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Reaction kinetics of amino acid and peptide alkyl radicals with GSH and ascorbate, the two most abundant endogenous antioxidants, were investigated by pulse radiolysis. Rate constants in the order of 10 6  M -1 s -1 were found. Alkyl radicals react at almost diffusion controlled rates and irreversibly with oxygen to form peroxyl radicals, and competition with this reaction is the benchmark for efficient repair in vivo. We consider repair of protein radicals and assume comparable rate constants for the reactions of GSH/ascorbate with peptide alkyl radicals and with alkyl radicals on a protein surface. Given physiological concentrations of oxygen, GSH and ascorbate, protein peroxyl radicals will always be a major product of protein alkyl radicals in vivo. Therefore, if they are formed by oxidative stress, protein alkyl radicals are a probable cause for biological damage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05±0.05)×10(5) M(-1) s(-1), while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, β-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.

The reduction of oxidized glutathione GSSG by hydrated electrons and hydrogen atoms to form GSSG•⁻ is quantitative. The radical anion dissociates into GS• and GS⁻, and the S-centered radical subsequently abstracts a hydrogen intramolecularly. We observe sequential development of UV absorbance signatures that indicate the formation of both α- and β-carbon-centered radicals. From experiments performed at pH 2 and pH 11.8, we determined forward and reverse rate constants for the overall equilibrium between sulfur-centered and carbon-centered radicals: k(forward) = 3·10⁵ s⁻¹, k(reverse) = 7·10⁵ s⁻¹, and K = 0.4. Furthermore, on the basis of the differences between the kinetics traces at 240 and 280 nm, we estimate that α- and β-carbon-centered radicals are formed at a surprising ratio of 1:3. The ratios found at pH 2 also apply to pH 7, with the conclusion that the equilibrium ratio of S-centered:β-centered:α-centered radicals is, very approximately, 8:3:1. The formation of carbon-centered radicals could lead to irreversible damage in proteins via the formation of carbon-carbon bonds or backbone fragmentation.

Laser flash photolysis experiments shows that the rate constant for the reaction of the glutathione thiyl radical with nitrogen monoxide to give S-nitrosoglutathione is lower than 2.8+/-0.6 x 10(7)M(-1)s(-1). The conversion of the thiyl radical to its carbon-centred form at 10(3)s(-1) exceeds the formation of S-nitrosoglutathione when physiological concentrations of nitrogen monoxide are taken into account.

Current studies provide evidence that proteins are initial targets of ROS (reactive oxygen species) in biological systems and that the damaged proteins can in turn damage other cell constituents. This study was designed to test the possibility that protein radicals generated by ROS can oxidize GSH and assess the probability of this reaction in vivo by measurement of the rate constant of this reaction. Lysozyme radicals were generated by hydroxyl and azide radicals in steady-state gamma ray radiolysis. In the absence of dioxygen, a range of protein carbon-centred amino acid radicals were produced by the hydroxyl radicals, and defined tryptophan radicals by the azide radicals. In the presence of dioxygen, each carbon-centred radical was converted to a protein peroxyl radical. Each of the peroxyl radicals was able to oxidize a molecule of GSH, regardless of its location in the protein. The peroxyl radicals were 10 and 20 times more effective GSH oxidants than the carbon-centred radicals produced randomly in the lysozyme, or the defined tryptophan lysozyme radicals respectively. We obtained for the first time the rate constant of reaction between a protein free-radical and GSH. Lysozyme tryptophan carbon radicals generated by nanosecond pulse radiolysis and flash photolysis oxidized GSH with a rate constant of (1.05+/-0.05)x10(5) M(-1) x s(-1). Overall, the results are consistent with the hypothesis that protein radicals may be important intermediates in the pathway linking oxidative stress and damage in living organisms and emphasize the strongly enhancing role of dioxygen in this process.