Differential scanning calorimetry of the irreversible denaturation of Rapana thomasiana (marine snail, Gastropod) hemocyanin

The thermal denaturation of the hemocyanin from gastropod Rapana thomasiana (RtH) at neutral pH was studied by means of differential scanning calorimetry (DSC). 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 (Tm) at 83 and 90 °C, were detected by DSC using buffer 20 mM MOPS, containing 0.1 M NaCl, 5 mM CaCl2 and 5 mM MgCl2, pH 7.2. Both Tm were dependent on the scanning rate, suggesting that the thermal denaturation of RtH is a kinetically controlled process. The activation energy (EA) of 597±20 kJ mol−1 was determined for the main transition (at 83 °C). EA for the second transition was 615±25 kJ mol−1. The Tm and ΔHcal values for the thermal denaturation of RtH were found to be independent of the protein concentration, signifying that the dissociation of the protein into monomers does not take place before the rate-determining state of the process of thermal unfolding.