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  • Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803.

Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803.

Bio-protocol (2021-03-05)
Anabella Aguilera, Charlotte Steelheart, Matías Alegre, Federico Berdun, Graciela Salerno, Carlos Bartoli, Gabriela Pagnussat, María Victoria Martin
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Dithiothreitol, ≥98% (HPLC), ≥99.0% (titration)
Sigma-Aldrich
5,5′-Dithiobis(2-nitrobenzoic acid), ≥98%, BioReagent, suitable for determination of sulfhydryl groups
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, ≥93% (HPLC)
Glass beads, acid-washed, 150-212 μm (70-100 U.S. sieve)
Sigma-Aldrich
L-Glutathione oxidized, ≥98% (HPLC)
Sigma-Aldrich
Glutathione Reductase from baker's yeast (S. cerevisiae), ammonium sulfate suspension, 100-300 units/mg protein (biuret)
Sigma-Aldrich
L-Ascorbic acid, BioXtra, ≥99.0%, crystalline
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%