Thermal stability of extracellular hemoglobin of Glossoscolex paulistus: determination of activation parameters by optical spectroscopic and differential scanning calorimetric studies

Abstract

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS), optical absorption spectroscopy (UV–VIS) and differential scanning calorimetry (DSC). At pH 7.0, cyanomet-HbGp is very stable, no oligomeric dissociation is observed, while denaturation occurs at 56 °C, 4 °C higher as compared to oxy-HbGp. The oligomeric dissociation of HbGp occurs simultaneously with some protein aggregation. Kinetic studies for oxy-HbGp using UV–VIS and DLS allowed to obtain activation energy (Ea) values of 278–262 kJ/mol (DLS) and 333 kJ/mol (UV–VIS). Complimentary DSC studies indicate that the denaturation is irreversible, giving endotherms strongly dependent upon the heating scan rates, suggesting a kinetically controlled process. Dependence on protein concentration suggests that the two components in the endotherms are due to oligomeric dissociation effect upon denaturation. Activation energies are in the range 200–560 kJ/mol. The mid-point transition temperatures were in the range 50–65 °C. Cyanomet-HbGp shows higher mid-point temperatures as well as activation energies, consistent with its higher stability. DSC data are reported for the first time for an extracellular hemoglobin.