Thermal denaturation and protein stability analysis of Haliotis rubra hemocyanin

Abstract

The thermal denaturation and stability of hemocyanin purified from the marine mollusc Haliotis rubra were investigated. The protein has been previously shown to have strong antiviral properties; however, its therapeutic efficacy has been limited by the stability of its formulation. Denaturation of the hemocyanin was found to be irreversible by the absence of an endotherm on rescanning calorimetric traces. Two denaturation transitions were observed at 78.9 and at 85.1 °C in a 100 mM Tris/HCl buffer at pH 7.6 at a scan rate of 1 K min−1. These denaturation peaks were found to be scan rate dependent, suggesting a kinetically rather than thermodynamically controlled denaturation process. The clear prominence of two distinct transitions has not been previously reported for any hemocyanin protein so far. The accurate deconvolution of these two peaks was due to the high resolution of the TA nano-DSC calorimeter used for this work. The activation energy for the first transition was determined to be 535 ± 65 kJ mol−1, which compares well with values reported previously for hemocyanins in related species. Scans performed at various protein concentrations showed no change in unfolding kinetics, suggesting that a change in oligomeric state does not occur at the onset of denaturation. While the irreversible unfolding of hemocyanins from other species has been reported previously, the denaturation temperature and kinetics vary dramatically from species to species, and the observed H. rubra hemocyanin denaturation has not been noted before.