Abstract
Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 μM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803.
Keywords: Cyanobacteria, Antioxidants, Glutathione, Ascorbic acid, Oxidative stress
Background
The redox status in the cell can be altered by multiple factors generating oxidative stress. We used this protocol to quantify GSH and AsA contents in Synechocystis sp. PCC 6803 exposed to heat (50 °C). As described for Arabidopsis thaliana, heat stress resulted in a decline in the content of both antioxidants and triggered cell death by ferroptosis (Distéfano et al., 2017; Aguilera et al., 2019 preprint). Although this protocol was set for Synechocystis sp. PCC 6803, it can be applied to determine GSH and AsA contents in other cyanobacteria. In cyanobacteria AsA contents are very low, uM range in normal conditions and pM under some treatments. For that reason we propose this senstitive method with an improved cell lysate procedure.
Materials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
This research was funded by grants to M.V. Martin from Agencia Nacional de Promoción Científica y Técnica Argentina (PICT 1956 and PICT 0173) and to G.C. Pagnussat from Agencia Nacional de Promoción Científica y Técnica Argentina (PICT-2017-0201). This protocol was adapted from previously published studies (Griffith, 1980; Bartoli et al., 2006; Narainsamy et al., 2016).
Competing interests
The authors declare no competing financial interests.
References
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