Measurement of NO and NO Synthase

Ben Avi Weissman1, Steven S. Gross2

1 Israel Institute for Biological Research, Ness Ziona, Israel, 2 Cornell University Medical College, New York, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.13
DOI:  10.1002/0471142301.ns0713s05
Online Posting Date:  May, 2001
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Abstract

Nitric oxide (NO) is a key biosignaling molecule produced in both peripheral tissues and the central nervous system by a family of enzymes known as nitric oxide synthases (NOSs). NOSs convert L‐arginine to stoichiometric quantities of NO and L‐citrulline using molecular oxygen and NADPH as cofactors. Techniques for measurement of NO and NOS activity are essential to demonstrate the role of NO and NO‐derived species in biological systems. This unit describes two methods for detection of NO: a direct method employing chemiluminescent detection and one based on quantification of the stable oxidation products with detection using the Greiss reagent. Additionally, NOS activity can be quantified by measuring the conversion of radiolabeled L‐arginine to radiolabeled L‐citrulline.

     
 
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Table of Contents

  • Basic Protocol 1: Chemiluminescent Detection of NO
  • Support Protocol 1: Preparation of a Reducing Agent for Conversion of Nitrite to NO
  • Support Protocol 2: Preparation of Nitrate Reducing Agent
  • Support Protocol 3: Deproteination of Samples for Nitrate/Nitrite Assay
  • Basic Protocol 2: Nitrite and Nitrite/Nitrate Assay by the Griess Method
  • Basic Protocol 3: L‐Citrulline Assay for Nitric Oxide Synthase (NOS) Activity
  • Support Protocol 4: Preparation and Regeneration of Cation‐Exchange Columns
  • Support Protocol 5: Purification of Radiolabeled L‐Arginine
  • Support Protocol 6: Nitric Oxide Synthase (NOS) Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Chemiluminescent Detection of NO

  Materials
  • Acidified KI or NaI for nitrite reduction (see protocol 2) or acidified VCl 3 for nitrite/nitrate reduction (see protocol 3)
  • NO gas cylinder and stainless steel regulator (100 to 200 ppm NO in N 2, for preparation of NO gas standards) and N 2 gas cylinder and regulator (Matheson Gas Products)
  • 10 µM sodium nitrite
  • 10 µM sodium nitrate
  • Samples for analysis
  • Antifoam B (Sigma)
  • Chemiluminescence detection system (Fig. and Fig. ) consisting of:
  •  Glass purge vessel with gas‐tight Swagelock connections (Fig. ; available from Sievers Instruments as #ASM03292)
  •  Helium gas tank and regulator
  •  Acid trap with gas bubbler, containing 1 M NaOH (optional)
  •  Chemiluminescence detector for NO x (e.g., Sievers Instruments Model 280A)
  •  Vacuum pump
  •  Recirculating heating water bath
  •  Gas‐sampling tube (glass, with stopcocks on either end and a septum for extraction with a gas‐tight Hamilton syringe)
  •  Teflon/silicone septa for purge vessel
  •  Data collection system (i.e., chart recorder, integrator, computer)
  •  Ringstand, clamps, Swagelock connectors, Teflon ferrules, and stainless steel or PFA connection tubing
  • 10, 25, 50, and 100 µl gas‐tight Hamilton syringes

Support Protocol 1: Preparation of a Reducing Agent for Conversion of Nitrite to NO

  • Glacial acetic acid
  • KI or NaI
  • Antifoam B (Sigma)

Support Protocol 2: Preparation of Nitrate Reducing Agent

  Materials
  • VCl 3 (Aldrich)
  • Concentrated HCl
  • 50‐ml volumetric flask (dry)
  • Equipment for refluxing (optional): three‐necked flask, condenser, nitrogen bubbler, thermometer, and electric heating mantle
  • Filter paper

Support Protocol 3: Deproteination of Samples for Nitrate/Nitrite Assay

  Materials
  • 100% ethanol (absolute ethanol; anhydrous)
  • Sample for analysis

Basic Protocol 2: Nitrite and Nitrite/Nitrate Assay by the Griess Method

  Materials
  • Griess reagents A and B (see recipe)
  • 100 µM sodium nitrite standard stock solution (dissolve 69 mg NaNO 2 in 10 ml H 2O)
  • 100 µM sodium nitrate standard stock solution (dissolve 85 mg NaNO 3 in 10 ml H 2O)
  • Nitrate reductase buffer (see recipe)
  • 200 mM recipeTris⋅Cl, pH 7.6 ( appendix 2A)
  • Nitrate reductase (from Aspergillus; Sigma, cat. no. N‐7265)
  • L‐lactic dehydrogenase (from rabbit muscle; Sigma, cat. no. L‐2500)
  • 500 mM sodium pyruvate
  • 96‐well microtiter plates (flat‐bottom or round‐bottom)
  • Repeating pipettor (e.g., Repipettor from Eppendorf) and 5, 1.25, and 0.5 ml reservoir tips (e.g., Combitips from Eppendorf)
  • Microtiter‐plate reader with 540‐ or 550‐nm filter (maximum absorbance, 546 nm)

Basic Protocol 3: L‐Citrulline Assay for Nitric Oxide Synthase (NOS) Activity

  Materials
  • Assay buffer (see recipe),ice‐cold
  • Assay mix 1 and 2 (see reciperecipes) ice‐cold
  • 4 mM ‐NG‐nitro‐L‐arginine, ice‐cold
  • Test compounds in H 2O, ice‐cold
  • 20 mM EGTA, ice‐cold
  • 20 mM EGTA/20 mM NG‐nitro‐L‐arginine, ice‐cold
  • Stop buffer: 50 mM HEPES, pH 5.5, containing 5 mM recipeEDTA, ice‐cold ( appendix 2A)
  • Assay tubes: 12 × 75–mm disposable borosilicate glass test tubes
  • Preequilibrated Dowex AG 50X‐8 columns or Dowex AG 50X‐8 resin slurry preequilibrated with stop buffer (see protocol 7)
  • Additional reagents and equipment for preparation of NOS samples (see protocol 9) and protein determination (see CPMB UNIT and appendix 1A in this manual)
CAUTION: Radioactive materials require special handling; all column eluates must be considered radioactive waste and disposed of appropriately.

Support Protocol 4: Preparation and Regeneration of Cation‐Exchange Columns

  Materials
  • Dowex AG 50X‐8, H+ or Na+ form (100 to 200 mesh recommended; Bio‐Rad)
  • 0.5 N HCl
  • 0.5 N NaOH
  • Stop buffer: 50 mM HEPES, pH 5.5, containing 5 mM recipeEDTA, ice‐cold ( appendix 2A)
  • 500‐ml glass beaker
  • pH test paper (e.g., Color pHast; EM Science)
  • Disposable columns (e.g., polystyrene, 0.4 × 4.0–cm; Bio‐Rad)

Support Protocol 5: Purification of Radiolabeled L‐Arginine

  Materials
  • Stop buffer: 50 mM HEPES, pH 5.5, containing 5 mM recipeEDTA, ice‐cold ( appendix 2A)
  • Radiolabeled precursor: L‐[3H]arginine or L‐[14C]arginine (NEN Life Science Products, Amersham, or Sigma)
  • High‐water‐capacity scintillation cocktail
  • 0.5 M NH 4OH, ice‐cold
  • 2% ethanol solution
  • Preequilibrated Dowex AG 50X‐8 columns (see protocol 7)
  • 12 × 75–mm glass test tubes

Support Protocol 6: Nitric Oxide Synthase (NOS) Preparation

  Materials
  • Cells or tissue of interest
  • Phosphate‐buffered saline (PBS; appendix 2A; optional)
  • Homogenization buffer (see recipe) with and without 1 M KCl
  • Assay buffer (see recipe) with and without DTT
  • Rubber policeman or Teflon cell scraper (if preparing samples from cultured cells in Petri dishes or flasks, respectively)
  • Tissue homogenizer (e.g., Polytron from Brinkmann) or sonicator
  • High‐speed centrifuge
  • Dowex AG 50X‐8 columns (Na+‐form; Bio‐Rad): prepare as in protocol 7 but use 20 to 50 mesh resin and preequilibrate in assay buffer recipe without DTT
  • Additional reagents and equipment for protein determination (see CPMB UNIT and appendix 1A in this manual)
NOTE: All the following procedures should be performed at 0° to 4°C.
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Figures

Videos

Literature Cited

Literature Cited
   Bredt, D.S. and Snyder, S.H. 1990. Isolation of nitric oxide synthetase, a calmodulin‐requiring enzyme. Proc. Natl. Acad. Sci. U.S.A. 87:682‐685.
   Fontijn, A., Sabadell, A.J., and Ronco, R.J. 1970. Homogenous chemiluminescent measurement of nitric oxide: Implications for continuous selective monitoring of gaseous air pollutants. Anal. Chem. 42:575‐579.
   Furchgott, R.F. and Zawadzki, J.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373‐376.
   Griess, J.P. 1864. On a new series of bodies in which nitrogen is substituted for hydrogen. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 154:667‐731.
   Gross, S.S. 1996. Microtiter plate assay for determining the kinetics of nitric oxide synthesis. Methods Enzymol. 286:159‐168.
   Hattori, Y., Campbell, E.B., and Gross, S.S. 1994. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle: Role in the regeneration of arginine for nitric oxide synthesis. J. Biol. Chem. 269:9405‐9408.
   Ignarro, L.J., Buga, B.M., Wood, K.S., Byrns, R.E., and Chaudhuri, G. 1987. Endothelium‐derived relaxing factor produced and released by artery and vein is nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 84:9265‐9269.
   Ishii, K., Sheng, H., Warner, T.D., Forstermann, U., and Murad, F. 1991. A simple and sensitive bioassay method for detection of EDRF with RFL‐6 rat lung fibroblasts. Am. J. Physiol. 261:H598‐603.
   Kikuchi, K., Nagano, T., Hayakawa, H., Hirata, Y., and Hirobe, M. 1993. Detection of nitric oxide production from a perfused organ by a luminol‐H2O2 system. Anal. Chem. 65:1794‐1799.
   Malinski, T., Bailey, F., Zhang, Z.G., and Chopp, M. 1993. Nitric oxide measured by a porphyrinic microsensor in rat brain after transient cerebral artery occlusion. J. Cereb. Blood Flow Metab. 13:355‐358.
   Nathan, C. and Xie, Q.W. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269:13725‐13728.
   Palmer, R.M.J., Ferrige, A.G., and Moncada, S. 1987. Nitric oxide release accounts for the biological activity of endothelium‐derived relaxing factor. Nature 327:524‐526.
   Salter, M., Knowles, R.G., Moncada, S. 1991. Widespread tissue distribution, species distribution and changes in activity of Ca2+‐dependent and Ca2+‐independent nitric oxide synthases. FEBS Lett. 291:145‐149.
   Schmidt, H.H., Hofmann, H., Schindler, U., Shutenko, Z.S., Cunningham, D.D., and Feelisch, M. 1997. No NO from NO synthase. Proc. Natl. Acad. Sci. U.S.A. 93:14492‐14497.
   Stuehr, D.J., Gross, S.S., Sakuma, I., Levi, R., and Nathan, C. 1989. Activated macrophages secrete a metabolite of arginine with the bioactivity of endothelium‐derived relaxing factor and the chemical reactivity of nitric oxide. J. Exp. Med. 169:1011‐1020.
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