Fluorometric Techniques for the Detection of Nitric Oxide and Metabolites

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 10.4
DOI:  10.1002/0471140856.tx1004s00
Online Posting Date:  May, 2001
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Abstract

Recently fluorometric techniques have been developed to determine NO and S‐nitrosothiols with improved sensitivity over spectrophotometric techniques based on similar chemistry. This unit contains protocols for detection and quantification of NO from chemical reactions and from cellular systems, and of S‐nitrosothiols in chemical, biochemical, and cellular experiments.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of NO from Chemical Reactions
  • Basic Protocol 2: Detection of NO in Cellular Systems
  • Basic Protocol 3: Detection of S‐Nitrosothiols
  • Basic Protocol 4: Determination of Nitrate by Fluorescent Techniques
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of NO from Chemical Reactions

  Materials
  • Cell culture medium with no phenol red or vitamins (see recipe)
  • 2,3‐Diaminonaphthylene (DAN)
  • Dimethyl formamide (DMF) or dimethylsulfoxide (DMSO)
  • 20 to 100 mM sodium phosphate, pH 7.4 ( appendix 2A)
  • Nitrogen or argon gas
  • NO gas
  • Concentrated NaOH (1 or 10 M; appendix 2A)
  • Sulfanilamide
  • N‐(1‐Naphthyl)ethylenediamine dihydrochloride (NEDD)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • NO‐generating system to be tested
  • Fluorescent cuvettes (preferably quartz)
  • Airtight 100‐µl syringe
  • NO gas cylinder
  • Fluorometer
  • Airtight syringe
  • Plastic tuberculin syringe
  • Syringe needles

Basic Protocol 2: Detection of NO in Cellular Systems

  Materials
  • Cells thought to produce NO
  • Cell culture medium (see recipe)
  • 2,3‐Diaminonaphthylene (DAN; see protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Concentrated NaOH (1 or 10 M; appendix 2A)

Basic Protocol 3: Detection of S‐Nitrosothiols

  Materials
  • Mercuric chloride or copper sulfate
  • Dimethyl formamide (DMF) or dimethylsulfoxide (DMSO)
  • 2,3‐Diaminonaphthylene (DAN)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Glutathione
  • Sodium nitrite
  • Hydrochloric acid (HCl)
  • Cell extracts
  • Buffer samples
  • Rubber gloves
  • Fluorometer

Basic Protocol 4: Determination of Nitrate by Fluorescent Techniques

  Materials
  • Hydrochloric acid (HCl)
  • Sample to be tested
  • 2,3‐Diaminonaphthylene (DAN)
  • Concentrated NaOH (1 or 10 M; appendix 2A)
  • 5000 U/ml lactate dehydrogenase (Sigma) diluted 1:10 in phosphate‐buffered saline (PBS; appendix 2A)
  • 100 mM sodium pyruvate
  • Hydrogen peroxide
  • 100 U/ml GSH peroxidase (Sigma)
  • 10 U/ml Aspergillus nitrate reductase
  • Flavin adenine dinucleotide (FAD)
  • β‐Nicotinamide adenine dinucleotide phosphate (NADPH)
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Figures

Videos

Literature Cited

Literature Cited
   Cook, J.A., Kim, S.‐M., Teaug, D., Krishna, M.C., Pacelli, R., Mitchell, J.B., Nims, R.W., Christodoulou, D., Miles, A.M., Grisham, M.B., and Wink, D.A. 1996. Convenient colorimetric and fluorometric assays for S‐nitrosothiols. Anal. Biochem. 238:150‐158.
   Granger, D.L., Taintor, R.R., Boockvar, K.S., Hibbs, J.B., and 1996. Measurement of nitrate and nitrite in biological system using nitrate reductase and Griess reaction. Methods Enzymol. 268:142‐151.
   Grisham, M.B., Johnson, G.G., and Lancaster, J.R. 1996. Quantitation of nitrate and nitrite in extracellular fluids. Methods Enzymol. 268:237‐246.
   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.
   Miles, A.M., Gibson, M., Krishna, M., Cook, J.C., Pacelli, R., Wink, D.A., Grisham, M.B., and 1995. Effects of superoxide on nitric oxide‐dependent N‐nitrosation reactions. Free Radic. Res. Commun. 233:379‐390.
   Miles, A.M., Wink, D.A., Cook, J.C., Grisham, M.B., and 1996. Determination of nitric oxide using fluorescence spectroscopy. Methods Enzymol. 268:105‐120.
   Nims, R.W. and Darbyshire, J.F., Saavedra, J.E., Christodoulou, D., Hanbauer, I., Cox, G.W., Grisham, M.B., Laval, J., Cook, J.A., Krishna, M.C., Wink, D.A., and 1995. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods: A Companion to Methods Enzymol. 7:48‐54.
   Nims, R.W., Cook, J.C., Krishna, M.C., Christodoulou, D., Poore, C.M.B., Miles, A.M., Grisham, M.B., Wink, D.A., and 1996. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods Enzymol. 268:93‐105.
   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, 1997. J. Biol. Chem. 272:9922‐9932.
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