A three-state heat-denaturation of bovine α-lactalbumin

The heat-denaturation of bovine α-lactalbumin (α-La) was studied using tryptophan and tyrosine fluorescence intensity measurements at 2–80 °C. The solvent used was sodium cacodylate buffer (10 mM, pH 6.0) with 1 mM EDTA or 9 mm CaCl2. The denaturation of apo-α-La conformed to a two-state reaction with a melting temperature (Tm) of 34.5 (± 0.7) °C. In contrast, the heat-unfolding of holo-α-La was consistent with a three-state reaction. The Tm for holo-α-La heat-unfolding was 47 (± 2·6) °C or 67·5 (± 1·3) °C as determined from tyrosine or tryptophan fluorescence changes, respectively. Such results suggest that Ca2+ binding preferentially stabilises one domain of α-La against heat-denaturation.