This work reports, for the first time, a complete analysis of the binding of Pb(II) ions to mammalian Zn-metallothionein (MT) and its individual domains (α and β) by means of ESI TOF MS. The use of highly pure recombinant Zn-MT and high resolution MS allowed the identification of the different heteronuclear Pb,Zn-MT and homonuclear Pb-MT complexes formed by Zn/Pb replacement in the initial Zn-MT complexes at pH 7. The results obtained showed, both for the whole Zn7-MT and for its individual Zn4-α and Zn3-β fragments, a partial substitution of the Zn initially bound to give rise to the formation of both Pb,Zn-MT and Pb-MT species. In the case of the whole Zn7-MT, the addition of seven equivalents of Pb(II) give rise to the formation of several PbxZny-MT species (x + y = 7 or 8) as well as Pb8-MT and Pb9-MT coexisting with the initial Zn7-MT species. The addition of a five-fold excess of Pb(II) showed the formation of species containing a higher amount of lead(II), PbxZny-MT species (x = 8-12, y = 0-1). In both cases, the evolution with time showed the disappearance of the species containing lead and concomitantly only detection of the initial Zn-MT clusters within 48 h. This behaviour was also observed for the Zn4-α and Zn3-β aggregates, confirming the reversibility of the Pb2+ displacement of Zn2+ ions in the three forms of MT. A similar result was obtained at acidic pH, when the apo-form was the major species in solution, this therefore confirming the low affinity of MT for Pb(II), even in the absence of zinc. The results obtained were consistent with the increase of MT synthesis in response to Pb2+ administered to rats reported in the literature, since the initial release of Zn(II) would promote the biosynthesis of MT, in agreement with the function of this ion as primary activator of the MTF-1 transcription factor; and could explain the difficulty of recovering homonuclear Pb-MT species from lead-treated organisms. © 2006.
|Publication status||Published - 30 Apr 2007|
- ESI TOF MS
- Recombinant mouse Zn -metallothionein 1 7
- α and β Zn complexes