Metal ion binding to the N and A conformers of bovine alpha-lactalbumin.

The binding of Ca2+, Zn2+, Tb3+, and Mn2+ to metal-free bovine alpha-lactalbumin (apo-BLA) was studied by both analytical gel filtration using isotopic metal ions and by fluorescence titration. In the absence of other metal ions, Ca2+ binds at a single site on apo-BLA. The pH dependence of pKa for calcium binding indicates that carboxylate groups of aspartic and/or glutamic residues are coordinating groups for the metal ions and that histidine residues are most likely absent from the site. An analysis of the (Ca2+) dependence of the equilibrium constant for the N-A conformational change indicates the absence of binding of this metal ion to the A conformer. Binding of zinc occurs at two sites on apo-BLA (pKa 5.05 and 2.78). Occupancy of the higher affinity site stabilizes the A state while binding at the second site has been shown to give rise to a time-dependent conformational change leading to an "expanded A state" (Kronman, M. J. and Bratcher, S. C. (1984) J. Biol. Chem. 259, 10887-10895). There are three binding sites for Tb3+ on apo-BLA with the occupancy of the site of highest affinity leading to the N conformation. Binding of terbium at a second site reduces the affinity of binding at the first one. Binding of terbium at the third site induces a time-dependent transformation to the "expanded A state" (see above for reference). There are three binding sites for Mn2+. A quantitative resolution of the affinities for each of these sites is precluded by the dependence of binding affinity on association of the Mn2+-liganded protein. At apo-BLA concentrations of the order of 4 microM (fluorescence titration), pKa for binding at the site with highest affinity is 5.8, more than an order of magnitude higher than seen at protein concentrations approaching 1 mM (Murakami, K., and Berliner, L. J. (1983) Biochemistry 22, 3370-3374). Binding of Mn2+ to apo-BLA was also found to be time-dependent in contrast with that of Ca2+ which appeared to be instantaneous. Measurements with Ca2+, Mn2+, and apo-BLA in experiments with simultaneous mixing of components revealed little if any competition of binding, i.e. Ca2+ binding was little effected and Mn2+ binding was strongly inhibited over nearly a 2000-fold range of concentrations of the latter ion. With sequential mixing of components (pre-equilibration of protein with Mn2+), markedly increased binding of Mn2+ was observed and binding of Ca2+ at two sites was seen.