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

Abstract

The binding of Cd 2+, Zn 2+, Cu 2+, Ni 2+, Co 2+, Mn 2+, and Mg 2+ 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 (K d < 10 −7 M) with apo HoSF at pH 7.5 were found for the various M 2+ studied: high stoichiometric binding (30–54 M 2+/HoSF) for Cd 2+, Zn 2+, Cu 2+, with two protons released per metal bound; intermediate binding (16 M 2+/HoSF) for Ni 2+ and Co 2+, with one proton released per metal bound; and low levels of binding (2–12 M 2+/HoSF) for Mn 2+, Mg 2+, and Fe 2+, with <0.5 protons released per metal bound. M 2+ binding to apo HoSF was nearly abolished at pH 5.5, except for Fe 2+ and Cu 2+, which remained unaffected by pH alteration. Holo HoSF bound much higher levels of M 2+, a result directly attributable to the presence of phosphate binding sites. This conclusion was confirmed by decreased binding of M 2+ to HoSF reconstituted in the absence of phosphate and by native holo HoSF with phosphate chemically removed. The binding of Cd 2+ to apo HoSF was 54 per HoSF, but in the presence of developing core, the amount bound decreased to about 30 Cd 2+/HoSF. This result indicated that Cd 2+ and developing core were competing for the same sites on the HoSF interior, suggesting that 24 of the Cd 2+ were bound to the inside surface. No other M 2+ studied bound to the interior of HoSF by this criterion. Several of the M 2+ appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF.