Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10739321 | Free Radical Biology and Medicine | 2005 | 8 Pages |
Abstract
S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.
Keywords
S-nitrosoglutathioneCysNOEF-2S-nitroso-N-acetylpenicillamineMMTSS-nitrosocysteine2-MEBAECsS-nitrosothiolsN-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamideBiotin-HPDPDTPAGSNOECLSDSHBSSMALDI-TOFN-ethylmaleimide2-mercaptoethanolAscorbateSDS-polyacrylamide gel electrophoresisSDS-PAGEenhanced chemiluminescenceDiethylenetriamine pentaacetic acidFree radicalsmatrix-assisted laser desorption ionization-time of flightsodium dodecyl sulfateBovine aortic endothelial cellsSNAPElongation factor 2methyl methanethiosulfonateHank’s balanced salt solutionNEMNitric oxideProteomics
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Authors
Yanhong Zhang, Agnes Keszler, Katarzyna A. Broniowska, Neil Hogg,