Structural and spectroscopic changes of human hemoglobin during iron-mediated oxidative stress

Biochemical studies have shown that iron produces reactive oxygen species via Haber-Weiss and Fenton reactions. The goal of this study is to examine the role of iron in oxidation of human hemoglobin and its structural changes in erythrocytes. In this experimental study, blood samples from healthy subjects were incubated aerobically with the iron containing metal catalyzed oxidation (MCO) system in the presence of 0.036, 0.7, 0.14, 0.28, 0.57, 1.14, 2.28, 4.55, 9.09, and 18.18 micromole of iron. Structural changes in Hb were followed by spectrophotometric analysis from 300 to 650 nm. In addition, carbonyl assay was performed for estimation of protein oxidation in globin chains. Based on the results, oxy-Hb decreased up to 68% in iron-treated erythrocytes. Decrease in the absorbance ratio (A577, A542 wavelength) indicated the conversion of oxy-Hb to met-Hb. Also, met-Hb concentration was 4.7 fold of hemichrome. After 24 hours of incubation, oxyHb concentration decreased up to 50% and metHb concentration increased up to 85%. Moreover, increase in iron concentration resulted in significant carbonyl formation in hemoglobin. These findings indicate that Hb oxidation instead of its oxygenation leads to anemia and hypoxia. The findings of this study may be directly applicable to oxidation states during hemolytic diseases and iron treatment.