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Assay of NOS Activity by the Measurement of Conversion of Oxyhemoglobin to Methemoglobin by NO

  • Mark Salter
  • Richard G. Knowles
Part of the Methods in Molecular Biology™ book series (MIMB, volume 100)

Abstract

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP+ (reviewed in ref. 1). This chapter describes the measurement of NOS activity by utilizing the conversion of oxyhemoglobin to methemoglobin by NO while monitoring the absorption difference between the wavelengths 401 and 421 nm or at just 401 nm. This assay was first described by Feelisch and Noack (2) and has since been modified to allow not only the measurement of NOS activity in vitro but also the degree of in vivo inhibition of neuronal (n) NOS (and also possibly endothelial (e) NOS) by ex vivo analysis after the administration of slowly dissociating inhibitors such as Nω-nitro-L-arginine (L-NNA) and its methyl ester, L-NAME (3,4). Using the protocol described, this technique is sensitive (limit of detection approx 20 pmol/min per g tissue), specific for NOS in conjunction with the use of NOS inhibitors, applicable to most preparations (with the exception of samples containing large amounts of hemoglobin) and suitable for continual monitoring.

Keywords

Methyl Ester Flavin Adenine Dinucleotide Nicotinamide Adenine Dinucleotide Phosphate Sodium Dithionite Flavin Adenine Dinucleotide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249–258.PubMedGoogle Scholar
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    Feelisch, M. and Noack, E. A. (1987) Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur. J. Pharmacol. 139, 19–30.PubMedCrossRefGoogle Scholar
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    Salter, M., Knowles, R. G., and Moncada, S. (1991) Widespread tissue distribution, species distribution and changes in activity of calcium-dependent and calcium-independent nitric oxide synthases. FEBS Lett. 291, 145–149.PubMedCrossRefGoogle Scholar
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    Salter, M., Duffy, C., and Hazelwood, R. (1995) Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results. Neuropharmacology 34, 327–334.PubMedCrossRefGoogle Scholar
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    Feelisch, M., Kubitzek, D., and Werringloer, J. (1996). The oxyhaemoglobin assay in Methods in Nitric Oxide Research, (Feelisch, M. and Stammler, J. S., eds.), Wiley, London, pp. 455–478.Google Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Mark Salter
    • 1
  • Richard G. Knowles
    • 2
  1. 1.Lead Discovery GroupGlaxo-Wellcome ResearchStevenageUK
  2. 2.Enzyme Pharmacology GroupGlaxo-Wellcome ResearchStevenageUK

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