Nitric oxide (NO) is produced by three distinct isoforms of nitric oxide synthase and functions as a key signaling molecule in physiology and pathophysiology.^1,2 NO transduces its effects by reacting either directly with heme and non-heme centers of proteins or indirectly via further oxidation to various reactive nitrogen species (RNS).^3,4Nitrosylation refers to the binding of an NO group to a transition metal, as typified in the activation of soluble guanylate cyclase, or to a thiol (SH) group of protein cysteine residues resulting in the formation of an S-NO moiety. This latter reaction, termed S-nitrosylation, is mediated by reactive nitrogen species of higher oxidation states of NO, such as NO₂ and N2O3.^5,6 S-Nitrosylation is a reversible, and seemingly specific, post-translational modification that regulates the activity of a large number of targets, including metabolic, structural, cytoskeletal, and signaling proteins.⁷ Cayman’s S-Nitrosylated Protein Detection Assay employs a modification of the Jaffrey et al. 'Biotin-switch' method to allow for the direct visualization of S-nitrosylated proteins in whole cells or tissues, as well as by western blot analysis.^8,9 Using this method, free SH groups are first blocked and any S-NO bonds present in the sample are then cleaved. Biotinylation of the newly formed SH groups provides the basis for visualization using streptavidin-based colorimetric or fluorescence detection.

1 Alderton, W.K., Cooper, C.E., and Knowles, R.G. Nitric oxide synthases: Structure, function, and inhibition. Biochem J 357 593-615 (2001).

2 Bredt, D.S. Endogenous nitric oxide synthesis: Biological functions and pathophysiology. Free Radic Res 31 577-596 (1999).

3 Beckman, J.S., and Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and the ugly. Am J Physiol 271 C1424-C1437 (1996).

4 Kelm, M. Nitric oxide metabolism and breakdown. Biochim Biophys Acta 1411 273-289 (1999).

5 Mannick, J.B., and Schonhoff, C.M. Nitrosylation: The next phosphorylation? Arch Biochem Biophys 408 1-6 (2002).

6 Martínez-Ruiz, A., and Lamas, S. S-nitrosylation: A potential new paradigm in signal transduction. Cardiovasc Res 62 43-52 (2004).

7 Stamler, J.S., Lamas, S., and Fang, F.C. Nitrosylation: The prototypic redox-based signaling mechanism. Cell 106 675-683 (2001).

8 Ckless, K., Reynaert, N.L., Taatjes, D.J., et al. In situ detection and visualization of S-nitrosylated proteins following chemical derivatization: Identification of Ran GTPase as a target for S-nitrosylation. Nitric Oxide 11 216-227 (2004).

9 Jaffrey, S.R., Erdjument-Bromage, H., Ferris, C.D., et al. Protein S-nitrosylation: A physiological signal for neuronal nitric oxide. Nat Cell Biol 3(2) 193-197 (2001 Feb 2).

Alderton, W.K., Cooper, C.E., and Knowles, R.G. Nitric oxide synthases: Structure, function, and inhibition. Biochem J 357 593-615 (2001).

Martínez-Ruiz, A., and Lamas, S. S-nitrosylation: A potential new paradigm in signal transduction. Cardiovasc Res 62 43-52 (2004).

Bredt, D.S. Endogenous nitric oxide synthesis: Biological functions and pathophysiology. Free Radic Res 31 577-596 (1999).

Stamler, J.S., Lamas, S., and Fang, F.C. Nitrosylation: The prototypic redox-based signaling mechanism. Cell 106 675-683 (2001).

Mannick, J.B., and Schonhoff, C.M. Nitrosylation: The next phosphorylation? Arch Biochem Biophys 408 1-6 (2002).

Jaffrey, S.R., Erdjument-Bromage, H., Ferris, C.D., et al. Protein S-nitrosylation: A physiological signal for neuronal nitric oxide. Nat Cell Biol 3(2) 193-197 (2001 Feb 2).

Beckman, J.S., and Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and the ugly. Am J Physiol 271 C1424-C1437 (1996).

Kelm, M. Nitric oxide metabolism and breakdown. Biochim Biophys Acta 1411 273-289 (1999).

Ckless, K., Reynaert, N.L., Taatjes, D.J., et al. In situ detection and visualization of S-nitrosylated proteins following chemical derivatization: Identification of Ran GTPase as a target for S-nitrosylation. Nitric Oxide 11 216-227 (2004).

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