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Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress.

Publication ,  Journal Article
Forrester, MT; Foster, MW; Stamler, JS
Published in: J Biol Chem
May 11, 2007

Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 11, 2007

Volume

282

Issue

19

Start / End Page

13977 / 13983

Location

United States

Related Subject Headings

  • Sunlight
  • Sulfhydryl Compounds
  • Protein Tyrosine Phosphatases
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Photolysis
  • Oxidative Stress
  • Oxidation-Reduction
  • Nitrogen
  • Nitric Oxide Synthase
  • Nitric Oxide
 

Citation

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Forrester, M. T., Foster, M. W., & Stamler, J. S. (2007). Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress. J Biol Chem, 282(19), 13977–13983. https://doi.org/10.1074/jbc.M609684200
Forrester, Michael T., Matthew W. Foster, and Jonathan S. Stamler. “Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress.J Biol Chem 282, no. 19 (May 11, 2007): 13977–83. https://doi.org/10.1074/jbc.M609684200.
Forrester, Michael T., et al. “Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress.J Biol Chem, vol. 282, no. 19, May 2007, pp. 13977–83. Pubmed, doi:10.1074/jbc.M609684200.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 11, 2007

Volume

282

Issue

19

Start / End Page

13977 / 13983

Location

United States

Related Subject Headings

  • Sunlight
  • Sulfhydryl Compounds
  • Protein Tyrosine Phosphatases
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Photolysis
  • Oxidative Stress
  • Oxidation-Reduction
  • Nitrogen
  • Nitric Oxide Synthase
  • Nitric Oxide