Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition |
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| Institution: | 1. Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8140, New Zealand;2. Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, 8092 Zurich, Switzerland;1. Lviv Polytechnic National University, S. Bandera Str., 12, 79013, Lviv, Ukraine;2. Institute for Problems of Material Science NASU, Chernivtsy Branch, Vilde Str., 5, 58001, Chernivtsy, Ukraine;3. Institute for Nuclear Research, Hungarian Academy of Sciences (ATOMKI), H-4026, Debrecen, Bem sqr. 18/c, Hungary;1. Istituto di Tecnologie Avanzate per l''Energia “Nicola Giordano”, via S. Lucia Sopra Contesse 5, 98126 Messina, Italy;2. Dipartimento di Scienze Chimiche, University of Messina Viale Ferdinando Stagno D''Alcontres n.31, 98166 Villaggio S. Agata, Messina, Italy;3. Istituto per lo Studio dei Materiali Nanostrutturati, c/o Dipartimento di Scienze Chimiche Viale Ferdinando Stagno D''Alcontres n.31, 98166 Villaggio S. Agata, Messina, Italy;1. Cyclotron Laboratory, Vrije Universiteit Brussel (VUB), Brussels, Belgium;2. Institute of Nuclear Research, Hungarian Academy of Sciences (ATOMKI), Debrecen, Hungary;1. Department of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland;2. Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland;1. Nan Hu College, Jiaxing University, Zhejiang 314001, China;2. College of Mechanical and Electrical Engineering, Jiaxing University, Jiaxing 314001, China;1. Lviv Polytechnic National University, 12 Bandera St, 79013 Lviv, Ukraine;2. Institute for Nuclear Research, Hungarian Academy of Sciences (ATOMKI), H-4026 Debrecen, Bemter 18/c, Hungary;3. Institute for Problems of Material Science NASU, Chernivtsy Branch, 5 Vilde St, 58001 Chernivtsy, Ukraine |
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| Abstract: | Tyrosine (Tyr) residues are major sites of radical generation during protein oxidation. We used insulin as a model to study the kinetics, mechanisms, and products of the reactions of radiation-induced or enzyme-generated protein-tyrosyl radicals with superoxide to demonstrate the feasibility of these reactions under oxidative stress conditions. We found that insulin-tyrosyl radicals combined to form dimers, mostly via the tyrosine at position 14 on the α chain (Tyr14). However, in the presence of superoxide, dimerization was largely outcompeted by the reaction of superoxide with insulin-tyrosyl radicals. Using pulse radiolysis, we measured a second-order rate constant for the latter reaction of (6±1) × 108 M?1 s?1 at pH 7.3, representing the first measured rate constant for a protein-tyrosyl radical with superoxide. Mass-spectrometry-based product analyses revealed the addition of superoxide to the insulin-Tyr14 radical to form the hydroperoxide. Glutathione efficiently reduced the hydroperoxide to the corresponding monoxide and also subsequently underwent Michael addition to the monoxide to give a diglutathionylated protein adduct. Although much slower, conjugation of the backbone amide group can form a bicyclic Tyr-monoxide derivative, allowing the addition of only one glutathione molecule. These findings suggest that Tyr-hydroperoxides should readily form on proteins under oxidative stress conditions where protein radicals and superoxide are both generated and that these should form addition products with thiol compounds such as glutathione. |
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| Keywords: | Insulin oxidation Superoxide radical Tyrosyl radical Protein hydroperoxide Pulse radiolysis Glutathionylation Free radicals |
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