Comparative Studies of Protein Modification Mediated by Fenton-like Reactions of Iron, Hematin, and Hemoglobin: Generation of Different Reactive Oxidizing Species

  • Kim, Young-Myeong (Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University) ;
  • Kim, Sung-Soo (Department of Pharmacology, College of Medicine, Kangwon National University) ;
  • Kang, Gu (Department of Pathology, College of Medicine, Kangwon National University) ;
  • Yoo, Yeong-Min (Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University) ;
  • Kim, Ki-Mo (Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University) ;
  • Lee, Mi-Eun (Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University) ;
  • Han, Jeong-A (Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University) ;
  • Hong, Sun-Joo (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • Received : 1997.11.13
  • Published : 1998.03.31

Abstract

TThe reactive oxygen species oxidatively modify the biological macromolecules, including proteins, lipids, and nucleic acids. Iron- and heme-mediated Fenton-like reactions produce different pro-oxidants. However, these reactive products have not been clearly characterized. We examined the nature of the oxidizing species from the different iron sources by measuring oxidative protein modification and spectroscopic study. Hemoglobin (Hb) and methemoglobin (metHb) were oxidatively modified in $O{\array-\\\dot{2}}$ and $H_{2}O_{2}$ generating systems. Globin and bovine serum albumin (BSA) were also modified by iron, iron-EDTA, hematin, and Hb in an $O{\array-\\\dot{2}}$ generating system. In a $H_{2}O_{2}$ generating system, the iron- and iron-EDTA-mediated protein modifications were markedly reduced while the Hb-and hematin-mediated modifications were slightly increased. In the $O{\array-\\\dot{2}}$ generating system, the iron- and iron-EDTA-mediated protein modifications were strongly inhibited by superoxide dismutase (SOD) or catalase, but heme- and Hb-mediated protein modifications were inhibited only by catalase and slightly increased by SOD. Mannitol, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), deoxyribose, and thiourea inhibited the iron-EDTA-mediated protein modification. Mannitol and DMPO, however, did not exhibit significant inhibition in the hematin-mediated modification. Desferrioxamine (DFO) inhibited protein modification mediated by iron, but cyanide and azide did not, while the hematin-mediated protein modification was inhibited by cyanide and azide, but not significantly by DFO. The protein-modified products by iron and heme were different. ESR and UV-visible spectroscopy detected the DMPO spin adduct of the hydroxyl radical and ferryl ion generated from iron-EDTA and metHb, respectively. These results led us to conclude that the main oxidizing species are hydroxyl radical in the iron-EDTA type and the ferry I ion in the hematin type, the latter being more effective for protein modification.

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