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Volume 272, Number 19, Issue of May 9, 1997 pp. 12328-12341
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Nitric Oxide Prevents Oxidative Damage Produced by tert-Butyl Hydroperoxide in Erythroleukemia Cells via Nitrosylation of Heme and Non-heme Iron
ELECTRON PARAMAGNETIC RESONANCE EVIDENCE

(Received for publication, June 28, 1996, and in revised form, February 26, 1997)

Nikolai V. Gorbunov ^§ , Jack C. Yalowich ^** , Arunasri Gaddam ^§ , Padmakumari Thampatty ^** , Vladimir B. Ritov ^§ , Elena R. Kisin ^§ , Nabil M. Elsayed and Valerian E. Kagan ^§**

From the Departments of ^§ Environmental and Occupational Health and  Pharmacology, University of Pittsburgh, and ^** Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15238, and the  Department of Respiratory Research, Division of Medicine, Walter Reed Army Institute of Research, Washington, D. C. 20307

We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2,2-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.

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