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Detection and quantification of nitric oxide–derived oxidants in biological systems

  • Matías N. Möller
    Affiliations
    Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay
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  • Natalia Rios
    Footnotes
    Affiliations
    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

    Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Madia Trujillo
    Affiliations
    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

    Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Rafael Radi
    Affiliations
    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

    Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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  • Ana Denicola
    Affiliations
    Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay

    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay
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  • Beatriz Alvarez
    Correspondence
    To whom correspondence should be addressed: Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. Tel./Fax: 598-25250749; E-mail: [email protected]
    Affiliations
    Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay

    Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay
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  • Author Footnotes
    1 Supported in part by a doctoral fellowship from Universidad de la República (CAP).
Open AccessPublished:August 12, 2019DOI:https://doi.org/10.1074/jbc.REV119.006136
      The free radical nitric oxide (NO) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO-derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO, and we discuss the approaches currently available for the analysis of NO, nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO/ONOOH), as well as peroxynitrite-derived hydroxyl (HO) and carbonate anion (CO3•−) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2), nitrate (NO3), nitrosyl–metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state–of–the–art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved.

      Introduction

      Soon after the discovery of nitric oxide (NO) as a physiological mediator in the vascular, nervous, and immune systems, it became evident that this moderately-reactive free radical can give rise to secondary species, many of which are oxidizing, nitrosating, or nitrating agents toward biomolecules (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      • Heinrich T.A.
      • da Silva R.S.
      • Miranda K.M.
      • Switzer C.H.
      • Wink D.A.
      • Fukuto J.M.
      Biological nitric oxide signalling: chemistry and terminology.
      ,
      • Ignarro L.J.
      Biosynthesis and metabolism of endothelium-derived nitric oxide.
      • Radi R.
      Nitric oxide, oxidants, and protein tyrosine nitration.
      ).
      The species formed downstream from NO (i.e. NO-derived oxidants) include nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO/ONOOH), as well as hydroxyl (HO) and carbonate anion (CO3•−) radicals formed from peroxynitrite (Fig. 1). These species are short-lived (half-lives are typically in the millisecond to microsecond range) and are frequently referred to in general as “reactive nitrogen species.” However, this term should be used with caution because in a similar way as “reactive oxygen species,” with which NO-derived species are usually grouped, this term may give the inaccurate idea that there exists only one ill-defined species that has a particular type of reactivity and targets all biomolecules (
      • Forman H.J.
      • Augusto O.
      • Brigelius-Flohe R.
      • Dennery P.A.
      • Kalyanaraman B.
      • Ischiropoulos H.
      • Mann G.E.
      • Radi R.
      • Roberts 2nd., L.J.
      • Vina J.
      • Davis K.J.
      Even free radicals should follow some rules: a guide to free radical research terminology and methodology.
      ,
      • Winterbourn C.C.
      Reconciling the chemistry and biology of reactive oxygen species.
      ). In contrast, the different species have unique reactivities and, depending on the particular properties of each, they may lead to oxidation, nitrosation, or nitration. As a further argument against the use of the term “reactive nitrogen species,” some of the species formed downstream from NO (e.g. CO3•−) do not contain nitrogen. Finally, researchers in the nitrogen fixation field might argue that the reactive nitrogen species are those formed in the activation of nitrogen in the nitrogenase-catalyzed process of ammonia formation. Thus, in line with proposals in the free radical research field (
      • Forman H.J.
      • Augusto O.
      • Brigelius-Flohe R.
      • Dennery P.A.
      • Kalyanaraman B.
      • Ischiropoulos H.
      • Mann G.E.
      • Radi R.
      • Roberts 2nd., L.J.
      • Vina J.
      • Davis K.J.
      Even free radicals should follow some rules: a guide to free radical research terminology and methodology.
      ,
      • Winterbourn C.C.
      Reconciling the chemistry and biology of reactive oxygen species.
      ), we suggest that the name of the identified species should be used whenever possible. When the species that are being referred to are unknown, we suggest using the term “NO-derived oxidants.”
      Figure thumbnail gr1
      Figure 1Nitric oxide and its biologically relevant derivatives. Nitric oxide can give rise to several species. Reaction with superoxide (O2•−) generates peroxynitrite (ONOO); with oxyhemoglobin (HbO2), nitrate (NO3); with oxygen (O2), nitrogen dioxide (NO2); with strong one-electron reductants, nitroxyl (HNO); with liganded iron(II) (Fe(II)L2), dinitrosyl iron complexes (DNICs); with thiyl radical (RS), S-nitrosothiol (RSNO); and with NO2, dinitrogen trioxide (N2O3). Many of these products are reactive and yield further products. Peroxynitrite at neutral pH will protonate and generate NO3, as well as NO2 and hydroxyl radicals (HO) in 30% yield. In the presence of carbon dioxide (CO2), peroxynitrite will generate NO3, as well as NO2 and carbonate anion radical (CO3•−) in 33% yield. In the presence of reductants, peroxynitrite will be reduced to nitrite (NO2) or NO2. Nitrogen dioxide can react with tyrosyl radicals (Tyr) to generate 3-nitrotyrosine (NO2–Tyr) or with a reductant to form NO2. Dinitrogen trioxide can be rapidly hydrolyzed to NO2, it can be formed by NO2 in acidic pH, and it can react with thiols (RSH) to generate RSNO. In this figure, stoichiometries are not always strict, and protons are sometimes omitted for simplicity.
      The preferential targets of NO-derived oxidants in biological systems are typically located in close proximity (in the micrometer distance range) and determined by a combination of factors, including kinetic aspects of rate constants multiplied by target concentration, compartmentalization, and membrane permeability. Some of the NO-derived oxidants are good one-electron oxidants that start oxygen-dependent chain reactions in both aqueous and lipidic compartments, which may amplify the effects (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ).
      In many cases, the formation of NO-derived oxidants is linked to the presence of partially-reduced oxygen species, as exemplified by peroxynitrite, which is formed from the reaction of NO with the superoxide radical (O2•−). Thus, the formation of NO-derived oxidants is frequently related to inflammation, in which increased formation of NO through the inducible nitric-oxide synthase converges with increased formation of O2•− and other oxidants. In fact, the high reactivity of some of the species derived from NO make them part of the weaponry that immune cells use in their battles against microorganisms (
      • Piacenza L.
      • Trujillo M.
      • Radi R.
      Reactive species and pathogen antioxidant networks during phagocytosis.
      ). In addition to their cell-damaging activity, they can have signaling roles. The recognition that hydrogen peroxide (H2O2) can act as second messenger (
      • Sundaresan M.
      • Yu Z.X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.
      ) and that signaling actions can be extended to species derived from NO (
      • Marine A.
      • Krager K.J.
      • Aykin-Burns N.
      • Macmillan-Crow L.A.
      Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells.
      ) have expanded the traditional view of oxidative stress as a misbalance between oxidant formation and antioxidant action to include the view of a disruption in regulatory pathways.
      Because of their high reactivity, the species derived from NO have relatively short half-lives that impede their detection in biological systems through direct spectroscopic techniques. Thus, the analytical strategies used to demonstrate the formation of a certain species in a particular biological context are based either on the measurement of downstream stable products or on the use of probes that react with the species. Because these strategies are not always specific for a certain species, the modulation of the formation or decay pathways of precursors and products provides complementary evidence. For example, the modulation of NO and O2•− formation, which are the precursors of peroxynitrite, should accompany the results obtained through the detection of the stable product 3-nitrotyrosine or through the use of peroxynitrite probes. The modulation of NO formation can be carried out using nitric oxide synthase inhibitors, among other strategies.
      In the following sections, we briefly describe the reactive species derived from NO in a biological milieu and NO itself, as well as some of the stable end products (Fig. 1). We examine methodologies used for their detection and quantification, focusing on strategies aimed at assessing their involvement in biological processes.

      Nitric oxide

      The discovery of nitric oxide, a free radical, as an endogenously generated effector molecule, was a paradigm shift in biological signaling. Nitric oxide (NO, IUPAC names nitrogen monoxide, oxidonitrogen(), or oxoazanyl) is a diatomic free radical produced in animals mainly by the enzymes nitric oxide synthases (NOS)
      The abbreviations used are: NOS
      nitric oxide synthase
      EPR
      electron paramagnetic resonance
      GC
      gas chromatography
      LC
      liquid chromatography
      MS
      mass spectrometry
      MALDI
      matrix-assisted laser desorption/ionization
      DCF
      2′,7′-dichlorofluorescein
      DCFH2
      2′,7′-dichlorodihydrofluorescein
      CBA
      coumarin boronic acid
      CBE
      coumarin boronic ester
      Fl-B
      fluorescein-boronate
      FlAmBE
      fluorescein-dimethylamide boronate
      FBBE
      4-(pinacol boronate)benzyl-derivative of fluorescein methyl ester
      DNIC
      dinitrosyl iron complex
      DAN
      diaminonaphthalene
      DAF
      diaminofluorescein
      DAF-FM
      4-amino-5-methylamino-2′,7′-difluorofluorescein
      DAF-2–DA
      4,5-diaminofluorescein diacetate
      PTIO
      2-phenyl-4,4,5,5-tetramethylimidazoline-1-yloxyl-3-oxide
      Fe(DETC)2
      iron diethyldithiocarbamate
      ICAT
      isotope-coded affinity tag
      SILAC
      stable isotope labeling by amino acids in cell culture
      TEMPO
      (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl
      Fl
      fluorescein
      ABTS
      2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
      COH
      7-hydroxycoumarin
      DMPO
      5,5-dimethyl-1-pyrroline-N-oxide.
      . Nitric oxide has a low dipole moment (0.159 D (
      • Lide D.R.
      Permittivity (Dielectric Constant) of Gases.
      , so it has weak intermolecular interactions and it is a gas at 1 atm and 25 °C. It is only sparingly soluble in water (1.94 ± 0.03 mm (
      • Zacharia I.G.
      • Deen W.M.
      Diffusivity and solubility of nitric oxide in water and saline.
      ) but is about 10 times more soluble in organic solvents (
      • Shaw A.W.
      • Vosper A.J.
      Solubility of nitric oxide in aqueous and nonaqueous solvents.
      ). The partition coefficient in membrane models and human low-density lipoprotein is 4.4–3.4 at 25 °C (
      • Möller M.
      • Botti H.
      • Batthyany C.
      • Rubbo H.
      • Radi R.
      • Denicola A.
      Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein.
      ). The diffusion through cell membranes is very rapid (
      • Möller M.
      • Botti H.
      • Batthyany C.
      • Rubbo H.
      • Radi R.
      • Denicola A.
      Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein.
      • Denicola A.
      • Souza J.M.
      • Radi R.
      • Lissi E.
      Nitric oxide diffusion in membranes determined by fluorescence quenching.
      ,
      • Möller M.N.
      • Denicola A.
      Diffusion of nitric oxide and oxygen in lipoproteins and membranes studied by pyrene fluorescence quenching.
      • Subczynski W.K.
      • Lomnicka M.
      • Hyde J.S.
      Permeability of nitric oxide through lipid bilayer membranes.
      ). The permeability coefficients of lipid membranes to NO range from 18 to 73 cm s−1 (
      • Denicola A.
      • Souza J.M.
      • Radi R.
      • Lissi E.
      Nitric oxide diffusion in membranes determined by fluorescence quenching.
      ,
      • Subczynski W.K.
      • Lomnicka M.
      • Hyde J.S.
      Permeability of nitric oxide through lipid bilayer membranes.
      ), similar to that of an equally thick layer of water.
      Unlike several other free radical species, NO is not a one-electron oxidant (E0′ (NO, H+/HNO) ∼−0.55 V at pH 7) (
      • Bartberger M.D.
      • Liu W.
      • Ford E.
      • Miranda K.M.
      • Switzer C.
      • Fukuto J.M.
      • Farmer P.J.
      • Wink D.A.
      • Houk K.N.
      The reduction potential of nitric oxide (NO) and its importance to NO biochemistry.
      ,
      • Shafirovich V.
      • Lymar S.V.
      Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide.
      ). It does not abstract hydrogen atoms, and it does not add to unsaturated bonds. Importantly, NO does not react directly with thiols (RSH). Among the main targets of NO in biological systems are metal centers. Coordination to the ferrous heme in soluble guanylate cyclase is responsible for many physiological effects of NO (
      • Enemark J.H.
      • Feltham R.D.
      Principles of structure, bonding, and reactivity for metal nitrosyl complexes.
      • Goodrich L.E.
      • Paulat F.
      • Praneeth V.K.
      • Lehnert N.
      Electronic structure of heme-nitrosyls and its significance for nitric oxide reactivity, sensing, transport, and toxicity in biological systems.
      ,
      • Toledo Jr., J.C.
      • Augusto O.
      Connecting the chemical and biological properties of nitric oxide.
      • Radi R.
      Reactions of nitric oxide with metalloproteins.
      ). Reaction with oxyhemoglobin to form NO3 is an important sink of NO (
      • Doyle M.P.
      • Hoekstra J.W.
      Oxidation of nitrogen oxides by bound dioxygen in hemoproteins.
      ,
      • Gardner P.R.
      Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases.
      ). Other relevant targets of NO are other free radical species, in particular O2•− (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). Nitric oxide can also react with oxygen, and this is analyzed in the next section on autoxidation.
      The effects of NO are exerted either via direct reactions with biological targets or indirectly via NO-derived oxidants. Dysregulation of NO homeostasis has been linked to neurodegeneration, cardiovascular disease, cancer, and inflammation. Therefore, the detection and quantification of NO and its derived oxidants in vitro and in vivo are relevant to understanding the molecular bases of physiological as well as pathological processes.

      Nitric oxide autoxidation

      The reaction of NO with oxygen (O2), termed autoxidation, is a complex process that gives different products and intermediates relevant to the detection of NO and several of its derived species. This process is considered to be too slow to be of relevance under most physiological conditions. In the first step, two molecules of NO and one molecule of O2 give two molecules of NO2 (Equation 1). Next, NO2 reacts with NO reversibly to give N2O3 (Equation 2) (
      • Treinin A.
      • Hayon E.
      Absorption spectra and reaction kinetics of NO2, N2O3, and N2O4 in aqueous solution.
      ,
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ). In water, in the absence of other targets, N2O3 is subsequently hydrolyzed to two molecules of NO2 (Equation 3).
      2NO+O22NO2
      (Eq. 1)


      NO2+NON2O3
      (Eq. 2)


      N2O3+H2O2NO2+2H+
      (Eq. 3)


      The rate of decomposition of NO is second order in NO and first order in O2 concentrations, and in water the final stoichiometry is four molecules of NO per O2, so that the rate equation is expressed as in Equation 4.
      d[NO]dt=4k[NO]2[O2]
      (Eq. 4)


      The limiting reaction in the autoxidation of NO is the reaction with O2 (Equation 1); trapping subsequent products has no effect on the overall rate (
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ). The rate constant of the process is third order (k ∼2 × 106 m−2 s−1 (
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ,
      • Ford P.C.
      • Wink D.A.
      • Stanbury D.M.
      Autoxidation kinetics of aqueous nitric oxide.
      )). In aqueous solutions, the forward reaction of N2O3 formation (Equation 2) is very fast (k = 1.1 × 109 m−1 s−1), and the dissociation has a rate constant k = 8 × 104 s−1 (
      • Graetzel M.
      • Taniguchi S.
      • Henglein A.
      Pulsradiolytische untersuchung der NO-oxydation und des gleichgewichts N2O3 NO + NO2 in waessriger loesung.
      ). The hydrolysis of N2O3 is also rapid in water and can be further accelerated by salts such as phosphate and bicarbonate (
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ,
      • Lewis R.S.
      • Tannenbaum S.R.
      • Deen W.M.
      Kinetics of N-nitrosation in oxygenated nitric oxide solutions at physiological pH: role of nitrous anhydride and effects of phosphate and chloride.
      ,
      • Caulfield J.L.
      • Singh S.P.
      • Wishnok J.S.
      • Deen W.M.
      • Tannenbaum S.R.
      Bicarbonate inhibits N-nitrosation in oxygenated nitric oxide solutions.
      ).
      The autoxidation of NO is slow in vivo because the rate of NO decay is second order in NO concentration, which is expected to be in the nanomolar range under normal conditions. This reaction can be accelerated in hydrophobic environments such as lipid membranes, lipoproteins, and proteins (
      • Liu X.
      • Miller M.J.
      • Joshi M.S.
      • Thomas D.D.
      • Lancaster J.R.
      Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes.
      ,
      • Möller M.N.
      • Li Q.
      • Vitturi D.A.
      • Robinson J.M.
      • Lancaster Jr., J.R.
      • Denicola A.
      Membrane “Lens” effect: focusing the formation of reactive nitrogen oxides from the NO/O2 reaction.
      • Möller M.N.
      • Denicola A.
      Acceleration of the autoxidation of nitric oxide by proteins.
      ) due to the increase in concentration of both NO and O2 because of their hydrophobicity (
      • Möller M.
      • Botti H.
      • Batthyany C.
      • Rubbo H.
      • Radi R.
      • Denicola A.
      Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein.
      ,
      • Möller M.N.
      • Li Q.
      • Chinnaraj M.
      • Cheung H.C.
      • Lancaster Jr., J.R.
      • Denicola A.
      Solubility and diffusion of oxygen in phospholipid membranes.
      ). This so-called “lens effect” may be of relevance where NO concentrations are significantly increased, especially in sites of inflammation.

      Detection of nitric oxide

      Nitric oxide is difficult to measure in vivo because of its short half-life (typically in the range of 0.1–10 s), reactivity, and low steady-state concentration (i.e. nanomolar to micromolar). Nonetheless, several strategies have been developed to measure NO or its derived species in vitro or in vivo that involve the use of absorbance, fluorescence, electron paramagnetic resonance (EPR), and electrochemistry (Fig. 2). Furthermore, NO can be measured by chemiluminescence, a methodology that can be adapted to also measure other species. These methods are described in the following sections.
      Figure thumbnail gr2
      Figure 2Detection of nitric oxide. A, oxidation of oxyhemoglobin. Methemoglobin can be detected spectrophotometrically. B, electrochemical sensor. NO is oxidized to nitrosonium cation (NO+), which is converted to nitrite (NO2). The current is directly proportional to NO concentration. C, reaction of NO with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-yloxyl-3-oxide (PTIO) to yield NO2 and PTI, the conversion can be followed by EPR. D, fluorogenic probes for NO-derived species. The 4,5-diaminofluorescein diacetate (DAF-2–DA) is cell-membrane–permeable, and the acetyl groups are removed by intracellular esterases to yield the nonfluorescent DAF-2 that then reacts with NO-derived species to yield the fluorescent triazole derivative DAF-2 T. At right are shown related fluorescent probes diaminonaphthalene (DAN) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM). The mechanisms of triazole formation from diamino-fluorogenic probes involve two possible routes: one is the direct nitrosation by N2O3 to give an intermediary N-nitrosamine that then diazotizes and reacts with the second amine to yield the triazole; the other mechanism requires the oxidation of the diamino probe by NO-derived and other one-electron oxidants, followed by the reaction of the radical with NO to form the N-nitrosamine. Either of these pathways implicate NO but with different stoichiometries. E, ozone-based chemiluminescence. F, reaction catalyzed by nitric oxide synthase to generate NO. l-Arginine is first hydroxylated to Nω-hydroxy-l-arginine with O2 and NADPH as cosubstrates. In the second step, this intermediate is oxidized by a second O2 and 0.5 eq of NADPH to give l-citrulline and NO. G, bioassays to measure downstream physiological actions of NO.

      Oxyhemoglobin oxidation

      The identification of the endothelial-derived relaxing factor as NO back in 1987 (
      • Ignarro L.J.
      • Buga G.M.
      • Wood K.S.
      • Byrns R.E.
      • Chaudhuri G.
      Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
      ) was in part made by the change in the UV-visible spectrum of deoxyhemoglobin to form nitrosyl hemoglobin, with a corresponding shift of the Soret band from 433 to 406 nm. Nonetheless, the reaction mostly used to quantify NO in vitro is with oxyhemoglobin, which is stable in air.
      Oxyhemoglobin (Fe(II)(Hb)O2) is oxidized by NO to yield NO3 and methemoglobin (Fe(III)(Hb)), which can be measured spectrophotometrically (Fig. 2A). The absorption of the Soret band is strong; thus the sensitivity of this method is relatively high (submicromolar). The maximum absorbance changes are observed at 401 (increased by reaction with NO) and 421 nm (decreased), with an isosbestic point at 411 nm. If multiple wavelengths can be measured, the reaction can be followed by the absorbance difference of 401–421 nm (Δϵ401–421 = 77 mm−1 cm−1) (
      • Murphy M.E.
      • Noack E.
      Nitric oxide assay using hemoglobin method.
      ). If there is interference at these wavelengths, the absorbance at 577 nm can be used (Δϵ577 = 10 mm−1 cm−1) or even both absorbances at 577 and 630 nm to calculate oxy- and methemoglobin concentrations before and after addition of NO (
      • Winterbourn C.C.
      Oxidative reactions of hemoglobin.
      ). Due to the fast reaction with NO (k = 3.7 × 107 m−1 s−1) (
      • Eich R.F.
      • Li T.
      • Lemon D.D.
      • Doherty D.H.
      • Curry S.R.
      • Aitken J.F.
      • Mathews A.J.
      • Johnson K.A.
      • Smith R.D.
      • Phillips Jr., G.N.
      • Olson J.S.
      Mechanism of NO-induced oxidation of myoglobin and hemoglobin.
      ), oxyhemoglobin is also often used as NO scavenger. Nitrite can also oxidize oxy- to methemoglobin but at much lower rates (although autocatalytically), so when high concentrations of NO2 are expected, like with the use of NO donors for long-time periods, the contribution of NO2 to hemoglobin oxidation should be considered. Peroxynitrite can also oxidize oxyhemoglobin; thus, addition of superoxide dismutase is recommended as a control to prevent peroxynitrite formation from NO and O2•− so that the methemoglobin formed can be associated with NO. In addition, a potential O2•−-dependent redox cycling of hemoglobin can be avoided.

      Electrochemical sensor

      Electrodes specific for NO are commercially available, but many research laboratories make their own. Typically, they consist of a filament made of carbon or platinum and a coating to provide specificity that either attracts NO (
      • Malinski T.
      • Taha Z.
      Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor.
      ) or excludes other oxidizable species (
      • Friedemann M.N.
      • Robinson S.W.
      • Gerhardt G.A.
      o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide.
      ). At the anode, NO is oxidized by one-electron to nitrosonium cation (NO+), which is converted to NO2 (Fig. 2B). The current generated from NO oxidation is directly proportional to NO concentration with a 10 nm detection limit (a calibration curve should be run with each experiment). The electrode is covered with a gas-permeable membrane that allows diffusion of NO but not NO2 or other charged species. Temperature should be kept constant considering that solubility of NO gas is very temperature-sensitive. Microelectrodes have been designed (<1 mm) that allow direct detection in cells in real time (
      • Cha W.
      • Tung Y.C.
      • Meyerhoff M.E.
      • Takayama S.
      Patterned electrode-based amperometric gas sensor for direct nitric oxide detection within microfluidic devices.
      ).

      Electron paramagnetic resonance (EPR)

      Although NO is a free radical, i.e. it has an unpaired electron, it is difficult to detect directly by EPR, and spin-trapping techniques have to be used. Nitronyl nitroxides (with nitrone and nitroxide functional groups) are used as NO probes (
      • Nagano T.
      • Yoshimura T.
      Bioimaging of nitric oxide.
      ). They react with NO to give an iminonitroxide (Fig. 2C) with a dramatic change in the EPR spectrum that can be followed in a continuous and quantitative way. For example, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-yloxyl-3-oxide (PTIO) or its water-soluble analogue carboxy-PTIO react with NO with second-order rates constants of 104 m−1 s−1 and a change in the EPR spectrum from five to seven lines (
      • Akaike T.
      • Yoshida M.
      • Miyamoto Y.
      • Sato K.
      • Kohno M.
      • Sasamoto K.
      • Miyazaki K.
      • Ueda S.
      • Maeda H.
      Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/NO through a radical reaction.
      ,
      • Goldstein S.
      • Russo A.
      • Samuni A.
      Reactions of PTIO and carboxy-PTIO with NO, NO2, and O2•−.
      ).
      Due to its fast reaction with NO, carboxy-PTIO is often used as a scavenger of NO; however, care should be taken because NO2 is a product of the reaction and has its own reactivities. In addition, biological reductants like thiols, ascorbate, or O2•− can nonspecifically reduce the nitroxides. Encapsulation of PTIO in liposomes has been used to avoid reduction (
      • Akaike T.
      • Maeda H.
      Quantitation of nitric oxide using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO).
      ).
      Hydrophobic and hydrophilic nitroxides are available that allow detection of NO at different depths of a biological membrane. Collisions of NO with spin labels located in water or in membranes alter both the linewidth and the spin-lattice relaxation time that can be used to qualitatively and quantitatively measure NO (
      • Subczynski W.K.
      • Lomnicka M.
      • Hyde J.S.
      Permeability of nitric oxide through lipid bilayer membranes.
      ,
      • Singh R.J.
      • Hogg N.
      • Mchaourab H.S.
      • Kalyanaraman B.
      Physical and chemical interactions between nitric oxide and nitroxides.
      ).
      Colloid iron diethyldithiocarbamate (Fe(DETC)2) or N-methyl-d-glucamine dithiocarbamate are reliable spin traps for NO detection. They form iron nitrosyl complexes with characteristic three-line EPR spectra (gav = 2.04; aN = 1.27 mT) at room temperature that are stable in the presence of oxygen (
      • Vanin A.F.
      EPR characterization of dinitrosyl iron complexes with thiol-containing ligands as an approach to their identification in biological objects: an overview.
      ). Dinitrosyl iron complexes (DNIC) with thiol-containing ligands have been detected in animal and bacterial cells by EPR. These complexes are formed in vivo in the paramagnetic (EPR-active) mononuclear as well as diamagnetic (EPR-silent) binuclear forms. The amount of NO can calculated from the EPR amplitude signal because the linewidth of the NO-Fe(DETC)2 EPR spectrum may vary considerably with variations in the amount of Fe(DETC)2 in membrane lipids and the amount of Fe(III) present (
      • Vanin A.F.
      EPR characterization of dinitrosyl iron complexes with thiol-containing ligands as an approach to their identification in biological objects: an overview.
      ,
      • Lancaster Jr., J.R.
      • Hibbs Jr., J.B.
      EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages.
      ).
      In addition, deoxyhemoglobin or other hemeproteins in the reduced Fe(II) form react with NO to form nitrosyl–heme complexes that besides having characteristic UV-visible spectra are paramagnetic and can be followed by EPR. However, because of the instability of the complexes, the EPR spectra should be run at low temperatures (77 K) (
      • Dikalov S.
      • Fink B.
      ESR techniques for the detection of nitric oxide in vivo and in tissues.
      • Giorgio S.
      • Linares E.
      • Capurro Mde L.
      • de Bianchi A.G.
      • Augusto O.
      Formation of nitrosyl hemoglobin and nitrotyrosine during murine leishmaniasis.
      ,
      • Hall D.M.
      • Buettner G.R.
      In vivo spin trapping of nitric oxide by heme: electron paramagnetic resonance detection ex vivo.
      • Wang Q.Z.
      • Jacobs J.
      • DeLeo J.
      • Kruszyna H.
      • Kruszyna R.
      • Smith R.
      • Wilcox D.
      Nitric oxide hemoglobin in mice and rats in endotoxic shock.
      ).

      Fluorogenic probes

      Fluorogenic probes have been developed that specifically react with NO-derived species (i.e. N2O3) to yield fluorescent products, such as diaminonaphthalene (DAN) and diaminofluorescein (DAF) derivatives (Fig. 3D). The most popular of this kind is 4,5-diaminofluorescein (DAF-2), where nitrosation results in the highly-fluorescent triazole DAF-2 T (λexc = 495 nm, λem = 515 nm). The esterified diacetate derivative (DAF-2-DA) is also commercially available. It is highly membrane-permeable and detects intracellular nitrosation of the probe.
      Figure thumbnail gr3
      Figure 3Chemiluminescence detection of nitric oxide and derived species. This sensitive method allows the determination of NO and several related species in gas or liquid phase. The system includes a purge vessel where the sample is injected. An inert gas transports NO from the purge vessel to a detector where ozone (O3) reacts with NO yielding NO2 in the excited state, which decays to the basal state emitting light. Different reagents can be used in the purge vessel to selectively convert certain analytes into NO. A neutral buffer is used if NO as such is to be measured. A triiodide/acid solution reduces most derivatives to NO, and thus it is useful for total quantitation. The sample can be pretreated with reagents that will trap specific species. Thus, the amount of NO2 in a complex sample is obtained by the difference between untreated sample and sample pretreated with sulfanilamide and acid to trap NO2. RSNO can be removed by pretreatment with Hg(II), sulfanilamide, and acid. DNICs are sensitive to the same treatment but differently from RSNO; they decay over time (10–12 h). The remaining signal after treatment with Hg(II), sulfanilamide, and acid derives mostly from N-nitrosamines, and to a lesser degree from nitrosyl–heme. Alternatively, the purge vessel can contain Cu(II) and cysteine, which does not reduce NO2 to NO but effectively reduces RSNO to NO, and it also releases NO from nitrosyl-hemoglobin and N-nitrosamines. When analyzing nitrosation of hemoglobin or other heme-proteins, carbon monoxide (CO) is added to avoid reaction of NO with heme-proteins.
      The pH should be carefully controlled because DAF-2 T fluorescence is pH-sensitive (
      • Itoh Y.
      • Ma F.H.
      • Hoshi H.
      • Oka M.
      • Noda K.
      • Ukai Y.
      • Kojima H.
      • Nagano T.
      • Toda N.
      Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry.
      ). Other potential artifacts are divalent cations, in particular calcium, which was reported to significantly increase the fluorescent signal from DAF-2, as well as the incident light (
      • Broillet M.
      • Randin O.
      • Chatton J.
      Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging.
      ). Multiple and long exposures to excitation light, instead of causing photobleaching of the dye, potentiate the fluorescence response. Thus, minimum periods of light exposure are recommended. The use of 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) is favored because of its increased photostability, stability to pH, and reactivity toward NO-derived species (
      • Itoh Y.
      • Ma F.H.
      • Hoshi H.
      • Oka M.
      • Noda K.
      • Ukai Y.
      • Kojima H.
      • Nagano T.
      • Toda N.
      Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry.
      ). The sensitivity of DAF-FM is 1.4 times higher than that of DAF-2. This increase of sensitivity is thought to result from the higher rate of the reaction with nitrosating NO+ equivalents due to the electron-donating effect of the methyl group (
      • Nagano T.
      • Yoshimura T.
      Bioimaging of nitric oxide.
      ).
      The proposed mechanism of triazole formation involves N2O3 reacting with an amine to form an intermediate N-nitrosamine that at neutral pH can diazotize and then react with the second amine to yield the triazole (Fig. 3D) (
      • Ralt D.
      • Wishnok J.S.
      • Fitts R.
      • Tannenbaum S.R.
      Bacterial catalysis of nitrosation: involvement of the nar operon of Escherichia coli.
      ,
      • Kojima H.
      • Urano Y.
      • Kikuchi K.
      • Higuchi T.
      • Hirata Y.
      • Nagano T.
      Fluorescent indicators for imaging nitric oxide production.
      ). Alternatively, a radical intermediate of the diamino-probe is formed by NO2 or other strong oxidants (e.g. radicals derived from peroxynitrite or peroxidases/H2O2) that then react with NO (Fig. 3D) (
      • Jourd'heuil D.
      Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide.
      ). These probes show some specificity issues, in that the triazole is not an exclusive product of NO, and fluorescent products can be derived from peroxynitrite, nitroxyl (HNO), and ascorbic acid, complicating the interpretation of results (
      • Espey M.G.
      • Xavier S.
      • Thomas D.D.
      • Miranda K.M.
      • Wink D.A.
      Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms.
      ,
      • Zhang X.
      • Kim W.-S.
      • Hatcher N.
      • Potgieter K.
      • Moroz L.L.
      • Gillette R.
      • Sweedler J.V.
      Interfering with nitric oxide measurements 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid.
      ). Nonetheless, the role of NO can be confirmed in cells using NOS inhibitors, NO scavengers, and also by HPLC to isolate DAF-triazole (
      • Zhang X.
      • Kim W.-S.
      • Hatcher N.
      • Potgieter K.
      • Moroz L.L.
      • Gillette R.
      • Sweedler J.V.
      Interfering with nitric oxide measurements 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid.
      ,
      • Cortese-Krott M.M.
      • Rodriguez-Mateos A.
      • Kuhnle G.G.
      • Brown G.
      • Feelisch M.
      • Kelm M.
      A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins.
      • Hogg N.
      • Zielonka J.
      • Kalyanaraman B.
      ).
      Novel genetically-encoded fluorescent NO biosensors have been developed (
      • Newman R.H.
      • Fosbrink M.D.
      • Zhang J.
      Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells.
      ). For example, one sensor has a fusion between a fluorescent protein and a bacteria-derived NO domain that selectively binds NO via a nonheme Fe(II) center. Once NO binds, the domain gets closer to the fluorescent protein and quenches its emission (
      • Eroglu E.
      • Gottschalk B.
      • Charoensin S.
      • Blass S.
      • Bischof H.
      • Rost R.
      • Madreiter-Sokolowski C.T.
      • Pelzmann B.
      • Bernhart E.
      • Sattler W.
      • Hallström S.
      • Malinski T.
      • Waldeck-Weiermair M.
      • Graier W.F.
      • Malli R.
      Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics.
      ).

      Ozone-based chemiluminescence detection of nitric oxide and related species

      The chemiluminescence detection of NO and several other related species presents very good sensitivity and reproducibility and has become the gold standard method for quantification (
      • Hogg N.
      • Zielonka J.
      • Kalyanaraman B.
      ,
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ). The sensitivity is in the nanomolar range. The method can be used with any type of gas or liquid sample, including cell lysates and tissue homogenates (
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ,
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ).
      The sample is injected into a purge vessel containing a given reactant such as triiodide. This vessel has fritted glass at the base and is purged at a constant flow rate with nitrogen or helium gas. The NO that was injected (or generated) in the vessel is carried by this inert gas to the detector (
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ). The NO in the carrier gas passes first through a reaction cell where ozone is constantly introduced. The reaction with ozone (O3) generates NO2 in the excited state (NO2*) that is then carried by the inert gas flow to the detection cell where red and near IR light emission from NO2* decay to the basal state is measured (
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ,
      • Clough P.N.
      • Thrush B.A.
      Mechanism of chemiluminescent reaction between nitric oxide and ozone.
      ). The intensity of emission is directly proportional to the amount of NO (Fig. 2E).
      This method is not only useful to the study of NO but also of other oxidation products that can be converted to NO through different methods, such as NO2, NO2, S-nitrosothiols, nitrosyl–metal complexes, and N-nitrosamines (
      • Hogg N.
      • Zielonka J.
      • Kalyanaraman B.
      ,
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ,
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ). The reactant used in the purge vessel determines what species can be quantified (Fig. 3). If buffer at neutral pH is used, only NO as such will give a signal (
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ). One of the most popular reactants for chemiluminescent detection of NO and its derivatives is an acidic triiodide solution (
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ). It consists of iodine plus iodide and acetic acid (
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • MacArthur P.H.
      • Shiva S.
      • Gladwin M.T.
      Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.
      ,
      • Basu S.
      • Wang X.
      • Gladwin M.T.
      • Kim-Shapiro D.B.
      Chemiluminescent detection of S-nitrosated proteins: comparison of tri-iodide, copper/CO/cysteine, and modified copper/cysteine methods.
      ). The triiodide that forms in this solution can convert NO2, S-nitrosothiols, N-nitrosamines, and nitrosyl–metal complexes to NO (
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ). In the case of a biological sample that contains a mixture of these species, several tubes are prepared that include the parent sample, then one with acidic sulfanilamide to trap NO2, and another that also includes HgCl2 to decompose S-nitrosothiols (
      • Samouilov A.
      • Zweier J.L.
      Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.
      ,
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ). The difference in the measured NO with the different treatments indicate how much NO2 and S-nitrosothiol were in the sample.
      Additional methods for more selective chemical reduction of S-nitrosothiols include copper-based assays where the reactant in the purge vessel consists of a buffer at neutral pH and Cu(II) plus an excess of cysteine (
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ,
      • Basu S.
      • Wang X.
      • Gladwin M.T.
      • Kim-Shapiro D.B.
      Chemiluminescent detection of S-nitrosated proteins: comparison of tri-iodide, copper/CO/cysteine, and modified copper/cysteine methods.
      ). In this case the copper is reduced to Cu(I) by cysteine, which then reduces S-nitrosothiols to NO and thiol. The use of Hg(II) is recommended to discriminate the signal from N-nitrosamines (
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ). For applications in blood, a modification of the method that includes carbon monoxide has been developed that prevents capture of NO by hemoglobin (
      • Doctor A.
      • Platt R.
      • Sheram M.L.
      • Eischeid A.
      • McMahon T.
      • Maxey T.
      • Doherty J.
      • Axelrod M.
      • Kline J.
      • Gurka M.
      • Gow A.
      • Gaston B.
      Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients.
      ).

      Nitric oxide synthase activity

      The NOS enzymes catalyze the oxidation of arginine to NO and stoichiometric amounts of citrulline (Fig. 2F) (
      • Stuehr D.J.
      • Santolini J.
      • Wang Z.-Q.
      • Wei C.-C.
      • Adak S.
      Update on mechanism and catalytic regulation in the NO synthases.
      ). Therefore, the rate of NO formation can be estimated from the rate of citrulline formation from arginine and saturating concentrations of NOS cofactors (NADPH, FAD, FMN, tetrahydrobiopterin, and calcium/calmodulin). Radiolabeled arginine is used, and the reaction is stopped with EDTA, which binds calcium and inactivates the enzyme. The radiolabeled citrulline product is separated from arginine by cation-exchange chromatography (cationic arginine is retarded and zwitterionic citrulline is eluted) and measured in a liquid scintillation counter (
      • Bredt D.S.
      • Snyder S.H.
      Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.
      ). Because citrulline in the cell could be derived from non-NOS pathways, controls should be performed with addition of a NOS inhibitor as well as omission of NADPH.
      There are commercially available kits to follow NOS activity indirectly, by measuring the time course of NO2 formation spectrophotometrically using the Griess reaction described below.

      Bioassays for nitric oxide

      The production of NO in mammalian cells can be detected indirectly by measuring its biological activities like vasodilation, platelet aggregation, and guanylate cyclase activation (Fig. 2G).

      Cyclic GMP

      In the cellular context, cyclic GMP (cGMP) is formed not only by guanylate cyclases stimulated by NO (NO-GC or soluble GC) but also by the membrane natriuretic peptide receptor-coupled guanylate cyclases (GC-A and GC-B). Therefore, to measure levels of cGMP as an indirect measurement of NO, controls with inhibitors of NOS should be included. The different methods used to determine cGMP have been recently reviewed in Ref.
      • Russwurm M.
      • Koesling D.
      Measurement of cGMP-generating and -degrading activities and cGMP levels in cells and tissues: focus on FRET-based cGMP indicators.
      and include radiolabeled [α-32P]GTP (
      • Russwurm M.
      • Koesling D.
      Purification and characterization of NO-sensitive guanylyl cyclase.
      ), a cGMP antibody in a commercially available enzyme-linked immunosorbent assay (ELISA), and fluorescence-based cGMP indicators (
      • Honda A.
      • Adams S.R.
      • Sawyer C.L.
      • Lev-Ram V.
      • Tsien R.Y.
      • Dostmann W.R.
      Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator.
      ).

      Vessel relaxation

      The seminal studies that introduced NO to the biological scenario as a critical regulator of blood flow were related to its identification as an endothelium-derived relaxing factor (
      • Ignarro L.J.
      • Buga G.M.
      • Wood K.S.
      • Byrns R.E.
      • Chaudhuri G.
      Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
      ,
      • Palmer R.M.
      • Ferrige A.G.
      • Moncada S.
      Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
      ). This function is explained by the formation of NO from endothelial NOS, subsequent diffusion to the underlying smooth muscle, and activation of soluble guanylate cyclase, which initiates a signaling cascade that ultimately leads to vasodilation and increased blood flow. Thus, a method amply used by physiologists and pharmacologists to detect production of NO consists of measuring tension in isolated vascular preparations treated with agonist and antagonists of NO-dependent signaling (
      • Palmer R.M.
      • Ferrige A.G.
      • Moncada S.
      Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
      ,
      • Isbell T.S.
      • Koenitzer J.R.
      • Crawford J.H.
      • White C.R.
      • Kraus D.W.
      • Patel R.P.
      Assessing NO-dependent vasodilatation using vessel bioassays at defined oxygen tensions.
      ).

      Inhibition of platelet aggregation

      A way to test NO production is to follow inhibition of platelet aggregation after activation. It is a very simple, inexpensive method first described in 1962 (
      • Born G.V.
      Aggregation of blood platelets by adenosine diphosphate and its reversal.
      ). Washed human platelets are equilibrated at 37 °C in a turbidometric platelet aggregometer in the absence and presence of a system that produces NO. An activator like thrombin is added to induce aggregation, and turbidimetry is followed with time (
      • Zucker M.B.
      Platelet aggregation measured by the photometric method.
      ).

      Nitrogen dioxide

      Nitrogen dioxide (NO2) is a reddish-brown free radical gas that forms part of air pollution. In biological systems, there are different endogenous sources of NO2. These include: (a) NO autoxidation (see section above); (b) NO2 oxidation, a reaction that is catalyzed by different heme-dependent peroxidases (Equations 57) and Cu,Zn-superoxide dismutase (
      • Brennan M.L.
      • Wu W.
      • Fu X.
      • Shen Z.
      • Song W.
      • Frost H.
      • Vadseth C.
      • Narine L.
      • Lenkiewicz E.
      • Borchers M.T.
      • Lusis A.J.
      • Lee J.J.
      • Lee N.A.
      • Abu-Soud H.M.
      • Ischiropoulos H.
      • Hazen S.L.
      A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species.
      ,
      • van der Vliet A.
      • Eiserich J.P.
      • Halliwell B.
      • Cross C.E.
      Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity.
      • Singh R.J.
      • Goss S.P.
      • Joseph J.
      • Kalyanaraman B.
      Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2: role of nitrogen dioxide free radical.
      ) or performed nonenzymatically by strong one-electron oxidants such as CO3•−, HO or peroxyl radicals (ROO) (
      • El-Agamey A.
      Laser flash photolysis of new water-soluble peroxyl radical precursor.
      ,
      • Adams G.E.
      • Boag J.W.
      • Michael B.D.
      Reactions of the hydroxyl radical. Part 2. Determination of absolute rate constants.
      • Huie R.E.
      • Shoute L.C.T.
      • Neta P.
      Temperature dependence of the rate constants for reactions of the carbonate radical with organic and inorganic reductants.
      ); and (c) homolysis of the peroxo bond of peroxynitrous acid (ONOOH) or of short-lived adducts formed from the reaction of peroxynitrite (ONOO) with carbon dioxide (CO2), with carbonyl-containing compounds, or with metal centers (
      • Merényi G.
      • Lind J.
      • Goldstein S.
      The rate of homolysis of adducts of peroxynitrite to the C=O double bond.
      ,
      • Lymar S.V.
      • Khairutdinov R.F.
      • Hurst J.K.
      Hydroxyl radical formation by O–O bond homolysis in peroxynitrous acid.
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • Radi R.
      Carbon dioxide-catalyzed peroxynitrite reactivity- the resilience of the radical mechanism after two decades of research.
      ).
      peroxidase-heme+H2O2peroxidase-compoundI+H2O
      (Eq. 5)


      peroxidase-compoundI+NO2peroxidase-compoundII+NO2
      (Eq. 6)


      peroxidase-compoundII+NO2peroxidase-heme+NO2
      (Eq. 7)


      The solubility of NO2 in water is low. The reported Henry coefficient (∼1.4 × 10−2 m atm−1 at 20 °C) (
      • Schwartz S.E.
      • White W.H.
      ,
      • Cheung J.L.
      • Li Y.Q.
      • Boniface J.
      • Shi Q.
      • Dacidovits P.
      • Worsnop D.R.
      • Jayne J.T.
      • Kolb C.E.
      Heterogeneous interactions of NO2 with aqueous surfaces.
      ) is quite uncertain due to its rapid (k = 4.5 × 108 m−1 s−1 (
      • Grätzel M.
      • Henglein A.
      • Lilie Ja.
      • Beck G.
      Pulse radiolytic study of some elementary processes of nitrite ion oxidation and reduction.
      )) dimerization to dinitrogen tetroxide (N2O4). The latter has ∼100-fold increased solubility in water (
      • Schwartz S.E.
      • White W.H.
      ) and rapidly hydrolyzes to NO2 and NO3 (k = 1000 s−1) (
      • Schwartz S.E.
      • White W.H.
      ,
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.
      • De Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ). However, under most physiological conditions where NO2 concentrations are low (<1 μm), dimerization, which is a reversible process with a Keq = 7 × 104 m−1, is outcompeted by bimolecular reactions of NO2 with different targets, some of which are far more concentrated (
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.
      • De Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ,
      • Squadrito G.L.
      • Postlethwait E.M.
      On the hydrophobicity of nitrogen dioxide: could there be a “lens” effect for NO2 reaction kinetics?.
      • Wardman P.
      ). The partition coefficients in organic solvents indicate that NO2 is slightly hydrophobic, although less than NO, which suggests a minor “lens effect” for NO2 reaction kinetics in membranes or other hydrophobic biological systems (
      • Squadrito G.L.
      • Postlethwait E.M.
      On the hydrophobicity of nitrogen dioxide: could there be a “lens” effect for NO2 reaction kinetics?.
      ,
      • Signorelli S.
      • Möller M.N.
      • Coitiño E.L.
      • Denicola A.
      Nitrogen dioxide solubility and permeation in lipid membranes.
      ).
      The reduction potential of the NO2/NO2 pair is 0.99 V at pH 7 (
      • Koppenol W.H.
      • Moreno J.J.
      • Pryor W.A.
      • Ischiropoulos H.
      • Beckman J.S.
      Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide.
      ). Thus, it is a good one-electron oxidant. According to kinetic considerations, NO2 is predicted to react mostly with thiol-containing molecules (kRS− ∼108 m−1 s−1) (
      • Ford E.
      • Hughes M.N.
      • Wardman P.
      Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.
      ) and ascorbate (k = 1.8–3.5 × 107 m−1 s−1 at pH 7.4) (
      • May J.M.
      Vitamin C transport and its role in the central nervous system.
      ), whereas urate (k = 2 × 107 m−1 s−1 at pH 7.4) is a main target in plasma (
      • Ford E.
      • Hughes M.N.
      • Wardman P.
      Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.
      ). In hydrophobic media, NO2 can initiate lipid peroxidation (
      • Pryor W.A.
      • Lightsey J.W.
      Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous acid.
      ). Furthermore, it can add to alkene double bonds in a fast and reversible process to form nitroalkyl radicals, which eventually undergo cis-trans–isomerization (
      • Jiang H.
      • Kruger N.
      • Lahiri D.R.
      • Wang D.
      • Vatèle J.M.
      • Balazy M.
      Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids. Detection of trans-arachidonic acids in vivo.
      ) or form nitro-derivatives. The biological formation, characteristics, and relevance of nitrated fatty acids has been reviewed (
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.
      • De Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ,
      • Freeman B.A.
      • Baker P.R.
      • Schopfer F.J.
      • Woodcock S.R.
      • Napolitano A.
      • d'Ischia M.
      Nitro-fatty acid formation and signaling.
      ,
      • Trostchansky A.
      • Bonilla L.
      • González-Perilli L.
      • Rubbo H.
      Nitro-fatty acids: formation, redox signaling, and therapeutic potential.
      ,
      • Schopfer F.J.
      • Cipollina C.
      • Freeman B.A.
      Formation and signaling actions of electrophilic lipids.
      ,
      • Turell L.
      • Steglich M.
      • Alvarez B.
      The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols.
      ). Finally, NO2 reacts at diffusion-controlled rates with other radical species, such as tyrosyl radicals in proteins, to form 3-nitrotyrosine (see section on 3-nitrotyrosine below). The reversible reaction with NO leads to the formation of the nitrosating species N2O3 (Equation 2).

      Detection of nitrogen dioxide

      The UV-visible absorption spectrum of NO2 shows a broad band peak at ∼400 nm with an absorption coefficient of 200 m−1 cm−1 in aqueous solution (
      • Treinin A.
      • Hayon E.
      Absorption spectra and reaction kinetics of NO2, N2O3, and N2O4 in aqueous solution.
      ). The low absorption coefficient, the low stability of the radical, and the need to make corrections for N2O4 and NO2 absorption limit the technique. Nitrogen dioxide is frequently detected and quantified by chemiluminescence methods, in which the intensity of the emitted light is proportional to the concentration of NO2. Some of these methods rely on the reaction of NO with ozone (see section above on ozone-based chemiluminescence detection of nitric oxide and related species) and therefore require that NO2 be reduced to NO using particular catalytic converters. The latter are usually nonspecific due to reduction of other nitrogen-containing compounds (
      • Clyne M.
      • Thrush B.
      • Wayne R.
      Kinetics of the chemiluminescent reaction between nitric oxide and ozone.
      ,
      • Hampl V.
      • Waters C.L.
      • Archer S.L.
      ). Nitrogen dioxide can also be converted to NO photolytically using UV-LED irradiation (
      • Reed C.
      • Evans M.J.
      • Di Carlo P.
      • Lee J.D.
      • Carpenter L.J.
      Interferences in photolytic NO2 measurements: explanation for an apparent missing oxidant?.
      ). In addition, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) in alkaline solution reacts with NO2 giving rise to intense chemiluminescence, although other one-electron oxidants can also lead to light emission (
      • Kelly T.J.
      • Spicer C.W.
      • Ward G.F.
      An assessment of the luminol chemiluminescence technique for measurement of NO2 in ambient air.
      ).
      Detection of NO2 in cells and tissues requires different methodologies. Because NO2 is a strong one-electron oxidant, it can react with typical redox probes such as 2′,7′-dichlorodihydrofluorescein (DCFH2). In experimental designs, addition of NO2 may be useful to convert other one-electron oxidants to NO2.
      One strategy depends on the ability of NO2 to rapidly combine with free or protein tyrosyl radicals to form 3-nitrotyrosine (
      • Prütz W.A.
      • Mönig H.
      • Butler J.
      • Land E.J.
      Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins.
      ). This is analyzed in the section below on 3-nitrotyrosine. Furthermore, nitration of green fluorescent protein (GFP) leads to a decrease in its intrinsic fluorescence and was used to evaluate NO2 formation. Although the decrease in fluorescence intensity is not specific for nitration, it can be utilized in combination with pharmacological modulation of NO levels to indicate NO2 formation (
      • Espey M.G.
      • Xavier S.
      • Thomas D.D.
      • Miranda K.M.
      • Wink D.A.
      Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms.
      ).
      Finally, because of the radical nature of NO2, EPR has been utilized either by direct detection of NO2 in salt matrices and low temperatures or by using spin traps such as nitromethane at alkaline pH or nitrone compounds (
      • Augusto O.
      • Bonini M.G.
      • Amanso A.M.
      • Linares E.
      • Santos C.C.
      • De Menezes S.L.
      Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology.
      ,
      • Rowlands J.R.
      • Gause E.M.
      Reaction of nitrogen dioxide with blood and lung components. Electron spin resonance studies.
      ,
      • Pace M.D.
      • Kalyanaraman B.
      Spin trapping of nitrogen dioxide radical from photolytic decomposition of nitramines.
      ,
      • Astolfi P.
      • Greci L.
      • Panagiotaki M.
      Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid.
      ). Nitroso spin traps do not trap NO2 (
      • Astolfi P.
      • Greci L.
      • Panagiotaki M.
      Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid.
      ).
      Due to the short half-life of NO2 in aqueous solutions even in the absence of other targets, as a result of dimerization and hydrolysis of N2O4, the study of the kinetics of NO2 reactions requires the use of very fast methodologies that allow measurements to be made in the microsecond time scale, such as pulse radiolysis. Furthermore, the low absorption coefficient of NO2 limits its direct detection so that product monitoring or competition kinetics need to be used. Competition with 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation to ABTS+ is frequently employed due to the high extinction coefficient of the latter radical at 414 nm (3.6 × 104 m−1 cm−1) (
      • Ford E.
      • Hughes M.N.
      • Wardman P.
      Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.
      ,
      • Carballal S.
      • Trujillo M.
      • Cuevasanta E.
      • Bartesaghi S.
      • Möller M.N.
      • Folkes L.K.
      • García-Bereguiain M.A.
      • Gutiérrez-Merino C.
      • Wardman P.
      • Denicola A.
      • Radi R.
      • Alvarez B.
      Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest.
      ).

      Dinitrogen trioxide and its detection

      Dinitrogen trioxide (N2O3) can be formed from NO2 reaction with NO (
      • Treinin A.
      • Hayon E.
      Absorption spectra and reaction kinetics of NO2, N2O3, and N2O4 in aqueous solution.
      ,
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ) and is considered an important intermediate in the autoxidation of NO (
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ) (see section on autoxidation above). It can also be formed from NO2 at acidic pH, with an equilibrium constant of 3 × 10−3 m−1 (Equation 8) (
      • Markovits G.Y.
      • Schwartz S.E.
      • Newman L.
      Hydrolysis equilibrium of dinitrogen trioxide in dilute acid solution.
      ).
      2HNO2N2O3+H2O
      (Eq. 8)


      As discussed in the section on autoxidation, N2O3 is rapidly hydrolyzed to NO2 (Equation 3), and this reaction is accelerated by certain salts, such as phosphate and bicarbonate (
      • Goldstein S.
      • Czapski G.
      Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates.
      ,
      • Lewis R.S.
      • Tannenbaum S.R.
      • Deen W.M.
      Kinetics of N-nitrosation in oxygenated nitric oxide solutions at physiological pH: role of nitrous anhydride and effects of phosphate and chloride.
      ,
      • Caulfield J.L.
      • Singh S.P.
      • Wishnok J.S.
      • Deen W.M.
      • Tannenbaum S.R.
      Bicarbonate inhibits N-nitrosation in oxygenated nitric oxide solutions.
      ). Therefore, the first approach to measure the production of N2O3 is by monitoring NO2 (see section below on nitrite and nitrate).
      N2O3 is considered an important nitrosating species in vitro because it reacts rapidly with thiolates and amines to give the corresponding nitrosated species (
      • Goldstein S.
      • Czapski G.
      Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: the nature of the nitrosating intermediates.
      ,
      • Vitturi D.A.
      • Minarrieta L.
      • Salvatore S.R.
      • Postlethwait E.M.
      • Fazzari M.
      • Ferrer-Sueta G.
      • Lancaster Jr., J.R.
      • Freeman B.A.
      • Schopfer F.J.
      Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride.
      ), but its role in biological nitrosation is uncertain because of the slow kinetics of NO autoxidation (
      • Goldstein S.
      • Czapski G.
      Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: the nature of the nitrosating intermediates.
      ,
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ). Because both NO2 and NO are slightly hydrophobic (
      • Squadrito G.L.
      • Postlethwait E.M.
      On the hydrophobicity of nitrogen dioxide: could there be a “lens” effect for NO2 reaction kinetics?.
      ,
      • Signorelli S.
      • Möller M.N.
      • Coitiño E.L.
      • Denicola A.
      Nitrogen dioxide solubility and permeation in lipid membranes.
      ), it was suggested that N2O3 formation should be accelerated in hydrophobic regions. However, the nitrosation of thiols buried in the hydrophobic regions actually decreases because the dissociation to the more reactive thiolate is disfavored (
      • Zhang H.
      • Andrekopoulos C.
      • Xu Y.
      • Joseph J.
      • Hogg N.
      • Feix J.
      • Kalyanaraman B.
      Decreased S-nitrosation of peptide thiols in the membrane interior.
      ). Anyway, the formation of S-nitrosothiols is not specific to N2O3, and other mechanisms may be more relevant (
      • Goldstein S.
      • Czapski G.
      Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: the nature of the nitrosating intermediates.
      ,
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ), so that S-nitrosothiols are not necessarily good indicators of N2O3 formation (see section below on S-nitrosothiols).
      Besides measuring NO2 and S-nitrosothiols, another method to detect N2O3 is to use fluorogenic probes such as diaminonaphthalene (DAN) and diaminofluorescein (DAF), which were discussed in the section on detection of nitric oxide and suffer from the same issues as S-nitrosothiols.

      Nitrosyl–metal complexes and their detection

      Intracellular dinitrosyl iron complexes (DNICs) are formed from NO, a ligand such as GSH, and loosely bound iron, also called labile or chelatable iron pool (
      • Toledo Jr., J.C.
      • Bosworth C.A.
      • Hennon S.W.
      • Mahtani H.A.
      • Bergonia H.A.
      • Lancaster Jr., J.R.
      Nitric oxide-induced conversion of cellular chelatable iron into macromolecule-bound paramagnetic dinitrosyliron complexes.
      ). Recent studies have shown that the exposure of cells to NO, either exogenous or endogenous, leads to the formation of more DNICs than S-nitrosothiols, in a 4:1 ratio (
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ). DNICs have been proposed to be relevant precursors in the nitrosation of thiols (
      • Bosworth C.A.
      • Toledo Jr., J.C.
      • Zmijewski J.W.
      • Li Q.
      • Lancaster Jr., J.R.
      Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide.
      ).
      The most selective technique to measure DNICs is through EPR (
      • Toledo Jr., J.C.
      • Bosworth C.A.
      • Hennon S.W.
      • Mahtani H.A.
      • Bergonia H.A.
      • Lancaster Jr., J.R.
      Nitric oxide-induced conversion of cellular chelatable iron into macromolecule-bound paramagnetic dinitrosyliron complexes.
      ,
      • Hogg N.
      Detection of nitric oxide by electron paramagnetic resonance spectroscopy.
      ). Mononuclear DNICs show a characteristic EPR spectrum (
      • Vanin A.F.
      EPR characterization of dinitrosyl iron complexes with thiol-containing ligands as an approach to their identification in biological objects: an overview.
      ,
      • Hogg N.
      Detection of nitric oxide by electron paramagnetic resonance spectroscopy.
      ). EPR has several advantages such as the ability to measure signals in optically opaque samples, a good sensitivity (200 nm), and the capacity to distinguish enzymatically generated NO by the change in the spectrum using [15N]arginine (
      • Hogg N.
      Detection of nitric oxide by electron paramagnetic resonance spectroscopy.
      ).
      DNICs made with GSH can also be analyzed by UV-visible spectrophotometry, provided there is a separation step such as HPLC. They show a spectrum with maximum absorbance below 200 nm and characteristic peaks at 310, 360, and 680 nm (ϵ = 9200, 7400, and 200 m−1 cm−1, respectively) for the diamagnetic binuclear form, or 390 nm (ϵ = 3900 m−1 cm−1) for the paramagnetic mononuclear form (
      • Vanin A.F.
      EPR characterization of dinitrosyl iron complexes with thiol-containing ligands as an approach to their identification in biological objects: an overview.
      ).
      Cellular DNICs can also be quantified through ozone-based chemiluminescence, using the triiodide method. Care should be taken in quantification because the signal is time-sensitive and decays within hours, and also because DNICs are sensitive to HgCl2, analogously to S-nitrosothiols (
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ). To distinguish between signals from S-nitrosothiols and DNICs, it was proposed to stabilize S-nitrosothiols in the cell lysate using a buffer containing diethylenetriaminepentaacetic acid and N-ethylmaleimide and to analyze the sample immediately after extraction and 20 h later to ensure the full decay of DNICs (
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ).
      Formation of protein nitrosyl–metal complexes is particularly relevant in red blood cells, because NO can react with deoxyhemoglobin to yield nitrosyl-hemoglobin. The detection of this product predominates at low oxygen tensions (
      • Dei Zotti F.
      • Lobysheva I.I.
      • Balligand J.-L.
      Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo.
      ) because deoxyhemoglobin will be more abundant and because oxyhemoglobin will rapidly decompose NO to NO3 (
      • Doyle M.P.
      • Hoekstra J.W.
      Oxidation of nitrogen oxides by bound dioxygen in hemoproteins.
      ). Nitrosyl-hemoglobin can be quantified in vitro through UV-visible spectrophotometry and shows absorption maxima at 403 and 575 nm (
      • Ignarro L.J.
      • Buga G.M.
      • Wood K.S.
      • Byrns R.E.
      • Chaudhuri G.
      Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
      ). In red blood cells, nitrosyl-hemoglobin is difficult to quantify by spectrophotometry where there is a mixture of different forms of hemoglobin absorbing at the same wavelength. EPR, in contrast, is specific for nitrosyl-hemoglobin and allows its quantification in packed red blood cells. The limit of quantification was calculated to be 200 nm. Under normal conditions, the amount of nitrosyl-hemoglobin in human blood is below the detection limit. However, patients exposed to 80 ppm NO inhalation treatment increased its nitrosyl-hemoglobin levels up to 2 μm (
      • Piknova B.
      • Gladwin M.T.
      • Schechter A.N.
      • Hogg N.
      Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation.
      ).

      S-Nitrosothiols

      The formation of S-nitrosothiols is undoubtedly linked to the formation of NO in biological systems. Nevertheless, the exact chemistry is still under debate. In fact, thiols or rather thiolates can be nitrosated by the products of NO autoxidation (see section above on autoxidation) in a direct mechanism by N2O3 or stepwise by NO2 and NO (Equation 9-11) (
      • Goldstein S.
      • Czapski G.
      Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: the nature of the nitrosating intermediates.
      ,
      • Broniowska K.A.
      • Hogg N.
      The chemical biology of S-nitrosothiols.
      ).
      RS+N2O3RSNO+NO2
      (Eq. 9)


      RS+NO2RS+NO2
      (Eq. 10)


      RS+NORSNO
      (Eq. 11)


      Although the formation of NO2 and N2O3 can be accelerated by hydrophobic regions in lipid membranes and even proteins (
      • Liu X.
      • Miller M.J.
      • Joshi M.S.
      • Thomas D.D.
      • Lancaster J.R.
      Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes.
      ,
      • Möller M.N.
      • Li Q.
      • Vitturi D.A.
      • Robinson J.M.
      • Lancaster Jr., J.R.
      • Denicola A.
      Membrane “Lens” effect: focusing the formation of reactive nitrogen oxides from the NO/O2 reaction.
      • Möller M.N.
      • Denicola A.
      Acceleration of the autoxidation of nitric oxide by proteins.
      ), autoxidation is still too slow to be biologically significant (
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ). Furthermore, in cells, oxygen inhibits rather than increases thiol nitrosation, arguing against a significant role for NO autoxidation in biological thiol nitrosation (
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ).
      Regarding the mechanisms of biological thiol nitrosation, there is evidence supporting the intermediacy of nitrosyl–iron complexes (DNICs) (
      • Keszler A.
      • Diers A.R.
      • Ding Z.
      • Hogg N.
      Thiolate-based dinitrosyl iron complexes: decomposition and detection and differentiation from S-nitrosothiols.
      ,
      • Bosworth C.A.
      • Toledo Jr., J.C.
      • Zmijewski J.W.
      • Li Q.
      • Lancaster Jr., J.R.
      Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide.
      ), as well as the intermediacy of cytochrome c (
      • Basu S.
      • Keszler A.
      • Azarova N.A.
      • Nwanze N.
      • Perlegas A.
      • Shiva S.
      • Broniowska K.A.
      • Hogg N.
      • Kim-Shapiro D.B.
      A novel role for cytochrome c: efficient catalysis of S-nitrosothiol formation.
      ).
      S-Nitrosothiols undergo further reactions with other thiols, such as trans-nitrosation, where the nitroso moiety is transferred regenerating the original thiol (
      • Hogg N.
      The kinetics of S-trans-nitrosation—a reversible second-order reaction.
      ). For instance, the trans-nitrosation from S-nitrosoglutathione to cysteine occurs with k = 140 m−1 s−1 (
      • Hogg N.
      The kinetics of S-trans-nitrosation—a reversible second-order reaction.
      ). Thioredoxin catalyzes both trans-nitrosation and denitrosation (
      • Sengupta R.
      • Holmgren A.
      Thioredoxin and thioredoxin reductase in relation to reversible S-nitrosylation.
      ). Alcohol dehydrogenase class III catalyzes the reduction of S-nitrosoglutathione efficiently and has therefore been called S-nitrosoglutathione reductase (
      • Jensen D.E.
      • Belka G.K.
      • Du Bois G.C.
      S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme.
      ).

      Detection of S-nitrosothiols

      Several methods have been developed to quantify total S-nitrosothiols and also to identify proteins that are nitrosated. S-Nitrosothiols show a UV spectrum with a maximum at 335 nm. The absorptivity for S-nitrosoglutathione at 335 nm is 922 m−1 cm−1; therefore, the sensitivity of the spectrophotometric analysis is low (above 50 μm) and depends on having a purified sample or on chromatographic or capillary electrophoresis separation (
      • Broniowska K.A.
      • Diers A.R.
      • Hogg N.
      S-Nitrosoglutathione.
      ).
      Another historically important method for S-nitrosothiols is by Saville (
      • Saville B.
      A scheme for the colorimetric determination of microgram amounts of thiols.
      ). In this method the S-nitrosothiol is treated with Hg(II). Tight binding to the thiolate releases NO+ that is rapidly hydrolyzed to NO2 (
      • Saville B.
      A scheme for the colorimetric determination of microgram amounts of thiols.
      ). The released NO2 is then measured by the Griess method. This method has micromolar sensitivity (see section below on detection of nitrite).
      Antibodies against S-nitrosocysteine have been used in immunohistochemical assays, Western blotting, and immunoprecipitation. However, specificity issues and the advent of biotin switch techniques that also allow mapping the modified cysteine within a protein have discouraged their use (
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ).
      The gold standard method to quantify S-nitrosothiols is ozone-based chemiluminescence that provides nanomolar sensitivity and is appropriate for most biological applications (Fig. 3). As discussed in the section on chemiluminescence, S-nitrosothiols can be quantified using the triiodide reaction and also using copper ions and reductants (
      • Diers A.R.
      • Keszler A.
      • Hogg N.
      Detection of S-nitrosothiols.
      ).
      It was early observed that several proteins could be S-nitrosated (
      • Stamler J.S.
      • Jaraki O.
      • Osborne J.
      • Simon D.I.
      • Keaney J.
      • Vita J.
      • Singel D.
      • Valeri C.R.
      • Loscalzo J.
      Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.
      ) but unbiased approaches to the S-nitrosoproteome were only possible after the introduction of the “biotin switch” method in 2001 (
      • Jaffrey S.R.
      • Snyder S.H.
      The biotin switch method for the detection of S-nitrosylated proteins.
      ). The original method involved blocking free thiols in S-nitrosated proteins with methyl methanethiosulfonate, then specifically reducing the nitrosated thiols with ascorbate, followed by reaction of these thiols with N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide. The biotinylated proteins could then be selectively captured by using the specific binding to immobilized streptavidin (
      • Jaffrey S.R.
      • Snyder S.H.
      The biotin switch method for the detection of S-nitrosylated proteins.
      ). Some issues with this method have been pointed out, namely that it is very difficult to ensure that all free thiols are effectively blocked in the first step, that ascorbate does not reduce S-nitrosothiols directly but through the intermediacy of metals in solution (
      • Wang X.
      • Kettenhofen N.J.
      • Shiva S.
      • Hogg N.
      • Gladwin M.T.
      Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins.
      ), and that no chemical trace is left to indicate that the thiol was effectively nitrosated.
      Relative quantification of protein S-nitrosation can be achieved through different means, including isotope-coded affinity tags (ICAT) and stable isotope labeling by amino acids in cell culture (SILAC). Both methods are based on using a light and a heavy isotope-containing tag. In ICAT, samples to compare are processed in parallel and tagged with biotin derivatives that include either light or heavy isotope linkers, and then mixed and further processed (
      • Chouchani E.T.
      • James A.M.
      • Methner C.
      • Pell V.R.
      • Prime T.A.
      • Erickson B.K.
      • Forkink M.
      • Lau G.Y.
      • Bright T.P.
      • Menger K.E.
      • Fearnley I.M.
      • Krieg T.
      • Murphy M.P.
      Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.
      ). SILAC involves adding either light or heavy isotope-containing arginine and lysine to control or stimulated cell cultures (
      • Zhou X.
      • Han P.
      • Li J.
      • Zhang X.
      • Huang B.
      • Ruan H.-Q.
      • Chen C.
      ESNOQ, proteomic quantification of endogenous S-nitrosation.
      ). The cell lysates from both cell cultures can then be mixed and processed as in the biotin switch method. If the same peptide is enriched from both control and treated samples, it will elute at the same time in the LC-MS analysis, but the mass spectra will differ by a known number of Da, and the relative amounts can be calculated from the intensities in the MS peaks (
      • Zhou X.
      • Han P.
      • Li J.
      • Zhang X.
      • Huang B.
      • Ruan H.-Q.
      • Chen C.
      ESNOQ, proteomic quantification of endogenous S-nitrosation.
      ).
      Other approaches to identify S-nitrosated proteins and the location of the modification include the use of organomercurial compounds to either trap or tag S-nitrosothiols, after blocking free thiols (
      • Raju K.
      • Doulias P.-T.
      • Tenopoulou M.
      • Greene J.L.
      • Ischiropoulos H.
      Strategies and tools to explore protein S-nitrosylation.
      ) or the selective reaction of S-nitrosothiols with derivatized phosphines to tag S-nitrosated peptides in one step through reductive ligation (
      • Zhang J.
      • Li S.
      • Zhang D.
      • Wang H.
      • Whorton A.R.
      • Xian M.
      Reductive ligation mediated one-step disulfide formation of S-nitrosothiols.
      ).

      Nitrite and nitrate

      Nitrite (NO2, IUPAC name dioxidonitrate(1−)) and nitrate (NO3, IUPAC name trioxidonitrate(1−)) were considered for a long time to be rather inert products of NO oxidation. The concentration of NO3 in plasma of fasting individuals is 20–40 μm, and it is considered to derive mostly from the reaction between NO and oxyhemoglobin, but also from the diet (
      • Lundberg J.O.
      • Weitzberg E.
      ). The concentration of NO2 in plasma is significantly lower (50–300 nm) because there are several processes by which it can be converted to NO or further oxidized to NO3 (
      • DeMartino A.W.
      • Kim-Shapiro D.B.
      • Patel R.P.
      • Gladwin M.T.
      Nitrite and nitrate chemical biology and signalling.
      ). Nitrate is concentrated in saliva and can be converted to NO2 by bacteria in the oral cavity (
      • Lundberg J.O.
      • Weitzberg E.
      ). Xanthine oxidase has also been shown to reduce NO3 to NO2, but it seems to be a minor contribution compared with the oral microbiome (
      • DeMartino A.W.
      • Kim-Shapiro D.B.
      • Patel R.P.
      • Gladwin M.T.
      Nitrite and nitrate chemical biology and signalling.
      ).
      Nitrite can be reduced to NO by different proteins, including deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, and aldehyde oxidase (
      • Kim-Shapiro D.B.
      • Gladwin M.T.
      Mechanisms of nitrite bioactivation.
      ). The reduction by deoxyhemoglobin is thought to be quantitatively the most important pathway for the generation of NO from NO2 and responsible for the NO-like effects of NO2 infusion in the presence of red blood cells (
      • Kim-Shapiro D.B.
      • Gladwin M.T.
      Mechanisms of nitrite bioactivation.
      ). The NO2 reductase activity of deoxyhemoglobin leads to the formation of NO and methemoglobin (Equation 12).
      NO2+Fe(II)(Hb)+H+NO+Fe(III)(Hb)+OH
      (Eq. 12)


      Detection of nitrite and nitrate

      There are several methods to detect NO2 in biological samples. The simplest method to measure NO2 is the Griess method, which sensitivity is in the micromolar range. The method is based on the diazotization of sulfanilamide by NO2 in acidic pH and the subsequent reaction with N-(1-naphthyl)ethylenediamine to yield an intensely pink-colored product with absorption maximum at 540 nm (Fig. 4) (
      • Tsikas D.
      Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the l-arginine/nitric oxide area of research.
      ). The measurement of NO3 is usually done by first converting it to NO2, either by using vanadium chloride or the enzyme NO3 reductase (
      • Tsikas D.
      Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the l-arginine/nitric oxide area of research.
      ). The analysis can readily be automated to measure NO2 and NO3 (
      • Green L.C.
      • Wagner D.A.
      • Glogowski J.
      • Skipper P.L.
      • Wishnok J.S.
      • Tannenbaum S.R.
      Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
      ). Lower concentrations of NO2 (down to 20 nm) can be quantified by the formation of the fluorescent triazole derivative of DAN (see section above on fluorescent detection). In this case, the reaction of NO2 with DAN is done at acidic pH (Fig. 3), and then the fluorescence of the product is measured at alkaline pH (
      • Misko T.P.
      • Schilling R.J.
      • Salvemini D.
      • Moore W.M.
      • Currie M.G.
      A fluorometric assay for the measurement of nitrite in biological samples.
      ).
      Figure thumbnail gr4
      Figure 4Detection of nitrite and nitrate with the Griess method. Sulfanilamide reacts with NO2 in acidic pH to yield a diazonium intermediate that subsequently reacts with N-(1-naphthyl)ethylenediamine to yield the intensely colored Griess product with absorption maximum at 540 nm. NO3 can be converted to NO2 with vanadium chloride or NO3 reductase to allow both NO3 and NO2 to be measured.
      Nitrite and nitrate can also be quantified by HPLC, capillary electrophoresis, and GC-MS (
      • Tsikas D.
      Review methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids.
      ,
      • Tsikas D.
      Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry.
      ). For low concentrations such as those often encountered in biological samples, the ozone-based chemiluminescence method (see section on chemiluminescence and Fig. 3) offers the required nanomolar sensitivity. In this case, the purge vessel needs to be filled with the triiodide acidic solution that converts NO2 to NO that is then carried to the detection cell by the carrier gas. Nitrate is measured by first reducing it chemically or enzymatically to NO2. The contribution of other species such as S-nitrosothiols to the signal is controlled by running samples treated with acidic sulfanilamide to trap all free NO2.

      Nitroxyl

      The product of the one-electron reduction of NO is HNO (nitroxyl, azanone, nitrosyl hydride, and hydrogen oxonitrate). The reduction potential of this process, E0′ (NO, H+/HNO) ∼−0.55 V at pH 7 (
      • Bartberger M.D.
      • Liu W.
      • Ford E.
      • Miranda K.M.
      • Switzer C.
      • Fukuto J.M.
      • Farmer P.J.
      • Wink D.A.
      • Houk K.N.
      The reduction potential of nitric oxide (NO) and its importance to NO biochemistry.
      ,
      • Shafirovich V.
      • Lymar S.V.
      Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide.
      ), is quite low, but high enough to make endogenous HNO formation a possibility. Biological studies are usually performed using nitroxyl donors (e.g. Angeli's salt). The ground state of HNO is a singlet in which all the electrons are spin-paired, whereas that of NO (nitroxyl anion, oxonitrate (1−)) is a triplet with two unpaired electrons (
      • Bartberger M.D.
      • Liu W.
      • Ford E.
      • Miranda K.M.
      • Switzer C.
      • Fukuto J.M.
      • Farmer P.J.
      • Wink D.A.
      • Houk K.N.
      The reduction potential of nitric oxide (NO) and its importance to NO biochemistry.
      ,
      • Shafirovich V.
      • Lymar S.V.
      Nitroxyl and its anion in aqueous solutions: spin states, protic equilibria, and reactivities toward oxygen and nitric oxide.
      ). Thus, deprotonation is spin-forbidden and slow, and the pKa of HNO is 11.4 (
      • Shafirovich V.
      • Lymar S.V.
      Spin-forbidden deprotonation of aqueous nitroxyl (HNO).
      ).
      Nitroxyl can react with soft electrophiles (
      • Bartberger M.D.
      • Liu W.
      • Ford E.
      • Miranda K.M.
      • Switzer C.
      • Fukuto J.M.
      • Farmer P.J.
      • Wink D.A.
      • Houk K.N.
      The reduction potential of nitric oxide (NO) and its importance to NO biochemistry.
      ). In vivo, the preferential reactions of HNO are with thiols and metal centers. For example, the reaction between HNO and GSH, which is present in millimolar concentrations inside cells, has a rate constant of 3.1 × 106 m−1 s−1 (
      • Smulik R.
      • Dębski D.
      • Zielonka J.
      • Michałowski B.
      • Adamus J.
      • Marcinek A.
      • Kalyanaraman B.
      • Sikora A.
      Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological implications.
      ). In addition, HNO can dimerize yielding nitrous oxide (N2O) and water (k = 8 × 106 m−1 s−1) (
      • Shafirovich V.
      • Lymar S.V.
      Spin-forbidden deprotonation of aqueous nitroxyl (HNO).
      ). Nitroxyl can also react with oxygen to form peroxynitrite with a rate constant of 1.8–2 × 104 m−1 s−1 at pH 7.4 (
      • Smulik R.
      • Dębski D.
      • Zielonka J.
      • Michałowski B.
      • Adamus J.
      • Marcinek A.
      • Kalyanaraman B.
      • Sikora A.
      Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological implications.
      ), but this process is relatively slow under biological conditions and has low relevance. More information on the biochemistry of HNO can be found in Refs.
      • Doctorovich F.
      • Bikiel D.E.
      • Pellegrino J.
      • Suárez S.A.
      • Martí M.A.
      Reactions of HNO with metal porphyrins: underscoring the biological relevance of HNO.
      • Bianco C.L.
      • Toscano J.P.
      • Bartberger M.D.
      • Fukuto J.M.
      The chemical biology of HNO signaling.
      ,
      • Fukuto J.M.
      • Bartberger M.D.
      • Dutton A.S.
      • Paolocci N.
      • Wink D.A.
      • Houk K.N.
      The physiological chemistry and biological activity of nitroxyl (HNO): the neglected, misunderstood, and enigmatic nitrogen oxide.
      • Miranda K.M.
      The chemistry of nitroxyl (HNO) and implications in biology.
      .

      Detection of nitroxyl

      Nitroxyl can be detected by observing the dimerization product, N2O, by gas chromatography (GC) (
      • Miranda K.M.
      The chemistry of nitroxyl (HNO) and implications in biology.
      ). It can also be detected by membrane inlet MS, in which HNO traverses a membrane before reaching the mass spectrometer (
      • Cline M.R.
      • Tu C.
      • Silverman D.N.
      • Toscano J.P.
      Detection of nitroxyl (HNO) by membrane inlet mass spectrometry.
      ) (Fig. 5, A and B).
      Figure thumbnail gr5
      Figure 5Detection of nitroxyl. A, detection based on the HNO dimerization product N2O by GC. B, membrane inlet MS for the detection of HNO and its decay products. C, reaction with metalloporphyrins for spectrophotometric detection. D, electrochemical sensor based on the reaction of HNO with a cobalt(III) porphyrin. E, fluorogenic probe based on the reaction of copper(II) to copper(I). F, reaction between HNO and a nitroxide TEMPO derivative to form NO and a hydroxylamine which, appropriately derivatized, is fluorescent. G, reaction between HNO and a 2-mercapto-2-methylpropionic acid fluorogenic derivative. H, reaction of HNO with an ester of 2-(diphenylphosphino)benzoic acid to give a benzamide phosphine oxide and an alcohol that, appropriately derivatized, is fluorescent. Some protons are omitted for simplicity.
      Iron(III) porphyrins react with HNO forming nitrosyl–iron(II) porphyrins (
      • Bari S.E.
      • Martí M.A.
      • Amorebieta V.T.
      • Estrin D.A.
      • Doctorovich F.
      Fast nitroxyl trapping by ferric porphyrins.
      ). The products can be detected both spectrophotometrically and by the typical three-line EPR signal of a ferrous nitrosyl complex under anaerobic conditions (
      • Adachi Y.
      • Nakagawa H.
      • Matsuo K.
      • Suzuki T.
      • Miyata N.
      Photoactivatable HNO-releasing compounds using the retro-Diels–Alder reaction.
      ). Manganese(III) porphyrins also react with HNO leading to a large shift in the UV-visible Soret band, which can be used for colorimetric detection of HNO (Fig. 5C) (
      • Martí M.A.
      • Bari S.E.
      • Estrin D.A.
      • Doctorovich F.
      Discrimination of nitroxyl and nitric oxide by water-soluble Mn(III) porphyrins.
      ).
      Cobalt(III) porphyrins react with HNO and constitute the basis of an amperometric electrochemical sensor for HNO (Fig. 5D). In the resting state, the polarized electrode (0.8 V) contains Co(III) porphyrin. When the porphyrin reacts with HNO it forms a Co(III)–NO complex that is oxidized releasing NO and the Co(III) porphyrin, ready for another cycle. The current intensity is proportional to HNO, and the sensitivity is in the nanomolar range. The success of the electrode is based on the fact that HNO reacts with Co(III) and not with Co(II) porphyrins, whereas NO reacts with Co(II) and not with Co(III). This is an advantage of Co(III) over Fe(III) porphyrins, which react both with NO and HNO (
      • Doctorovich F.
      • Bikiel D.E.
      • Pellegrino J.
      • Suárez S.A.
      • Martí M.A.
      Reactions of HNO with metal porphyrins: underscoring the biological relevance of HNO.
      ,
      • Suárez S.A.
      • Bikiel D.E.
      • Wetzler D.E.
      • Martí M.A.
      • Doctorovich F.
      Time-resolved electrochemical quantification of azanone (HNO) at low nanomolar level.
      ,
      • Suarez S.A.
      • Munoz M.
      • Bikiel D.E.
      • Marti M.A.
      • Doctorovich F.
      ).
      Nitroxyl can reduce Cu(II) to Cu(I) and NO. This is the basis of a group of fluorogenic probes in which the reduction of the metal ion is concomitant with the release of fluorescence quenching (Fig. 5E). The probes should be used with caution for the potential reduction by other reductants, as well as interference from hydrogen sulfide (H2S), S-nitrosothiols and oxygen (
      • Rosenthal J.
      • Lippard S.J.
      Direct detection of nitroxyl in aqueous solution using a tripodal copper(II) BODIPY complex.
      ,
      • Smulik-Izydorczyk R.
      • Dębowska K.
      • Pięta J.
      • Michalski R.
      • Marcinek A.
      • Sikora A.
      Fluorescent probes for the detection of nitroxyl (HNO).
      • Tennyson A.G.
      • Do L.
      • Smith R.C.
      • Lippard S.J.
      Selective fluorescence detection of nitroxyl over nitric oxide in buffered aqueous solution using a conjugated metallopolymer.
      ).
      Nitroxyl reacts with stable nitroxide free radicals such as (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO) with rate constants of 104–105 m−1 s−1 forming the hydroxylamine and NO (Fig. 5F) (
      • Miranda K.M.
      • Paolocci N.
      • Katori T.
      • Thomas D.D.
      • Ford E.
      • Bartberger M.D.
      • Espey M.G.
      • Kass D.A.
      • Feelisch M.
      • Fukuto J.M.
      • Wink D.A.
      A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system.
      ,
      • Samuni Y.
      • Samuni U.
      • Goldstein S.
      The use of cyclic nitroxide radicals as HNO scavengers.
      ). Fluorogenic TEMPO derivatives have been prepared in which the nitroxide group quenches the fluorescence, which is released when the nitroxide is converted to the hydroxylamine (
      • Smulik-Izydorczyk R.
      • Dębowska K.
      • Pięta J.
      • Michalski R.
      • Marcinek A.
      • Sikora A.
      Fluorescent probes for the detection of nitroxyl (HNO).
      ,
      • Cline M.R.
      • Toscano J.P.
      Detection of nitroxyl (HNO) by a prefluorescent probe.
      ). Due to the complex chemistry and to the potential to react with other reductants and oxidants, the use of the nitroxide probes in biological systems is limited.
      Nitroxyl reacts fast with thiols. The formation of GSH sulfinamide (GS(O)NH2) from the reaction of GSH with HNO can be used as footprint for HNO. An N-hydroxysulfenamide is formed as an intermediate, and the final sulfinamide can be separated and detected by HPLC or MS (Equation 13) (
      • Donzelli S.
      • Espey M.G.
      • Thomas D.D.
      • Mancardi D.
      • Tocchetti C.G.
      • Ridnour L.A.
      • Paolocci N.
      • King S.B.
      • Miranda K.M.
      • Lazzarino G.
      • Fukuto J.M.
      • Wink D.A.
      Discriminating formation of HNO from other reactive nitrogen oxide species.
      ,
      • Wong P.S.
      • Hyun J.
      • Fukuto J.M.
      • Shirota F.N.
      • DeMaster E.G.
      • Shoeman D.W.
      • Nagasawa H.T.
      Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.
      ).
      RSH+HNORSNHOHRS(O)NH2
      (Eq. 13)


      A probe has been developed that consists of an ester of 2-mercapto-2-methylpropionic acid and a fluorescent compound. The reaction of HNO with the thiol forms an N-hydroxysulfenamide intermediate that cyclizes releasing the fluorophore (Fig. 5G) (
      • Smulik-Izydorczyk R.
      • Dębowska K.
      • Pięta J.
      • Michalski R.
      • Marcinek A.
      • Sikora A.
      Fluorescent probes for the detection of nitroxyl (HNO).
      ,
      • Pino N.W.
      • Davis 3rd., J.
      • Yu Z.
      • Chan J.
      NitroxylFluor: a thiol-based fluorescent probe for live-cell imaging of nitroxyl.
      ).
      Nitroxyl reacts fast with arylphosphines to yield phosphine oxides and azaylides (Equation 14). The rate constants are in the order of 106 m−1 s−1 (
      • Reisz J.A.
      • Klorig E.B.
      • Wright M.W.
      • King S.B.
      Reductive phosphine-mediated ligation of nitroxyl (HNO).
      ,
      • Reisz J.A.
      • Zink C.N.
      • King S.B.
      Rapid and selective nitroxyl (HNO) trapping by phosphines: kinetics and new aqueous ligations for HNO detection and quantitation.
      ). The azaylides are indicative of the formation of HNO and can be detected by NMR and MS, although, depending on the phosphine used, they may hydrolyze to the corresponding phosphine oxide. Although arylphosphines are resistant to reductants, possible interference by S-nitrosothiols is a potential concern (
      • Miao Z.
      • King S.B.
      ).
      2R3P+HNOR3P=O+R3P=NH
      (Eq. 14)


      The azaylides are nucleophilic and can react with an adjacent electrophilic group such as an ester or a carbamate. When the azaylide attacks the carbonyl, alcohol is released, and a unique amide phosphine oxide product is formed (Fig. 5H). This product, as well as the alcohol, can serve as reporters for HNO. The hydrolysis of the probe should be controlled as well as possible interference from S-nitrosothiols (
      • Smulik-Izydorczyk R.
      • Dębowska K.
      • Pięta J.
      • Michalski R.
      • Marcinek A.
      • Sikora A.
      Fluorescent probes for the detection of nitroxyl (HNO).
      ,
      • Reisz J.A.
      • Zink C.N.
      • King S.B.
      Rapid and selective nitroxyl (HNO) trapping by phosphines: kinetics and new aqueous ligations for HNO detection and quantitation.
      ,
      • Kawai K.
      • Ieda N.
      • Aizawa K.
      • Suzuki T.
      • Miyata N.
      • Nakagawa H.
      A reductant-resistant and metal-free fluorescent probe for nitroxyl applicable to living cells.
      ).
      Despite the progress in the development of methods to measure HNO, the potential limitations should be carefully addressed. More than one method should be used, preferentially in combination with HPLC or MS detection of HNO-specific products (
      • Smulik-Izydorczyk R.
      • Dębowska K.
      • Pięta J.
      • Michalski R.
      • Marcinek A.
      • Sikora A.
      Fluorescent probes for the detection of nitroxyl (HNO).
      ).

      Peroxynitrite

      Peroxynitrite (ONOO) and peroxynitrous acid (ONOOH) are formed through the diffusion-controlled reaction between O2•− and NO (Equation 15). IUPAC names for ONOO and ONOOH are (dioxido)oxidonitrate(1−) and (hydridodioxido)oxidonitrogen, respectively. In this text, the term peroxynitrite is used for the mixture of ONOO and ONOOH, unless specified.
      NO+O2ONOO
      (Eq. 15)


      Peroxynitrite is a powerful one- and two-electron oxidant; the reduction potentials are E0′ (ONOOH, H+/NO2, H2O) = 1.6 V and E0′ (ONOOH, H+/NO2, H2O) = 1.3 V (
      • Koppenol W.H.
      • Kissner R.
      Can O=NOOH undergo homolysis?.
      ). Peroxynitrous acid can traverse membranes through simple diffusion, whereas ONOO can use anion channels. The anion is a good nucleophile, and ONOOH can act as an electrophile. In buffer, ONOOH (pKa 6.8, Equation 16) can decay to nitric acid (HNO3) plus a 30% fraction of HO and NO2 radicals (Equation 17), but this process (k = 0.9 s−1 at pH 7.4 and 37 °C) is relatively slow and has limited physiological significance. The most relevant biological targets for peroxynitrite are peroxiredoxins, GSH peroxidases, CO2, and metal centers (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ,
      • Huie R.E.
      • Shoute L.C.T.
      • Neta P.
      Temperature dependence of the rate constants for reactions of the carbonate radical with organic and inorganic reductants.
      ). The peroxiredoxins are thiol-dependent peroxidases that constitute the most efficient peroxynitrite scavengers known to date, with rate constants of ∼0.1–10 × 107 m−1 s−1 and high concentrations in different cellular compartments (
      • Perkins A.
      • Poole L.B.
      • Karplus P.A.
      Tuning of peroxiredoxin catalysis for various physiological roles.
      ). In addition, peroxynitrite can react fast (k ∼104 m−1 s−1 at pH 7.4) (
      • Denicola A.
      • Freeman B.A.
      • Trujillo M.
      • Radi R.
      Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations.
      ) with CO2, which is abundant in tissues (1.2 mm), leading to the formation of secondary radicals, CO3•− and NO2, in 33% yield (Equation 18) (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • Radi R.
      Carbon dioxide-catalyzed peroxynitrite reactivity- the resilience of the radical mechanism after two decades of research.
      ).
      ONOO+H+ONOOH
      (Eq. 16)


      ONOOH0.7HNO3+0.3NO2+0.3HO
      (Eq. 17)


      ONOO+CO2[ONOOCO2]0.67NO3+0.33NO2+0.33CO3
      (Eq. 18)


      The reactions with metal centers are diverse. Peroxynitrite can be reduced by one electron yielding NO2 as the metal center is oxidized, or by two electrons yielding NO2. In addition, some hemeproteins (e.g. methemoglobin) catalyze peroxynitrite isomerization to NO3, whereas others (e.g. Fe(III) cytochrome c) do not react at all (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ).

      Peroxynitrite detection

      Because of its short life in cells and tissues (∼1 ms) (
      • Ferrer-Sueta G.
      • Radi R.
      Chemical biology of peroxynitrite: kinetics, diffusion, and radicals.
      ), peroxynitrite cannot be detected in biological samples through direct spectroscopic techniques. Nevertheless, the UV absorbance of ONOO302 = 1700 m−1 cm−1) (
      • Bohle D.S.
      • Hansert B.
      • Paulson S.C.
      • Smith B.D.
      Biomimetic synthesis of the putative cytotoxin peroxynitrite, ONOO, and its characterization as a tetramethylammonium salt.
      ) has proven to be very useful for the quantification of stock solutions in the laboratory at alkaline pH, as well as for following ONOO decay in stopped-flow kinetic experiments.
      One analytical approach for the detection of peroxynitrite is the use of probes that react with peroxynitrite itself or with its downstream radicals (NO2, CO3•−, and HO). Because the specificity of the probes is not always straightforward, particularly for the latter, the modulation of O2•− and NO formations, which are the precursors of peroxynitrite, should accompany the results obtained with probes. Another analytical approach to evidence the involvement of peroxynitrite in a certain biological process is the detection of nitrotyrosine, a stable product formed from the reaction of radicals derived from peroxynitrite with tyrosine residues. As in the case of the probes, confirmatory evidence is required. These approaches are described in the next sections.
      A growing number of small fluorogenic organic molecules designed and synthesized to detect peroxynitrite are reported constantly, having different selectivity and sensitivity toward this oxidant. The basic common characteristic is to have weak basal fluorescence, which is largely increased upon oxidation (
      • Prolo C.
      • Rios N.
      • Piacenza L.
      • Álvarez M.N.
      • Radi R.
      Fluorescence and chemiluminescence approaches for peroxynitrite detection.
      ,
      • Wardman P.
      Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.
      • Winterbourn C.C.
      The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells.
      ). In terms of the reaction mechanism, fluorogenic probes can be divided in two main groups: 1) probes that react with the radicals derived from peroxynitrite and yield a fluorescent end product by a radical mechanism; and 2) probes that react directly through a nucleophilic attack by peroxynitrite anion (ONOO) to a particular functional group of the electrophilic probe, releasing masked fluorescence. The probes that react directly with ONOO are potentially more straightforward, specific, and quantitative. They must react fast (>105–106 m−1 s−1) and outcompete other routes of decay. Besides, genetically-encoded fluorescent protein sensors for peroxynitrite have been described recently (
      • Chen Z.-J.
      • Ren W.
      • Wright Q.E.
      • Ai H.-W.
      Genetically encoded fluorescent probe for the selective detection of peroxynitrite.
      ,
      • Chen Z.-J.
      • Tian Z.
      • Kallio K.
      • Oleson A.L.
      • Ji A.
      • Borchardt D.
      • Jiang D.E.
      • Remington S.J.
      • Ai H.-W.
      The N–B interaction through a water bridge: understanding the chemoselectivity of a fluorescent protein based probe for peroxynitrite.
      ). They use similar principles as some of the chemical probes that lead to direct detection of this oxidant (i.e. boronate-based compounds, see below).
      Importantly, detection methods based on probes reveal only a minor fraction of total peroxynitrite, because a large proportion reacts with other targets in the cell. Moreover, the fraction trapped by the probe may vary with cell type or metabolic state according to the abundance of alternative targets (
      • Ríos N.
      • Prolo C.
      • Álvarez M.N.
      • Piacenza L.
      • Radi R.
      ). For a full review of chemical probes for peroxynitrite detection, see Refs.
      • Prolo C.
      • Rios N.
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      • Álvarez M.N.
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      Fluorescence and chemiluminescence approaches for peroxynitrite detection.
      ,
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      • Radi R.
      .

      Probes that react with the radicals derived from peroxynitrite

      Probes frequently used for oxidant detection in biological systems are reduced dyes like 2′,7′-dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR-123). Although extensively used, they present a series of limitations and caveats (
      • Forman H.J.
      • Augusto O.
      • Brigelius-Flohe R.
      • Dennery P.A.
      • Kalyanaraman B.
      • Ischiropoulos H.
      • Mann G.E.
      • Radi R.
      • Roberts 2nd., L.J.
      • Vina J.
      • Davis K.J.
      Even free radicals should follow some rules: a guide to free radical research terminology and methodology.
      ). The general reaction mechanism is a one-electron oxidation by potent one-electron oxidants such as those derived from peroxynitrite (NO2, CO3•−, and HO) (
      • Wrona M.
      • Patel K.
      • Wardman P.
      Reactivity of 2′,7′-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals.
      ) yielding a radical intermediate (DCF•−), which is afterward oxidized to highly resonant moieties responsible for the increase in fluorescence emission (DCF) (Fig. 6A). These probes do not react directly with peroxynitrite (
      • Winterbourn C.C.
      The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells.
      ,
      • Wardman P.
      Methods to measure the reactivity of peroxynitrite-derived oxidants toward reduced fluoresceins and rhodamines.
      ). Neither NO nor O2•− are able to oxidize either probe at significant yields; however, these radicals may react with the radical intermediate (DCF•−) in termination reactions giving nonfluorescent products (
      • Radi R.
      • Peluffo G.
      • Alvarez M.N.
      • Naviliat M.
      • Cayota A.
      Unraveling peroxynitrite formation in biological systems.
      ). In addition to peroxynitrite-derived radicals, other potent one-electron oxidants such as those produced from heme peroxidases and other metalloproteins in the presence of H2O2 can generate fluorescent DCF (
      • Wardman P.
      Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.
      ). Thiyl radicals (RS) derived from the oxidation of GSH can also oxidize DCFH2 with a significantly high rate constant of ∼107 m−1 s−1 at pH 7.4 (
      • Wrona M.
      • Patel K.B.
      • Wardman P.
      The roles of thiol-derived radicals in the use of 2′,7′-dichlorodihydrofluorescein as a probe for oxidative stress.
      ). Moreover, DCF-dependent fluorescence can be self-amplified by redox-cycling of the one-electron oxidized dye (
      • Forman H.J.
      • Augusto O.
      • Brigelius-Flohe R.
      • Dennery P.A.
      • Kalyanaraman B.
      • Ischiropoulos H.
      • Mann G.E.
      • Radi R.
      • Roberts 2nd., L.J.
      • Vina J.
      • Davis K.J.
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      ).
      Figure thumbnail gr6
      Figure 6Detection of peroxynitrite. A, mechanism of 2′,7′-dichlorodihydrofluorescein (DCFH2) oxidation. The reduced probe (DCFH2) is oxidized by peroxynitrite-derived radicals and other one-electron oxidants yielding a radical intermediate (DCF•−) that is oxidized by oxygen to yield fluorescent DCF. Thin arrows show alternative reactions and redox cycles. AH stands for ascorbate. B, boronate oxidation by peroxynitrite. The major pathway (>85%, above) consists of heterolytic cleavage of the peroxyl bond leading to phenol which, appropriately derivatized, is fluorescent. The minor radical pathway (below) involves homolytic cleavage of the peroxyl bond giving NO2 and a phenyl-type radical that yields the nitro-derivative (
      • Sikora A.
      • Zielonka J.
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      Reaction between peroxynitrite and boronates: EPR spin-trapping, HPLC analyses, and quantum mechanical study of the free radical pathway.
      ). C, structures of boronate-derived probes. CBA and CBE indicate a boronic acid or a boronic pinacolate ester attached to a coumarin scaffold, respectively (
      • Sikora A.
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      Direct oxidation of boronates by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite.
      ). Fl-B (
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      Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.
      ), FlAmBE (
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      ), and FBBE (
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      Characterization of fluorescein-based monoboronate probe and its application to the detection of peroxynitrite in endothelial cells treated with doxorubicin.