The experimental and theoretical evidence in support of the radical model has been recently reviewed Accordingly, it cannot diffuse through membranes , and it is confined to water-soluble compartments. It can react both by electron transfer or hydrogen abstraction mechanisms It can also participate in oxygen transfer reactions, particularly when reacting with itself or with other free radicals.
It also rapidly reacts with transition metal centers or organic cofactors , — This has been utilized for measuring the kinetics of the reaction of the radical with different compounds by pulse radiolysis techniques , , Due to the short half-life of the radical, which is in the microsecond range, direct detection requires rapid or fast flow methodologies, which are not always available or require high reactant amounts Alternatively, spin-trapping EPR methodologies have also been utilized.
Carbonate anion radical can also be detected by means of chemiluminescence. This spontaneous decay has a relatively slow rate constant 0. It can also be formed from photochemical processes e. Hydroxyl radical can also be detected with the use of probes. Often, the hydroxylation reaction can be followed by fluorescence. Various coumarins and many other probes have been developed recently and are reviewed in Refs. The fate of a certain species is determined by kinetics i.
Detection methods based on probes reveal only a fraction of the total species, because a large fraction reacts with targets and remains undetected. Knowledge of the rate constants and concentrations of probe and targets can help estimate the actual amount of oxidant produced from the amount detected.
This can be pharmacologically achieved through the use of NOS inhibitors such as N -nitro- l -arginine methyl ester or N G -monomethyl- l -arginine. Alternatively, genetically engineered cell lines or animals in which NOS are knocked out or overexpressed can be used Complementary evidence can be achieved with inhibitors of the formation of other precursors. Their elusive character imposes technical challenges in their detection and quantification. In general terms, the strategies are based either on the detection of stable end products or on the use of synthetic probes.
These methods are summarized in Table 1. Rigorous use of these strategies requires understanding of the mechanistic pathways involved. In many cases, probes are able to react with several species, and efforts are under way to expand the toolset with probes of suitable selectivity.
The evidence about the role of a particular species in a certain pathophysiological process should combine data obtained through more than one analytical or immunochemical procedure as well as complementary evidence coming from the modulation of the formation or decay pathways of the species. Summary of methods for the detection of nitric oxide—derived oxidants.
J Biol Chem. Published online Aug PMID: Author information Copyright and License information Disclaimer. Keywords: nitric oxide, nitrosative stress, oxidation—reduction redox , oxidative stress, oxygen radicals, nitrosylation, reactive nitrogen species, reactive oxygen species, 3-nitrotyrosine, nitration, nitrogen dioxide, nitrosation, peroxynitrite. Open in a separate window.
Figure 1. Nitric oxide The discovery of nitric oxide, a free radical, as an endogenously generated effector molecule, was a paradigm shift in biological signaling. Detection of nitric oxide Nitric oxide is difficult to measure in vivo because of its short half-life typically in the range of 0. Figure 2.
Figure 3. Detection of S-nitrosothiols Several methods have been developed to quantify total S -nitrosothiols and also to identify proteins that are nitrosated. Figure 4. Detection of nitroxyl Nitroxyl can be detected by observing the dimerization product, N 2 O, by gas chromatography GC Figure 5. Figure 6. Probes that react directly with peroxynitrite anion Another mechanistic possibility for the detection of peroxynitrite involves a nucleophilic attack by the oxidant to an electrophilic functional group supported on the probe, leading to fluorescence.
Figure 7. Antibody-based methods for 3-nitrotyrosine detection Nitration changes the immunogenicity of a protein through the generation of new epitopes. Analysis of free 3-nitrotyrosine by HPLC-based methods HPLC with UV-visible detection or fluorescence detection following derivatization with fluorogenic compounds has been employed to measure 3-nitrotyrosine. Analysis of 3-nitrotyrosine in proteins Quantitative data on the amount of 3-nitrotyrosine in a protein-containing sample can be obtained by extraction of total proteins followed by hydrolysis by chemical or enzymatic methods and, finally, analysis of free 3-nitrotyrosine by the procedures described above.
Some reflections on the detection of nitric oxide—derived oxidants The fate of a certain species is determined by kinetics i. Table 1 Summary of methods for the detection of nitric oxide—derived oxidants. References 1. Ferrer-Sueta G. Heinrich T.dufiqexumosi.ml
Detection and quantification of nitric oxide–derived oxidants in biological systems
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