NMR AND STOPPED-FLOW STUDIES OF METAL-ION BINDING TO ALPHA-LACTALBUMINS

H-1-NMR spectroscopy and stopped-flow techniques have been used to inv estigate the binding of a host of metal ions to alpha-lactalbumins fro m bovine, goat, and human sources. We have identified two H-3-NMR mark ers 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 e ither slow (La3+, LU(3+), Y3+, Sr2+, SC3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have de termined an affinity series for metal ion binding at this site, in whi ch lanthanides and Y3+ bind the strongest (Y3+ > La3+, LU(3+) > Ca2+ > Sr2+ > Cd2+ > pb(2+) > Ba2+ > SC3+). Several metal ions do not alter the H-1 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 dist inct site, termed the 'zinc site', and that His-68 is involved in meta l ion coordination. Finally, stopped-flow techniques using the indicat or Xylenol orange were employed to obtain lanthanide off-rates for bov ine, human, and goat cu-lactalbumins (bovine and goat alpha-LA: k(off) (s(-1))approximate to 0.2 to 0.01 from La3+ to LU(3+); human alpha-LA: k(off)(s(-1))approximate to 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 m etal ions are relatively constant. Data for the bovine and goat protei ns are virtually identical, while the off-rates for human cu-lactalbum in are appreciably slower. In addition, these rates are much slower th an the Ca2+ off-rate for the bovine protein (k(off) (s(-1))approximate to 2 to 5), determined using the fluorescent indicator, BAPTA.