Nitric Oxide pp 281-297 | Cite as

Thioredoxin-Dependent Decomposition of Protein S-Nitrosothiols

  • Sophie Kneeshaw
  • Steven H. Spoel
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)


The addition of nitric oxide to cysteine moieties of proteins results in the formation of S-nitrosothiols (SNO) that have emerged as important posttranslational signaling cues in a wide variety of eukaryotic processes. While formation of protein-SNO is largely nonenzymatic, the conserved family of Thioredoxin (TRX) enzymes are capable of selectively reducing protein-SNO. Consequently, TRX enzymes are thought to provide reversibility and specificity to protein-SNO signaling networks. Here, we describe an in vitro methodology based on enzymatic oxidoreductase and biotin-switch techniques, allowing for the detection of protein-SNO targets of TRX enzymes. We show that this methodology identifies both global and specific protein-SNO targets of TRX in plant cell extracts.

Key words

Biotin switch technique S-nitrosylation Denitrosylation Thioredoxin Protein-SNO reductase Nitric oxide 



This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 678511), a Royal Society University Research Fellowship (UF090321), and a Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISSF).


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Molecular Plant Sciences, School of Biological SciencesUniversity of EdinburghEdinburghUK

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