Metal ion binding to alpha-lactalbumin species.
A strong cation (calcium) binding site has been demonstrated to exist in several alpha-lactalbumin species; bovine, goat, human, and guinea pig. A metal ion induced conformational change occurs, resulting in a unique (10-14-nm) blue shift and relative quenching of Trp fluorescence for all species. Calcium ion binding to the alpha-lactalbumins yielded dissociation constants (Kdiss consistently in the 10(-10)--10(-12) M range, while Mn(II) binding was in the 20-30 microM range. Independent determinations of these cation binding equilibria were made by ESR measurements of free unliganded Mn(II) in titrations with the bovine species. One strong site (Kdiss = 30.5 microM) was found, which correlated directly with the fluorescence-associated cation binding, plus three weaker sites (Kdiss = 1.1, 5.0, and 5.0 mM, respectively). Several lanthanides as well as Mg(II) were found to displace Mn(II) from the strong site on bovine alpha-lactalbumin (as monitored by ESR) and to cause the identical fluorescence changes as found for Ca(II) and Mn(II) above. The importance of measuring these equilibria by both fluorescence and ESR was borne out by demonstrating the potential errors in estimating dissociation equilibria by the fluorescence method alone. Also, the errors in estimating Kdiss for samples containing partially metal bound apo-alpha-lactalbumin are described as well as rapid, sensitive methods for estimating the extent of metal-free protein and correctly accounting for residual bound metal in equilibrium calculations.
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004578:Electron Spin Resonance Spectroscopy; D006041:Goats; D006168:Guinea Pigs; D006801:Humans; D007768:Lactalbumin / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002412:Cations; D002104:Cadmium; D008345:Manganese; D007768:Lactalbumin; D008274:Magnesium; D002118:Calcium