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Measurement of NO and NO Synthase

互联网2013-12-31

1294

Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 VCl₃ for nitrite/nitrate reduction (see protocol 3 )
NO gas cylinder and stainless steel regulator (100 to 200 ppm NO in N₂ , for preparation of NO gas standards) and N₂ 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

VCl₃ (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 NaNO₂ in 10 ml H₂ O)
100 µM sodium nitrate standard stock solution (dissolve 85 mg NaNO₃ in 10 ml H₂ O)
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 ‐N^G ‐nitro‐L‐arginine, ice‐cold
Test compounds in H₂ O, ice‐cold
20 mM EGTA, ice‐cold
20 mM EGTA/20 mM N ^G ‐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‐[³ H]arginine or L‐[¹⁴ C]arginine (NEN Life Science Products, Amersham, or Sigma)
High‐water‐capacity scintillation cocktail
0.5 M NH₄ OH, 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

Figure 7.13.1 The reaction catalyzed by NOS.

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Figure 7.13.2 The chemiluminescence detector for measurement of gas‐phase NO. Inflow of gas from the purge vessel to the chemiluminescence detector is regulated with a needle valve. NO in the inflow gas mixes with ozone in a reaction chamber, producing excited state NO₂ *. The decay of NO₂ * results in a photon emission that is converted to an electric current by a photomultiplier tube (PMT). The amplified signal is sent to a chart recorder, integrator, or computerized data analysis system for processing.

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Figure 7.13.3 A gas sparging vessel that can be utilized for analysis of NO, NO/nitrite, and NO/nitrite/nitrate by chemiluminescence detection.

View Image

Figure 7.13.4 Griess reaction for detection of nitrite, and the sum of nitrite and nitrate, in biological samples.

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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.
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	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.
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	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.