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

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 (K_d < 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.