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Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H2O)(OH)]2+, and a highly luminescent Tb(III) reagent

  • Carmen M. Barnés
  • Stéphane Petoud
  • Seth M. Cohen
  • Kenneth N. Raymond
Original Article

Abstract.

The ability of ferritin as an Fe(II) detoxifier and Fe(III) storage protein is limited by its ability to recognize and incorporate Fe(II), which is then oxidized and mineralized at internal protein sites. The Cr(III) amine complex [Cr(N(CH2CH2NH2)3(H2O)(OH)]2+ [abbreviated as Cr(TREN)] is a kinetically inert inhibitor of iron incorporation and mineralization in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands. Competition studies between Cr(TREN) and Tb(III) binding have been performed in horse spleen ferritin (HoSF) to probe uptake of Fe(II). From these studies, we propose that Cr(TREN) inhibits Fe(II) uptake by obstructing the routes of metal uptake and by disrupting the early recognition events at the protein surface that precede metal ion uptake. Using an improved luminescence approach to quantify Tb(III) binding to the protein, we demonstrate that Tb(III) cannot interfere with Cr(TREN) binding to ferritin, but that Cr(TREN) dramatically inhibits Tb(III) binding. We show that bound Tb(III) serves as a reliable reporter for Cr(TREN) binding, as the latter efficiently quenches the Tb(III) luminescence via inter-ion energy transfer. Two types of Cr(TREN) binding sites were successfully distinguished from these competition experiments. A common Tb(III)/Cr(TREN) site was identified with stoichiometry of ~0.6 equivalents of metal cation per ferritin subunit. We propose that the sites along the three-fold channels and the ferroxidase sites are common binding sites for Tb(III) and Cr(TREN). The remaining Cr(TREN) (2.4 equivalents of metal ions/subunit) does not compete with Tb(III) but rather blocks Tb(III) access into the cavity and decreases the protein's affinity for Tb(III). Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0409-4. On that page (frame on the left side), a link takes you directly to the supplementary material.

Ferritin Iron uptake Iron storage Luminescence Terbium 

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Copyright information

© SBIC 2002

Authors and Affiliations

  • Carmen M. Barnés
    • 1
  • Stéphane Petoud
    • 1
  • Seth M. Cohen
    • 1
  • Kenneth N. Raymond
    • 1
  1. 1.Department of Chemistry, University of California, Berkeley, CA 94720, USA

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