Conformational assessment of human hemoglobin during iron induced oxidative stress.
Abstract
Background: Our previous studies demonstrated the oxidative effects of iron(1,2), menadione(3) and phylloquinone(4) on human erythrocytes and albumin. This work is interested in investigating the conformational changes of iron treated human hemoglobin (Hb).
Methods: Erythrocytes were incubated aerobically with the iron containing metal catalyzed oxidation system. Structural changes in Hb were followed by spectrophotometric analysis. Hb derivatives were measured according to the following equations:Oxy-Hb=119A577-39A630 -89A560, Met-Hb=28A577+307A630 -55A560 and Hemichrome=-133A577-114A630+233A560, Estimation of protein oxidation was performed by determination of carbonyl groups using 2,4 dinitrophenylhydrazine.
Results: Oxy-Hb were lower (12.56 mM vs. 18.59 mM) in iron-treated erythrocytes compared with control groups. A significant increase was observed in hemichrome concentration(6.64 mM vs. 0.28 mM). The absorbance ratio (A577/A542) in iron-treated erythrocytes was 0.93 compared with normal subjects (0.97). After iron treatment, carbonyl groups significantly increased from 1.1±0.5 to 2.9±0.2 nmol/mg protein in erythrocytes (p<0.05).
Conclusion: These results indicate Hb oxidation instead of its oxygenation resulted in structural changes and dysfunction. The relevance of our findings may be directly applicable to oxidative states during administration of iron and hemolytic diseases.
Key words: carbonyl, conformational assessment, hemoglobin, oxidative stress.
References
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