title: Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition.
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) × 10(8) 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.
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8140, New Zealand.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Das
authors.affiliation: Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, 8092 Zurich, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Nauser
authors.affiliation: Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, 8092 Zurich, Switzerland.
authors.forename: Willem H
authors.initials: WH
authors.lastname: Koppenol
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8140, New Zealand.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8140, New Zealand.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8140, New Zealand. Electronic address: peter.nagy@oncol.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Nagy
mesh_terms: D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D011675:Pulse Radiolysis; D013481:Superoxides / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D007328:Insulin; C114979:tyrosyl radical YZ; D013481:Superoxides; D014443:Tyrosine; D006861:Hydrogen Peroxide; D005978:Glutathione
keywords: Free radicals; Glutathionylation; Insulin oxidation; Protein hydroperoxide; Pulse radiolysis; Superoxide radical; Tyrosyl radical