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Molecular Biotechnology

, Volume 12, Issue 3, pp 275–279 | Cite as

A microtiter-plate assay of nitric oxide synthase activity

Protocol

Abstract

We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing the spectral shift in optical absorbence between the wavelengths 405 and 420 nm on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). This is a high-throughput assay permitting 96 or 384 simultaneous kinetic measurements and is ideal for the study of NOS inhibitors and their time dependence. It is also possible to measure enzyme rates under different conditions simultaneously for the study of the cofactor and substrate dependence of NOS preparations. The assay requires approximately 10 pmol/min of NOS activity to achieve a 1moD/min rate.

Index Entries

Nitric oxide synthases oxyhemoglobin microtiter-plate kinetic 

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References

  1. 1.
    Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249–258.PubMedGoogle Scholar
  2. 2.
    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, 9–30.CrossRefGoogle Scholar
  3. 3.
    Salter, M. and Knowles, R. G. (1989) Assay of NOS activity by the measurement of conversion of oxyhemoglobin to methemoglobin by NO, in Nitric Oxide Protocols (Titheridge, M.A., ed.), Humana Press, New Jersey, pp. 61–65.Google Scholar
  4. 4.
    Feelisch, M., Kubitzek, D., and Werringloer, J. (1996) The oxyhaemoglobin assay, in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, pp. 472, 473.Google Scholar
  5. 5.
    Gross, S.S., Jaffe, E.A., Levi, R., and Kilbourn, R.G. (1991) Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-dependent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem. Biophys. Res. Comm. 178 (3), 823–829.PubMedCrossRefGoogle Scholar
  6. 6.
    Stuehr, D. J. and Griffith, O. W. (1996) Purification, assay and properties of mammalian nitric oxide synthases, in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, p. 185.Google Scholar
  7. 7.
    Stuehr, D. J. and Griffith, O. W. (1996) Purification, assay and properties of mammalian nitric oxide synthases, in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, pp. 183–184.Google Scholar
  8. 8.
    Knowles, R.G. and Salter, M. (1989) Measurement of NOS activity by conversion of radiolabelled arginine to citrulline using ion-exchange separation, in Nitric Oxide Protocols (Titheridge, M.A., ed.), Humana Press, New Jersey, pp. 67–73.Google Scholar
  9. 9.
    Paterson, R.A., Eagles, P.A.M., Young, D.A.B., and Beddell, C.R. (1976) Rapid purification of large quantities of human hemoglobin with low phosphate content by counter-flow dialysis. Int. J. Biochem. 7 117, 118.CrossRefGoogle Scholar
  10. 10.
    Charles, I. C., Scorer, C. A., Angeles Moro, M., Fernandez, C., Chubb, A., Dawson, J., Foxwell, N., Knowles, R. G., and Baylis, S. A. (1996) Expression of the three human NO synthase isozymes. Methds Enzymol. 268, 449–460.CrossRefGoogle Scholar
  11. 11.
    Komori, Y., Hyun, J., Chiang, K., and Fukuto, J. M. (1995). The role of thiols in the apparent activation of rat brain nitric oxide synthase (NOS). J. Biochem 117, 923–927PubMedGoogle Scholar
  12. 12.
    Hofmann, H. and Schmidt, H. H. H. W. (1995). Thiol dependence of nitric oxide synthase. Biochemistry 34 (41), 13,443–13,452.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  1. 1.Glaxo-Wellcome Research and DevelopmentStevenage

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