Electrostatic effects control the stability and iron release kinetics of ovotransferrin

The contribution of electrostatic interactions to the stability of ovotransferrin-Fe³⁺ (oTf-Fe³⁺) complex has been assessed by equilibrium experiments that measure iron retention level of diferric-ovotransferrin (Fe₂oTf) as a function of pH and urea in the presence of salts (NaCl, Na₂SO₄, NaBr, NaNO₃) and sucrose at 25 °C. As [salt] is increased, the pH-midpoint for iron release increases monoexponentially and plateau at ∼0.4(±0.05) M NaCl/NaBr/NaNO₃ or ∼0.15(±0.03) M Na₂SO ₄. However, at pH 7.4, the urea-midpoints for iron release (based on fluorescence emission at 340 nm) and for unfolding of Fe₂oTf and apo-ovotransferrin (based on ellipticity values at 222 and 282 nm) decrease at low salt concentrations [≤0.1(±0.02) M Na₂SO₄ or ≤0.35(±0.15) M NaCl], but increase at higher salt concentrations. Furthermore, Na₂SO₄ has a greater effect than NaCl in increasing the urea-midpoints for iron release and unfolding. These results indicate that at low salt concentrations, the electrostatic effects destabilize the oTf-Fe³⁺ complex and also decrease the structural stability of the proteins. In contrast, at higher concentrations, salt ions behave according to Hofmeister series. At pH 5.6, as [salt] is increased, the rate constants for reductive iron release (Fe²⁺ release) and urea denaturation-induced iron release (Fe³⁺ release) from the N-lobe of oTf (Fe _NoTf) increase monoexponentially and plateau at ∼0.4(±0.1) M NaNO₃/NaCl or ∼0.2(±0.05) M Na₂SO ₄. These results suggest that the anion-binding-induced conformational change as well as the electrostatic screening of surface Coulombic interactions plays important role in accelerating the iron release from Fe_NoTf under endosomal pH conditions.