russian Academy of Sciences, Vavilov Street 28, 117813 GSP-1, Moscow, Russian Federation. grinberg@ineos.ac.ru
authors.forename: V Y
authors.initials: VY
authors.lastname: Grinberg
authors.forename: T V
authors.initials: TV
authors.lastname: Burova
authors.forename: T
authors.initials: T
authors.lastname: Haertlé
authors.forename: V B
authors.initials: VB
authors.lastname: Tolstoguzov
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D003627:Data Interpretation, Statistical; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010947:Plants, Toxic; D011487:Protein Conformation; D011489:Protein Denaturation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D014026:Nicotiana; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry*
chemical_list: D014360:Trypsin Inhibitor, Kunitz Soybean; D012273:Ribulose-Bisphosphate Carboxylase