Methods for Distinguishing Nitrosative and Oxidative Chemistry of Reactive Nitrogen Oxide Species Derived from Nitric Oxide

David A. Wink1, Sungmee Kim1, Allen Miles2, David Jourd'heuil2, Matthew B. Grisham2

1 National Cancer Institute, Bethesda, Maryland, 2 Louisiana State University Medical Center, Shreveport, Louisiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.8
DOI:  10.1002/0471140856.tx1008s03
Online Posting Date:  May, 2001
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NO‐derived intermediates formed under aerobic conditions may engage in complex chemical reactions with biologically important molecules. The outcomes of these reactions and their ultimate effect on biological systems depend on the selectivity of the species and the concentrations of different substances present and whether the reaction takes place in the gas or aqueous phase. In this unit conversion of two different compounds to fluorescent products is used to distinguish between oxidative and nitrosative chemistry of different reactive nitrogen oxide species.

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Table of Contents

  • Basic Protocol 1: Determination of Nitrosation Reactions
  • Basic Protocol 2: Determination of the Selectivity for DHR Oxidation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Determination of Nitrosation Reactions

  • 2,3‐diaminonapthylene (DAN; Aldrich)
  • Dimethylformamide (DMF) or dimethylsufoxide (DMSO)
  • Buffer or medium under investigation
  • PBS without calcium or magnesium ( appendix 2A)
  • 20 to 100 mM sodium phosphate, pH 7.4 ( appendix 2A)
  • Nitrogen or argon gas (see Fig. for setup)
  • NO gas cylinder (see Fig. for setup)
  • 1 M NaOH
  • Sulfanilamide
  • N‐(1‐naphthyl)ethylenediamine dihydrochloride (NEDD; Aldrich)
  • Substrate to be evaluated
  • Bubbling apparatus for nitrogen or argon and for NO (Fig. ), consisting of:
    •  Glass vials with rubber septa
    •  Tygon tubing
  • UV/visible spectrophotometer and quartz cuvettes
  • 100‐µl airtight syringe
  • Spectrofluorometer
  • 13 × 75–mm glass test tubes

Basic Protocol 2: Determination of the Selectivity for DHR Oxidation

  • 123 dihydrorhodamine (DHR)
  • Dimethylformamide (DMF)
  • PBS ( appendix 2A) or sample buffer
  • Oxidizing substances under investigation
  • 123 rhodamine
  • 13 × 75–glass test tubes
  • Spectrofluorometer
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Literature Cited

Literature Cited
   Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.H., and Freeman, B.A. 1990 Apparent hydroxyl radical production by peroxynitrites: Implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87:1620‐1624.
   Crow, J. 1997. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide Biol. Chem. 1:145‐157.
   Ford, P.C., Wink, D.A., and Stanbury, D.M. 1993. Autooxidation kinetics of aqueous nitric oxide. FEBS Lett. 326:1‐3.
   Huie, R.E. and Padmaja, S. 1993. The reaction of NO with superoxide. Free Radic. Res. Commun. 18:195‐199.
   Keefer, L.K., Nims, R.W., Davies, K.W., and Wink, D.A. 1996. NONOates (diazenolate‐2‐oxides) as nitric oxide dosage forms. Methods Enzymol. 268:281‐294.
   Kooy, N.W., Royall, J.A., Ischiropoulos, H., and Beckman, J.S. 1994. Peroxynitrite‐mediated oxidation of dihydrorhodamine 123. Free Radicals Biol. Med. 16:149‐156.
   Kroncke, K.‐D., Fechsel, K., Schmidt, T., Zenke, F.T., Dasting, I., Wesener, J.R., Bettermann, H., Breunig, K.D., and Kolb‐Bachofen, V. 1994. Nitric oxide destroys zinc‐finger clusters inducing zinc release from metallothionein and inhibition of the zinc finger‐type yeast transcription activator LAC9. Biochem. Biophys. Res. Commun. 200:1105‐1110.
   Miles, A.M., Gibson, M., Krishna, M., Cook, J.C., Pacelli, R., Wink, D.A., and Grisham, M.B. 1995. Effects of superoxide on nitric oxide–dependent N‐nitrosation reactions. Free Radical Res. 233:379‐390.
   Miles, A.M., Bohle, .D.S., Glassbrenner, P.A., Hansert, B., Wink, D.A., and Grisham, M.B. 1996. Modulation of superoxide‐dependent oxidation and hydroxylation reactions by nitric oxide. J. Biol. Chem. 271:40‐47.
   Nims, R.W., Darbyshire, J.F., Saavedra, J.E., Christodoulou, D., Hanbauer, I., Cox, G.W., Grisham, M.B., Laval, J., Cook, J.A., Krishna, M.C., and Wink, D.A. 1995. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods 7:48‐54.
   Pires, M., Ross, D.S., and Rossi, M.J. 1994. Kinetic and mechanistic aspects of the NO oxidation by O2 in aqueous phase. Int. J. Chem. Kinet. 26:1207‐1227.
   Pryor, W.A. and Squadrito, G.L. 1996. The chemistry of peroxynitrite and peroxynitrous acid: Products from the reaction of nitric oxide with superoxide. Am. J. Phys. 268:L699‐721.
   Williams, D.L.H. 1988. Reagents involved in Nitrosation. In Nitrosation (D.L.H. Williams, ed.) pp. 18‐37. Cambridge University Press, New York.
   Wink, D.A. and Laval, J. 1994. The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo. Carcinogenesis 15:2125‐2129.
   Wink, D.A., Darbyshire, J.F., Nims, R.W., Saveedra, J.E., and Ford, P.C. 1993. Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 6:23‐27.
   Wink, D.A., Nims, R.W., Darbyshire, J.F., Christodoulou, D., Hanbauer, I., Cox, G.W., Laval, F., Laval, J., Cook, J.A., Krishna, M.C., DeGraff, W., and Mitchell, J.B. 1994. Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH: Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 7:519‐525.
   Wink, D.A., Hanbauer, I., Grisham, M.B., Laval, F., Nims, R.W., Laval, J., Cook, J.C., Pacelli, R., Liebmann, J., Krishna, M.C., Ford, M.C., and Mitchell, J.B. 1996a. The chemical biology of NO: Insights into regulation, protective and toxic mechanisms of nitric oxide. Curr. Top. Cell. Regul. 34:159‐187.
   Wink, D.A., Grisham, M., Mitchell, J.B., and Ford, P.C. 1996b. Direct and indirect effects of nitric oxide: Biologically relevant chemical reactions in biology of NO. Methods Enzymol. 268:12‐31.
   Wink, D.A., Grisham, M.B., Miles, A.M., Nims, R.W., Krishna, M.C., Pacelli, R., Poore, C., and Cook, J.A. 1996c. Methods for the determination of selectivity of the reactive nitrogen oxide species for various substrates. Methods Enzymol. 268:120‐130.
   Wink, D.A., Cook, J.A., Kim, S., Vodovotz, Y., Pacelli, R., Kirshna, M.C., Russo, A., Mitchell, J.B., Jourd'heuil, D., Miles, A.M., and Grisham, M.B. 1997. Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide derived reactive intermediates. J. Biol. Chem. 272:11147‐11151.
   Wink, D.A., Feelisch, M., Vodovotz, Y., Fukuto, J., and Grisham, M.B. 1998. The chemical biology of NO: An update. In Reactive Oxygen Species in Biological Systems (D. Gilbert and C.A. Cotton, eds.) pp. 275‐292. Plenum, N.Y.
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