Differential scanning calorimetry of the irreversible denaturation of β-hemocyanin of Helix pomatia

The thermal denaturation of beta-hemocyanin from the gastropod Helix pomatia at neutral pH was studied by means of differential scanning calorimetry. The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. Two transitions with apparent transition temperatures (T-m) of 84 and 88 degrees C were detected by DSC in 20mM MOPS buffer containing 0.1 M NaCl, 5 mM CaCl2 and 5 mM MgCl2 at pH 7.2, and a heating rate of 1.0 K/min. Both T-m were dependent on the scanning rate, suggesting that the thermal denaturation of beta-HpH is a kinetically controlled process. T-m and specific enthalpy values (Delta H-cal) for the thermal denaturation of beta-HpH were found to be independent of the protein concentration indicating that the dissociation of the protein into monomers does not take place before the rate-determining step of the process of thermal unfolding. Successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Activation energy (EA) of 521 +/- 7 kJ mol(-1) was determined for the main transition (at 84 degrees C). E-A for the second transition was 459 +/- 8 kJ mol(-1).