Characteristics of the Binding of Ca 2 + and Other Divalent Metal Ions to Bovine a

Removal of the tightly bound Ca2+ ion from bovine a-lactalbumin (Hiraoka et al. (1980) Biochem. Biophys. Res. Commun. 95, 1098-1104) produces a pronounced conformational change, as indicated by fluorescence and absorbance changes. These changes closely resemble the changes that occur on acid denaturation of the native protein. The binding of ions to apo-a-lactalbumin at pH 7.4 has been examined by monitoring fluorescence changes and by direct binding measurements with 4SCaC12. The results indicate the presence of two Ca2+ binding sites on apo-bovine a-lactalbumin, a stronger binding site (K, of 2.7 X lo6 M-’) and a weaker site (& of 3.1 X lo4 M-’); the fluorescence changes on Ca2+ rebinding correlate with saturation of the stronger site. Mn2+ can also bind to restore a “native” structure but with a lower affinity (K, of 3.5 X lo5 M-’). Zn2+ and Co2+ do not produce this change, but Zn2+ (1m ~ ) greatly inhibits the binding of 4sCa2+ in the direct binding assay and produces a time-dependent displacement of Ca2+ from the native protein to an apo-protein-like conformation. Co2+ does not produce these effects. Studies with metal-depleted galactosyltransferase activated with Zn2+ or Coz+ and apo-a-lactalbumin or Caz+-saturated a-lactalbumin show that the Ca2+, Zn2+, and apoa-lactalbumin are all able to bind with galactosyltransferase to produce an active lactose synthase complex.