In Vitro Alkylation Methods for Assessing the Protein Redox State

  • Flavien Zannini
  • Jérémy Couturier
  • Olivier Keech
  • Nicolas RouhierEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1653)


Cysteines are important residues for protein structure, function, and regulation. Owing to their modified reactivity, some cysteines can undergo very diverse redox posttranslational modifications, including the reversible formation of disulfide bonds, a widespread protein regulatory process as well exemplified in plant chloroplasts for Calvin-Benson cycle enzymes. Both core- and peripheral-photorespiratory enzymes possess conserved cysteines, some of which have been identified as being subject to oxidative modifications. This is not surprising considering their presence in subcellular compartments where the production of reactive species can be important. However, in most cases, the types of modifications and their biochemical effect on protein activity have not been validated, meaning that the possible impact of these modifications in a complex physiological context, such as photorespiration, remains obscure.

We here describe a detailed set of protocols for alkylation methods that have been used so far to (1) study the protein cysteine redox state either in vitro by submitting purified recombinant proteins to reducing/oxidation treatments or in vivo by western blots on protein extracts from plants subject to environmental constraints, and its dependency on the two major reducing systems in the cell, i.e., the thioredoxin and glutathione/glutaredoxin systems, and (2) determine two key redox parameters, i.e., the cysteine pKa and the redox midpoint potential.

Key words

Alkylation Cysteine Oxidative modification pKa Redox potential 



The UMR1136 is supported by a grant overseen by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). The Swedish Research Council “VetenskapsRådet” (grant: 621-2014-4688) as well as the Kempe Foundations are acknowledged for their financial support to O.K.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Flavien Zannini
    • 1
  • Jérémy Couturier
    • 1
  • Olivier Keech
    • 2
  • Nicolas Rouhier
    • 1
    Email author
  1. 1.Faculté des Sciences et Technologies, UMR 1136 Interactions Arbres/MicroorganismesUniversité de Lorraine/INRAVandoeuvre-lès-NancyFrance
  2. 2.Department of Plant Physiology, Umeå Plant Science CentreUmeå UniversityUmeåSweden

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