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Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry.

Authors
  • Baranauskiene, Lina1
  • Matuliene, Jurgita
  • Matulis, Daumantas
  • 1 Institute of Biotechnology, Graiciūno 8, Vilnius, LT-02241, Lithuania. , (Lithuania)
Type
Published Article
Journal
Journal of Biochemical and Biophysical Methods
Publisher
Elsevier
Publication Date
Apr 24, 2008
Volume
70
Issue
6
Pages
1043–1047
Identifiers
DOI: 10.1016/j.jprot.2007.12.003
PMID: 18255160
Source
Medline
Language
English
License
Unknown

Abstract

The enthalpy of unfolding (DeltauH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 degrees C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 degrees C, the DeltauH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (DeltauCp) is dependent on temperature. The T-derivative of DeltauCp was equal to 100+/-30 J/(molxK2), with the result that the DeltauCp is equal to 15.8 kJ/(molxK) at 5 degrees C, 19.0 kJ/(molxK) at 37 degrees C and 21.8 kJ/(molxK) at 64 degrees C. The enthalpy of unfolding is zero at 17 degrees C. At lower temperatures, the Delta(u)H becomes exothermic. This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.

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