Abstract

The unfolding of bovine α-lactalbumin (BAL) in guanidine hydrochloride (GnHCl) was studied by UV difference and fluorescence spectrophotometry. When BAL was dissolved in calcium-free solvents, nonsuperimposable unfolding profiles were observed. Thus, it may be concluded that the two spectrophotometric techniques are sensitive to different BAL chromophores or levels of structural organisation.In a solvent containing calcium (0·1 m Tris-HCl buffer, pH 7·0; 9 mm calcium chloride) there was an increase in the molar UV difference absorbance change associated with GnHCl unfolding of BAL. Unfolding profiles were also biphasic, i.e. denaturation occurred in two stages at different GnHCl concentrations. This pattern of unfolding is different from the usual S-shaped profiles recorded for the three- or two-state BAL unfolding reaction. The results suggest that BAL possesses a more compact structure in the presence of calcium ions. The biphasic transition may arise from the loss of structure, which is unique to calcium-stabilised BAL.