A 3-STATE HEAT-DENATURATION OF BOVINE ALPHA-LACTALBUMIN

The heat-denaturation of bovine alpha-lactalbumin (alpha-La) was studi ed using tryptophan and tyrosine fluorescence intensity measurements a t 2-80 degrees 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-alpha- La conformed to a two-state reaction with a melting temperature (T-m) of 34.5 (+/- 0.7)degrees C. In contrast, the heat-unfolding of holo-al pha-La was consistent with a three-state reaction. The T-m for holo-al pha-La heat-unfolding was 47 (+/- 2.6)degrees C or 67.5 (+/- 1.3)degre es C as determined from tyrosine or tryptophan fluorescence changes, r espectively. Such results suggest that Ca2+ binding preferentially sta bilises one domain of alpha-La against heat-denaturation.