title: A possible origin of differences between calorimetric and equilibrium estimates of stability parameters of proteins.
abstract: To test the validity of thermodynamic parameters from the equilibrium method, we have studied the reversible heat-induced denaturations of lysozyme, ribonuclease A, cytochrome c and myoglobin at various pH values, using absorption spectral measurements. For each protein, if a linear temperature-dependence of the pre- and post-transition baselines is assumed for the analysis of the conformational-transition curve, the estimate of DeltaH (the enthalpy change on denaturation at T(m), the midpoint of denaturation) is significantly less than DeltaH, the value obtained by the calorimetric measurements. If the analysis of thermal-denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function, there exists an excellent agreement between DeltaH(m) values of all proteins obtained from equilibrium and calorimetric methods. The latter analysis is supported by the studies on model compounds, for measurements of absorption properties of tyrosine, tryptophan and haem as a function of temperature suggested that the temperature-dependencies of the optical properties are indeed non-linear. We have observed that for each protein the constant-pressure heat-capacity change on denaturation (DeltaC(p)) determined from the plots of DeltaH versus T(m) is not only independent of the method of analysis of the transition curve, but it is also in excellent agreement with calorimetric DeltaC(p). An important conclusion of this study is that for these proteins that exhibit two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.
authors.affiliation: Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi-110 025, India.
authors.forename: A
authors.initials: A
authors.lastname: Sinha
authors.forename: S
authors.initials: S
authors.lastname: Yadav
authors.forename: R
authors.initials: R
authors.lastname: Ahmad
authors.forename: F
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002151:Calorimetry / Q000379:methods*; D003574:Cytochrome c Group / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D003574:Cytochrome c Group; D009211:Myoglobin; D011506:Proteins; D014443:Tyrosine; D006418:Heme; D014364:Tryptophan; D012259:Ribonuclease, Pancreatic; D009113:Muramidase