NMR and stopped-flow studies of metal ion binding to α-lactalbumins

¹H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to α-lactalbumins from bovine, goat, and human sources. We have identified two ¹H-NMR markers diagnostic of metal ion binding to the high-affinity Ca²⁺-binding site of bovine α-lactalbumin, namely the signals corresponding to the δCH₃ groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca²⁺ bind to this site in either slow (La³⁺, Lu³⁺, Y³⁺, Sr²⁺, Sc³⁺) or fast (Cd²⁺, Ba²⁺, Pb²⁺) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y³⁺ bind the strongest (Y³⁺ > La³⁺, Lu³⁺ > Ca²⁺ > Sr²⁺ > Cd²⁺, Pb²⁺, Ba²⁺ > Sc³⁺). Several metal ions do not alter the ¹H spectrum of bovine α-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co²⁺ and Cu²⁺, bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination, Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat α-lactalbumins (bovine and goat α-LA: k(off) (s^-1) ~ 0.2 to 0.01 from La³⁺ to Lu³⁺; human α-LA: k(off) (s^-1) ~ 0.02 to 0.001 from La³⁺ to Lu³⁺). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human α-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca²⁺ off-rate for the bovine protein (k(off) (s^-1) ~ 2 to 5), determined using the fluorescent indicator, BAPTA.