title: A study of the Pb(II) binding to recombinant mouse Zn(7)-metallothionein 1 and its domains by ESI TOF MS.
abstract: 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 (alpha and beta) 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 Zn(7)-MT and for its individual Zn(4)-alpha and Zn(3)-beta 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 Zn(7)-MT, the addition of seven equivalents of Pb(II) give rise to the formation of several Pb(x)Zn(y)-MT species (x+y=7 or 8) as well as Pb(8)-MT and Pb(9)-MT coexisting with the initial Zn(7)-MT species. The addition of a five-fold excess of Pb(II) showed the formation of species containing a higher amount of lead(II), Pb(x)Zn(y)-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 48h. This behaviour was also observed for the Zn(4)-alpha and Zn(3)-beta aggregates, confirming the reversibility of the Pb(2+) displacement of Zn(2+) 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 Pb(2+) 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.
authors.affiliation: Equipe de Chimie Analytique, CNRS UMR 5034, Hélioparc, 2, av. Pr. Angot, F-64053 Pau, France.
authors.forename: Oscar
authors.initials: O
authors.lastname: Palacios
authors.forename: Angels
authors.initials: A
authors.lastname: Leiva-Presa
authors.forename: Sílvia
authors.initials: S
authors.lastname: Atrian
authors.forename: Ryszard
authors.initials: R
authors.lastname: Lobinski