JBIC Journal of Biological Inorganic Chemistry

, Volume 19, Issue 6, pp 1009–1024 | Cite as

Electrostatic effects control the stability and iron release kinetics of ovotransferrin

  • Sandeep Kumar
  • Deepak Sharma
  • Rajesh Kumar
  • Rajesh Kumar
Original Paper


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 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-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 (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.


Iron release Electrostatic interactions Anion-binding Conformational change Structural stability 



This work was supported by DST-SERC Fast Track Research Grant (to R.K., project No. SR/FT/CS-070/2009), a Ramalingaswami Re-entry Fellowship by DBT, India (to D.S., BT/RLF/RE-ENTRY-33-2010), and UGC major grant (to R.K., F. No. 41-258/2012 (SR), Government of India.

Supplementary material

775_2014_1145_MOESM1_ESM.pdf (57 kb)
Supplementary material 1 (PDF 57 kb)


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Copyright information

© SBIC 2014

Authors and Affiliations

  • Sandeep Kumar
    • 1
  • Deepak Sharma
    • 2
  • Rajesh Kumar
    • 1
  • Rajesh Kumar
    • 1
  1. 1.School of Chemistry and BiochemistryThapar UniversityPatialaIndia
  2. 2.Institute of Microbial Technology, Council of Scientific and Industrial ResearchChandigarhIndia

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