The thermal denaturation, isothermal chemical denaturation and thermal chemical denaturation of bovine immunoglobulin G (bIgG) at pH 7.4 were studied by differential scanning calorimetry (DSC) and fluorescence spectroscopy adopting guanidine hydrochloride (GuHCl) and urea as denaturant. The thermodynamic parameters of thermal and chemical denaturation were determined for bIgG using both methods. The experimental results of DSC and fluorescence spectroscopy showed that the thermal denaturation and thermal chemical denaturation of bIgG were both a complex irreversible process, and might follow a three-state mechanism. Isothermal chemical denaturation studies of bIgG were carried out in the presence of GuHCl or urea to observe that denaturant induced isothermal chemical denaturation of bIgG also followed a multistate process. The thermal denaturation measured in the presence of the denaturant displayed that the transition temperature and enthalpy change on denaturation of bIgG strongly depended on denaturant concentration, and GuHCl and urea interacted with bIgG primarily by hydrogen bonds, yielding a randomly coiled conformation in its unfolded state, while thermal denaturation produced an incompletely unfolded aggregation of bIgG. Therefore, both chemical denaturation methods can yield different structurally unfolded states of the bIgG.

Key words: bovine immunoglobulin G, protein folding, protein denaturation, chemical denaturant, differential scanning calorimetry, fluorescence spectroscopy