Detection of Nitric Oxide by Electron Paramagnetic Resonance Spectroscopy: Spin-Trapping with Iron-Dithiocarbamates

Part of the Methods in Molecular Biology book series (MIMB, volume 1424)


Electron paramagnetic resonance (EPR) spectroscopy is the ideal methodology to identify radicals (detection and characterization of molecular structure) and to study their kinetics, in both simple and complex biological systems. The very low concentration and short life-time of NO and of many other radicals do not favor its direct detection and spin-traps are needed to produce a new and persistent radical that can be subsequently detected by EPR spectroscopy.

In this chapter, we present the basic concepts of EPR spectroscopy and of some spin-trapping methodologies to study NO. The “strengths and weaknesses” of iron-dithiocarbamates utilization, the NO traps of choice for the authors, are thoroughly discussed and a detailed description of the method to quantify the NO formation by molybdoenzymes is provided.

Key words

Nitric oxide radical Electron paramagnetic resonance (EPR) Spin-trap Iron-dithiocarbamate Nitrite Xanthine oxidoreductase Aldehyde oxidoreductase 



Aldehyde oxidoreductase






5,5-Dimethyl-1-pyrroline N-oxide


Electron paramagnetic resonance












Mononitrosyl-iron complex




Nitric oxide radical (NO)


Spin-trap molecule




Xanthine oxidase



This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728.


  1. 1.
    Moncada S, Palmer RMJ, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109–142PubMedGoogle Scholar
  2. 2.
    Gusarov I, Nudler E (2005) NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria. Proc Natl Acad Sci U S A 102:13855–13860CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Neill S, Bright J, Desikan R, Hancock J, Harrison J, Wilson I (2008) Nitric oxide evolution and perception. J Exp Bot 59:25–35CrossRefPubMedGoogle Scholar
  4. 4.
    Wilson ID, Neill SJ, Hancock JT (2008) Nitric oxide synthesis and signalling in plants. Plant Cell Environ 31:622–631CrossRefPubMedGoogle Scholar
  5. 5.
    Gupta KJ, Kaiser WM (2010) Production and scavenging of nitric oxide by barley root mitochondria. Plant Cell Physiol 51:576–584CrossRefPubMedGoogle Scholar
  6. 6.
    Moreau M, Lindermayr C, Durner J, Klessig DF (2010) NO synthesis and signaling in plants -where do we stand? Physiol Plant 138:372–383CrossRefPubMedGoogle Scholar
  7. 7.
    Toledo JC, Augusto O (2012) Connecting the chemical and biological properties of nitric oxide. Chem Res Toxicol 25:975–989CrossRefPubMedGoogle Scholar
  8. 8.
    Maia L, Moura JJG (2014) How biology handles nitrite. Chem Rev 114:5273–5357CrossRefPubMedGoogle Scholar
  9. 9.
    Dalton LR (ed) (1985) EPR and advanced EPR studies of biological systems. CRC Press, Boca RatonGoogle Scholar
  10. 10.
    Palmer G (1985) The electron paramagnetic resonance of metalloproteins. Biochem Soc Trans 13:548–560CrossRefPubMedGoogle Scholar
  11. 11.
    Weil JA, Bolton JR, Wertz JE (1994) Electron paramagnetic resonance: elementary theory and practical applications. Wiley, New YorkGoogle Scholar
  12. 12.
    Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide and peroxynitrite: the good, the bad and the ugly. Am J Physiol 271:C1424–C1437PubMedGoogle Scholar
  13. 13.
    Koppenol WH (1998) The basic chemistry of nitrogen monoxide and peroxynitrite. Free Radic Biol Med 25:385–391CrossRefPubMedGoogle Scholar
  14. 14.
    Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434–456CrossRefPubMedGoogle Scholar
  15. 15.
    Feelisch M, Rassaf T, Mnaimneh S, Singh N, Bryan NS, Jourd’Heuil D, Kelm M (2002) Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J 16:1775–1785CrossRefPubMedGoogle Scholar
  16. 16.
    Taha ZH (2003) Nitric oxide measurements in biological samples. Talanta 61:3–10CrossRefPubMedGoogle Scholar
  17. 17.
    Bryan NS, Rassaf T, Maloney RE, Rodriguez CM, Saijo F, Rodriguez JR, Feelisch M (2004) Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc Natl Acad Sci U S A 101:4308–4313CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Perkins MJ (1980) Spin trapping. Adv Phys Org Chem 17:1–64Google Scholar
  19. 19.
    Janzen EG, Haire DL (1990) Two decades of spin trapping. Adv Free Rad Chem 1:253–295Google Scholar
  20. 20.
    Davies MJ, Timmins GS, Clark RJH, Hester RE (1996) EPR spectroscopy of biologically relevant free radicals in cellular, ex vivo, and in vivo systems. In: Clark RJH, Hester RE (eds) Biomedical applications of spectroscopy. John Wiley & Sons, New YorkGoogle Scholar
  21. 21.
    Berliner LJ, Khramtsov V, Fujii H, Clanton TL (2001) Unique in vivo applications of spin traps. Free Radic Biol Med 30:489–499CrossRefPubMedGoogle Scholar
  22. 22.
    Villamena FA, Zweier JL (2004) Detection of reactive oxygen and nitrogen species by EPR spin trapping. Antioxid Redox Signal 6:619–629CrossRefPubMedGoogle Scholar
  23. 23.
    Davies MJ (1993) Detection and identification of macromolecule-derived radicals by EPR spin trapping. Res Chem Intermed 19:669–679CrossRefGoogle Scholar
  24. 24.
    Clement JL, Gilbert BC, Rockenbauer A, Tordo P (2001) Radical damage to proteins studied by EPR spin-trapping techniques. J Chem Soc Perkin Trans 2:1463–1470CrossRefGoogle Scholar
  25. 25.
    Davies MJ, Hawkins CL (2004) EPR spin trapping of protein radicals. Free Radic Biol Med 36:1072–1086CrossRefPubMedGoogle Scholar
  26. 26.
    Akaike T, Yoshida M, Miyamoto Y, Sato K, Kohno M, Sasamoto K, Miyazaki K, Ueda S, Maeda H (1993) Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/.bul.NO (nitric oxide) through a radical reaction. Biochemistry 32:827–832CrossRefPubMedGoogle Scholar
  27. 27.
    Joseph J, Kalyanaraman B, Hyde JS (1993) Trapping of nitric oxide by nitronyl nitroxides: an electron spin resonance investigation. Biochem Biophys Res Commun 192:926–934CrossRefPubMedGoogle Scholar
  28. 28.
    Hogg N, Singh RJ, Joseph J, Neese F, Kalyanaraman B (1995) Reactions of nitric oxide with nitronyl nitroxides and oxygen: prediction of nitrite and nitrate formation by kinetic simulation. Free Radic Res 22:47–56CrossRefPubMedGoogle Scholar
  29. 29.
    Grätzel M, Taniguchi S, Henglein Ber A (1970) Pulsradiolytische Untersuchung der NO-Oxydation und des Gleichgewichts N2O3 → NO + NO2 in wäßriger Lösung. Ber Bunsen-Ges Phys Chem 74:488–492CrossRefGoogle Scholar
  30. 30.
    Treinin A, Hayon E (1970) Absorption spectra and reaction kinetics of NO2, N2O3, and N2O4 in aqueous solution. J Am Chem Soc 92:5821–5828CrossRefGoogle Scholar
  31. 31.
    Singh RJ, Hogg N, Joseph J, Konorev E, Kalyanaraman B (1999) The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch Biochem Biophys 361:331–339CrossRefPubMedGoogle Scholar
  32. 32.
    Zhang Y, Hogg N (2004) Formation and stability of S-nitrosothiols in RAW 2647 cells. Am J Physiol Lung Cell Mol Physiol 287:L467–L474CrossRefPubMedGoogle Scholar
  33. 33.
    Zhang Y, Hogg N (2002) Mixing artifacts from the bolus addition of nitric oxide to oxymyoglobin: implications for s-nitrosothiol formation. Free Radic Biol Med 32:1212–1219CrossRefPubMedGoogle Scholar
  34. 34.
    Piknova B, Gladwin MT, Schechter AN, Hogg N (2005) Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation. J Biol Chem 280:40583–40588CrossRefPubMedGoogle Scholar
  35. 35.
    Hille R, Olson JS, Palmer G (1979) Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin. J Biol Chem 254:12110–12120PubMedGoogle Scholar
  36. 36.
    Louro SR, Ribeiro PC, Bemski G (1981) EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition. Biochim Biophys Acta 670:56–63CrossRefPubMedGoogle Scholar
  37. 37.
    Kozlov AV, Staniek K, Nohl H (1999) Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett 454:127–130CrossRefPubMedGoogle Scholar
  38. 38.
    Huang KT, Keszler A, Patel N, Patel RP, Gladwin MT, Kim-Shapiro DB, Hogg N (2005) The reaction between nitrite and deoxyhemoglobin - reassessment of reaction kinetics and stoichiometry. J Biol Chem 280:31126–31131CrossRefPubMedGoogle Scholar
  39. 39.
    Vanin AE, Mordvintcev EI, Kleschyov AL (1984) Biotransformation of sodium nitroprusside into dinitrosyl iron complexes in tissue of ascites tumors of mice. Stud Biophys 102:135–143Google Scholar
  40. 40.
    Varich VJ, Vanin AE, Ovsyannikova LM (1987) Discovery of endogenous nitric oxide in the mouse liver by electron paramagnetic resonance. Biofizika 32:1064–1065Google Scholar
  41. 41.
    Mordvintcev P, Mülsch A, Busse R, Vanin A (1991) On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spectroscopy. Anal Biochem 199:142–146CrossRefPubMedGoogle Scholar
  42. 42.
    Kubrina LN, Caldwell WS, Mordvintcev EI, Malenkova IV, Vanin AF (1992) EPR evidence for nitric oxide production from guanidino nitrogen of L-arginine in animal tissues in vivo. Biochim Biophys Acta 1099:233–237CrossRefPubMedGoogle Scholar
  43. 43.
    Komarov A, Mattson D, Jones MM, Singh PK, Lai CS (1993) In vivo spin trapping of nitric oxide in mice. Biochem Biophys Res Commun 195:1191–1198CrossRefPubMedGoogle Scholar
  44. 44.
    Komarov AM, Lai CS (1995) Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy. Biochim Biophys Acta 1272:29–36CrossRefPubMedGoogle Scholar
  45. 45.
    Vanin AF, Huisman A, Van Faassen E (2002) Iron dithiocarbamate as spin trap for nitric oxide detection: pitfalls and successes. Methods Enzymol 359:27–42CrossRefPubMedGoogle Scholar
  46. 46.
    Fujii S, Yoshimura T, Kamada H (1996) Nitric oxide trapping efficiencies of water-soluble iron(III) complexes with dithiocarbamate derivatives. Chem Lett 1996:785–786CrossRefGoogle Scholar
  47. 47.
    Paschenko SV, Khramtsov VV, Scatchkov MP, Plyusnin VF, Bassenge E (1996) EPR and laser flash photolis studies of the reaction of nitric oxide with water soluble NO trap Fe(II)-proline-dithiocarbamate complex. Biochem Biophys Res Commun 225:577–584CrossRefPubMedGoogle Scholar
  48. 48.
    Pou S, Tsai P, Porasuphatana S, Halpern HJ, Chandramouli GV, Barth ED, Rosen GM (1999) Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies. Biochim Biophys Acta 1427:216–226CrossRefPubMedGoogle Scholar
  49. 49.
    Fujii S, Kobayashi K, Tagawa S, Yoshimura T (2000) Reaction of nitric oxide with the iron(III) complex of N-(dithiocarboxy)sarcosine: a new type of reductive nitrosylation involving iron(IV) as an intermediate. J Chem Soc Dalton Trans:3310–3315Google Scholar
  50. 50.
    Fujii S, Yoshimura T (2000) A new trend in iron–dithiocarbamate complexes: as an endogenous NO trapping agent. Coord Chem Rev 198:89–99CrossRefGoogle Scholar
  51. 51.
    Vanin AF, Liu XP, Samouilov A, Stukan RA, Zweier JL (2000) Redox properties of iron–dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems. Biochim Biophys Acta 1474:365–377CrossRefPubMedGoogle Scholar
  52. 52.
    Enemark JH, Feltham RD (1974) Principles of structure, bonding, and reactivity metal nitrosyl complexes. Coord Chem Rev 13:339–406CrossRefGoogle Scholar
  53. 53.
    Miilsch A, Vanin A, Mordvintcev R, Hauschildt S, Busse R (1992) NO accounts completely for the oxygenated nitrogen species generated by enzymic L-arginine oxygenation. Biochem J 288:597–603CrossRefGoogle Scholar
  54. 54.
    Xia Y, Zweier JL (1997) Direct measurement of nitric oxide generation from nitric oxide synthase. Proc Natl Acad Sci U S A 94:12705–12710CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Yoneyema H, Kosaka H, Ohnishi T, Kawazoe T, Mizoguchi K, Ichikawa Y (1999) Reaction of neuronal nitric oxide synthase with the nitric oxide spin-trapping agent, iron complexed with N-dithiocarboxysarcosine. Eur J Biochem 266:771–777CrossRefGoogle Scholar
  56. 56.
    Li H, Samouilov A, Liu X, Zweier JL (2001) Characterization of the magnitude and kinetics of xanthine oxidase catalyzed nitrite reduction: evaluation of its role in nitric oxide generation in anoxic tissues. J Biol Chem 276:24482–24489CrossRefPubMedGoogle Scholar
  57. 57.
    Huisman A, Vos I, van Faassen EE, Joles JA, Grone HJ, Martasek P, Zonneveld AJ, Vanin AF, Rabelink TJ (2002) Anti-inflammatory effects of tetrahydrobiopterin on early rejection in renal allografts: modulation of inducible nitric oxide synthase. FASEB J 16:1135–1137PubMedGoogle Scholar
  58. 58.
    Li H, Cui H, Kundu TK, Alzawahra W, Zweier JL (2008) Nitric oxide production from nitrite occurs primarily in tissues not in the blood: Critical role of xanthine oxidase and aldehyde oxidase. J Biol Chem 283:17855–17863CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Li H, Kundu TK, Zweier JL (2009) Characterization of the magnitude and mechanism of aldehyde oxidase-mediated nitric oxide production from nitrite. J Biol Chem 284:33850–33858CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Maia L, Moura JJG (2011) Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases. J Biol Inorg Chem 16:443–460CrossRefPubMedGoogle Scholar
  61. 61.
    Maia L, Moura JJG (2014) Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo. Biochemistry. doi: 10.1021/bi500987w Google Scholar
  62. 62.
    Tominaga T, Sato S, Ohnishi T, Ohnishi ST (1993) Potentiation of nitric oxide formation following with bilateral carotid occlusion and local cerebral ischemia in the rat: in vivo detection of the nitric oxide radical by electron paramagnetic resonance spin trapping. Brain Res 614:342–346CrossRefPubMedGoogle Scholar
  63. 63.
    Lai CS, Komarov AM (1994) Spin trapping of nitric oxide produced in vivo in septic-shock mice. FEBS Lett 345:120–124CrossRefPubMedGoogle Scholar
  64. 64.
    Zweier JL, Wang P, Kuppusamy P (1995) Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem 270:304–307CrossRefPubMedGoogle Scholar
  65. 65.
    Zweier JL, Wang P, Samouilov A, Kuppusamy P (1995) Enzyme-independent formation of nitric oxide in biological tissues. Nat Med 1:804–809CrossRefPubMedGoogle Scholar
  66. 66.
    Kuppusamy P, Wang P, Samouilov A, Zweier JL (1996) Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging. Magn Reson Med 36:212–218CrossRefPubMedGoogle Scholar
  67. 67.
    Yoshimura T, Yokoyama H, Fujii S, Takayama F, Oikawa K, Kamada H (1996) In vivo EPR detection and imaging of endogenous NO in LPS-treated mice. Nat Biotechnol 14:992–994CrossRefPubMedGoogle Scholar
  68. 68.
    Fujii H, Koscielniak J, Berliner LJ (1997) Determination and characterization of nitric oxide generation in mice in vivo L-band EPR spectroscopy. Magn Res Med 38:565–568CrossRefGoogle Scholar
  69. 69.
    Mikoyan VD, Kubrina LN, Serezhenkov VA, Stukan RA, Vanin AF (1997) Complexes of Fe2+ with diethyldithiocarbamate or N-methyl- D-glucamine dithiocarbamate as traps of nitric oxide in animal tissues: comparative investigations. Biochim Biophys Acta 1336:225–234CrossRefPubMedGoogle Scholar
  70. 70.
    Kotake Y, Moore DR, Sang H, Reinke LA (1999) Continuous monitoring of in vivo nitric oxide formation using EPR analysis in biliary flow. Nitric Oxide 3:114–122CrossRefPubMedGoogle Scholar
  71. 71.
    Tsuchiya K, Jiang JJ, Yoshizumi M, Tamaki T, Houchi H, Minakuchi K, Fukuzawa K, Mason RP (1999) Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD. Free Radic Biol Med 27:347–355CrossRefPubMedGoogle Scholar
  72. 72.
    Lu C, Koppenol WH (2005) Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate. Free Radic Biol Med 39:1581–1590CrossRefPubMedGoogle Scholar
  73. 73.
    Tsuchiya K, Takasugi M, Minakuchi K, Fukuzawa K (1996) Sensitive quantitation of nitric oxide by EPR spectroscopy. Free Radic Biol Med 21:733–737CrossRefPubMedGoogle Scholar
  74. 74.
    McGrath C, O’Connor C, Sangregodo C, Seddon J, Sinn E, Sowrey E, Young N (1999) Direct measurement of the high-spin and low-spin bond lengths and the spin state population in mixed spin state systems: an Fe K-edge XAFS study of iron(III) dithiocarbamate complexes. Inorg Chem Commun 2:536–539CrossRefGoogle Scholar
  75. 75.
    Tsuchiya K, Yoshizumi M, Houchi H, Mason R (2000) Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite. J Biol Chem 275:1551–1556Google Scholar
  76. 76.
    Kubrina LN, Mikoyan VD, Mordvintcev PI, Vanin AF (1993) Iron potentiates lipopolysaccharide-induced nitric oxide formation in animal organs. Biochim Biophys Acta 1176:240–244CrossRefPubMedGoogle Scholar
  77. 77.
    Vanin AF, Huisman A, Stroes ESG, Ruijter-Heijstek FC, Rabelink TJ, Faassen EE (2001) Antioxidant capacity of mononitrosyl-iron-dithiocarbamate complexes: implications for NO trapping. Free Radic Biol Med 30:813–824CrossRefPubMedGoogle Scholar
  78. 78.
    Tsuchihashi K, Kirima K, Yoshizumi M, Houchi H, Tamaki T, Mason RP (2002) The role of thiol and nitrosothiol compounds in the nitric oxide-forming reactions of the iron-N-methyl-D-glucamine dithiocarbamate complex. Biochem J 367:771–779CrossRefGoogle Scholar
  79. 79.
    Stamler JS, Singel DJ, Loscalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258:1898–1902CrossRefPubMedGoogle Scholar
  80. 80.
    Komarov AM, Wink DA, Feelisch M, Schmidt HHW (2000) Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase. Free Radic Biol Med 28:739–742CrossRefPubMedGoogle Scholar
  81. 81.
    Xia Y, Cardounel AJ, Vanin AF, Zweier JL (2000) Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. Free Radic Biol Med 29:793–797CrossRefPubMedGoogle Scholar
  82. 82.
    Cocco D, Calabrese L, Rigo A, Agrese E, Rotilio G (1981) Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase. J Biol Chem 256:8983–8986PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.UCIBIO, REQUIMTE, Departamento Química, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal

Personalised recommendations