Metal ion binding to apo, holo, and reconstituted horse spleen ferritin

S. Pead, E. Durrant, B. Webb, C. Larsen, D. Heaton, J. Johnson, G. D. Watt

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Abstract

The binding of Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ to apo, holo, reconstituted horse spleen ferritin (HoSF), and native holo HoSF with phosphate removed was measured by gel-exclusion chromatography. Three classes of strong binding interactions (Kd < 10-7 M) with apo HoSF at pH 7.5 were found for the various M2+ studied: high stoichiometric binding (30-54 M2+/HoSF) for Cd2+, Zn2+, Cu2+, with two protons released per metal bound; intermediate binding (16 M2+/HoSF) for Ni2+ and Co2+, with one proton released per metal bound; and low levels of binding (2-12 M2+/HoSF) for Mn2+, Mg2+, and Fe2+, with <0.5 protons released per metal bound. M2+ binding to apo HoSF was nearly abolished at pH 5.5, except for Fe2+ and Cu2+, which remained unaffected by pH alteration. Holo HoSF bound much higher levels of M2+, a result directly attributable to the presence of phosphate binding sites. This conclusion was confirmed by decreased binding of M2+ to HoSF reconstituted in the absence of phosphate and by native holo HoSF with phosphate chemically removed. The binding of Cd2+ to apo HoSF was 54 per HoSF, but in the presence of developing core, the amount bound decreased to about 30 Cd2+/HoSF. This result indicated that Cd2+ and developing core were competing for the same sites on the HoSF interior, suggesting that 24 of the Cd2+ were bound to the inside surface. No other M2+ studied bound to the interior of HoSF by this criterion. Several of the M2+ appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF.

Original languageEnglish (US)
Pages (from-to)15-27
Number of pages13
JournalJournal of Inorganic Biochemistry
Volume59
Issue number1
DOIs
StatePublished - Jul 1995

Bibliographical note

Funding Information:
This research was supported by Research Grant 5R01 DK36799-05 from the National Institutes of Health. SP, BW, and CL were partially supported by the Undergraduate Research Program of the College of Physical and Mathematical Sciences at Brigham Young Uniuersity.

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