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

1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-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 alpha-lactalbumin, namely the signals corresponding to the delta-CH3 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 alpha-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 alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 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 alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.