Electrostatic effects control the stability and iron release kinetics of ovotransferrin.
The contribution of electrostatic interactions to the stability of ovotransferrin-Fe(3+) (oTf-Fe(3+)) 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 Na2SO4. 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-Fe(3+) 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 (Fe(2+) release) and urea denaturation-induced iron release (Fe(3+) release) from the N-lobe of oTf (FeNoTf) increase monoexponentially and plateau at ~0.4(±0.1) M NaNO3/NaCl or ~0.2(±0.05) M Na2SO4. 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 FeNoTf under endosomal pH conditions.
School of Chemistry and Biochemistry, Thapar University, Patiala, 147004, India.
Sandeep
S
Kumar
Deepak
D
Sharma
Rajesh
R
Kumar
Rajesh
R
Kumar
D003207:Conalbumin / Q000737:chemistry*; D011992:Endosomes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D055672:Static Electricity
D003207:Conalbumin; D007501:Iron