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Thiyl radicals are prominent intermediates during biological conditions of oxidative stress and have been suggested to be involved in the mutagenic effects of thiols. While several enzymatic processes rely on the formation and selective reactions of protein thiyl radicals with substrates, such reactions may represent a source for biological damage when occurring uncontrolled during oxidative stress. For example, intramolecular hydrogen transfer reactions to protein cysteine thiyl radicals may lead to secondary amino acid oxidation products, which may represent starting points for protein aggregation and/or fragmentation. Here, we have used a kinetic NMR method to determine rate constants, ksc, for hydrogen transfer reactions between thiyl radicals and amino acid side chain C−H bonds at 37 °C. Rate constants cover a range between ksc ≤ 1 × 10³ M^-1 s^-1 (Val) and ksc = 1.6 × 10⁵ M^-1 s^-1 (Ser). On the basis of these values and earlier data, model calculations are performed, which will demonstrate that protein thiyl radicals may attack protein C−H bonds via intramolecular hydrogen transfer at physiological conditions, potentially resulting in irreversible protein damage.

Laser flash photolysis at 266 nm of 3-nitrotyrosine and N-acetyl-3-nitrotyrosine ethyl ester generates an oxidizing species, which shows all of the characteristics of a hydroxyl radical. This species reacts with Br^- to yield Br2^•-, via an intermediate, that is kinetically identified as HOBr^•-. Moreover, the formation of Br2^•- can be suppressed by methanol; competition kinetics yield relative rate constants for the reaction of the reactive species with Br^- and methanol that are similar to those for the hydroxyl radical. Parallel time-resolved UV/vis spectroscopy suggests the formation of phenoxyl radicals, consistent with the formation of hydroxyl radicals. Laser flash photolysis at 355 nm also generates reactive intermediates that oxidize Br^- to Br2^•- but appear not to be hydroxyl radicals.

Free radical damage of DNA is a well-known process affecting biological tissue under conditions of oxidative stress. Thiols can repair DNA-derived radicals. However, the product thiyl radicals may also cause biological damage. To obtain quantitative information on the potential reactivity with DNA components, we measured the rate constant for hydrogen abstraction by cysteamine thiyl radicals from thymine C5−CH3, k = (1.2 ± 0.8) × 10⁴ M^-1 s^-1, and thymidine-5‘-monophosphate, k = (0.9 ± 0.6) × 10⁴ M^-1 s^-1. Hence, the hydrogen abstraction from C5−CH3 occurs with rate constants similar to the hydrogen abstraction from the carbohydrate moieties. Especially at low oxygen concentration such as that found in skeletal muscle, such hydrogen abstraction processes by thiyl radicals may well compete against other dioxygen-dependent reactions. The rate constants for hydrogen abstraction at thymine C5−CH3 were compared to those with benzylic substrates, toluenesulfonic acid, and benzyl alcohol.

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (l.9 ± 0.2) × 10¹⁰ M^-1 s^-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO^- and ONOOH is formed with a stability constant of (1.0 ± 0.l) × 10⁴ M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent:  from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 ± 1.0 cm³ mol^-1 was calculated. This small value is not compatible with homolysis of the O−O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO^- to form [(HO)ONOO]^•- with a rate constant of 5.8 × l0⁹ M^-1 s^-1. This radical absorbs with a maximum at 420 nm (ε = 1.8 × 10³ M^-1 cm^-1) and decays by second-order kinetics, k = 3.4 × l0⁶ M^-1 s^-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.

The reaction of superoxide with nitrogen monoxide has been reinvestigated. Photolysis of alkaline peroxynitrite solutions results in the formation of superoxide and nitrogen monoxide, which subsequently react at a rate of (1.5 ± 0.1) × 10¹⁰ M^-1 s^-1. When hydrogen peroxide is photolyzed in the presence of nitrogen monoxide, hydroxyl radicals are formed; these react with hydrogen peroxide to form superoxide, which then reacts with the nitrogen monoxide present. For the reaction of nitrogen monoxide with superoxide, a rate constant of (2.0 ± 0.4) × 10¹⁰ M^-1 s^-1 is derived. Photolysis of nitrite gives rise to hydroxyl radicals and nitrogen monoxide. In the presence of formate and oxygen, the hydroxyl radicals subsequently form superoxide radicals, which, in turn, react with nitrogen monoxide. From these experiments, a rate constant of (1.3 ± 0.2) × 10¹⁰ M^-1 s^-1 is derived. We thus report a rate constant of (1.6 ± 0.3) × 10¹⁰ M^-1 s^-1, the weighted average of these three photolysis experiments.

A standard Gibbs energy of formation of 16.6 kcal mol^-1 has been reported for peroxynitrite [Merényi, G., and Lind, J. (1998) Chem. Res. Toxicol. 11, 243−246]. This value is based on the rate constants for the forward and backward rate constants of the equilibrium O2^•- + NO^•  ONOO^-. A rate constant of 0.017 s^-1 for the backward rate constant was determined by observing the formation of C(NO2)3^- when peroxynitrite was mixed with C(NO2)4. However, a similar rate constant is also observed in the presence of NO^•, which indicates that formation of C(NO2)3^- is due to a process other than the reduction of C(NO2)4 by O2^•-. Additionally, copper(II) nitrilotriacetate enhances the decay of ONOO^- at pH 9.3, without reduction of copper(II). The preferred thermodynamic values are therefore as follows:  ΔfH°(ONOO^-) = −10 ± 2 kcal mol^-1, ΔfG°(ONOO^-) = 14 ± 3 kcal mol^-1, S°(ONOO^-) = 31 eu, and E°‘(ONOOH/NO2^•, H2O) = 1.6 V at pH 7 [Koppenol, W. H., and Kissner, R. (1998) Chem. Res. Toxicol. 11, 87−90].