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

James M. Aramini, Toshifumi Hiraoki, Michael R. Grace, Thomas W. Swaddle, Emilia Chiancone, Hans J. Vogel

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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.

Original languageEnglish
Pages (from-to)72-82
Number of pages11
JournalBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Issue number1
Publication statusPublished - 7 Mar 1996
Externally publishedYes


  • Lanthanide series
  • Metal ion binding
  • NMR, H-
  • Stopped-flow analysis
  • α-Lactalbumin

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