Quantification and Localization of S-Nitrosothiols (SNOs) in Higher Plants

  • Juan B. Barroso
  • Raquel Valderrama
  • Alfonso Carreras
  • Mounira Chaki
  • Juan C. Begara-Morales
  • Beatriz Sánchez-Calvo
  • Francisco J. Corpas
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)

Abstract

S-nitrosothiols (SNOs) are a family of molecules produced by the reaction of nitric oxide (NO) with –SH thiol groups present in the cysteine residues of proteins and peptides caused by a posttranslational modification (PTM) known as S-nitrosylation (strictly speaking S-nitrosation) that can affect the cellular function of proteins. These molecules are a relatively more stable form of NO and consequently can act as a major intracellular NO reservoir and, in some cases, as a long-distance NO signal. Additionally, SNOs can be transferred between small peptides and protein thiol groups through S-transnitrosylation mechanisms. Thus, detection and cellular localization of SNOs in plant cells can be useful tools to determine how these molecules are modulated under physiological and adverse conditions and to determine their importance as a mechanism for regulating different biochemical pathways. Using a highly sensitive chemiluminescence ozone technique and a specific fluorescence probe (Alexa Fluor 488 Hg-link phenylmercury), the methods described in this chapter enable us to determine SNOs in an nM range as well as their cellular distribution in the tissues of different plant species.

Key words

Chemiluminescence Confocal laser scanning microscope Fluorescent probes Nitric oxide Reactive nitrogen species S-nitrosothiol 
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Notes

Acknowledgments

This work was supported by an ERDF-cofinanced grant from the Ministry of Science and Innovation (BIO2012-33904) and Junta de Andalucía (groups BIO192 and BIO286).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Juan B. Barroso
    • 1
  • Raquel Valderrama
    • 1
  • Alfonso Carreras
    • 1
  • Mounira Chaki
    • 1
  • Juan C. Begara-Morales
    • 1
  • Beatriz Sánchez-Calvo
    • 1
  • Francisco J. Corpas
    • 2
  1. 1.Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular BiologyUniversity of JaénJaénSpain
  2. 2.Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of PlantsEstación Experimental del Zaidín, CSICGranadaSpain

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