Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry
Lina Baranauskienė, Jurgita Matulienė, Daumantas Matulis
Institute of Biotechnology, Graičiu¯no 8, Vilnius, LT-02241, Lithuania
Received 25 September 2007; received in revised form 19 November 2007; accepted 31 December 2007
Abstract
The enthalpy of unfolding (ΔuH) 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 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the ΔuH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (ΔuCp) is dependent on
temperature. The T-derivative of ΔuCp was equal to 100 ± 30 J/(mol × K2), with the result that the ΔuCp is equal to 15.8 kJ/(mol × K) at 5 °C,
19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the ΔuH 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.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Acid-unfolding; Isothermal titration calorimetry; Differential scanning calorimetry; Carbonic anhydrase; Heat capacity of unfolding; Enthalpy of unfolding
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