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

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.