Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803
蓝藻 Synechocystis PCC 6803中抗坏血酸和谷胱甘肽含量的测定
(*contributed equally to this work) 发布: 2020年10月20日第10卷第20期 DOI: 10.21769/BioProtoc.3800 浏览次数: 4139
评审: Samik BhattacharyaDhiraj Kumar ChaudharyAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Nov 2019
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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 (蓝藻)
Antioxidants (抗氧化剂)
Glutathione (谷胱甘肽)
Ascorbic acid (抗坏血酸)
Oxidative stress (氧化应激)
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
- Pipette tips: 0.1-10 µl, 1-200 µl and 100-1,000 µl
- 1.5 ml microcentrifuge tubes
- 50 ml conical centrifuge tubes
- Sterile syringes: 1 ml and 10 ml
- Sterile acid-washed glass beads (150-212 µm) (Sigma-Aldrich, catalog number: G1145 )
- 0.22 µm nylon menbrane filters (e.g., GVS, catalog number: FJ13BNPNY002AD01 )
- Synechocystis PCC 6803 liquid cultures
- Ice
- Liquid nitrogen
- Sterile Mili-Q water
- Water for HPLC (Sigma-Aldrich, catalog number 900682)
- Bond Elut C18 cartridges (Agilent Technologies, catalog number: 12102028 )
- TFA (Trifluoroacetic acid) (Carlo Erba, catalog number: 411564 )
- DTT (Dithiothreitol) (Sigma-Aldrich, catalog number: D0632 )
- Methanol HPLC-grade (Sigma-Adrich, catalog number: 34860 )
- Phosphoric acid for HPLC (85%) (Sigma-Adrich, catalog number: 49685 )
- Potassium phosphate dibasic (Sigma-Aldrich, catalog number: P8281 )
- Potassium dihydrogen phosphate anhydrous (Sigma-Aldrich, catalog number: 543841 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 431788 )
- NADPH (β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate ≥93% (HPLC) (Sigma-Aldrich, catalog number: N1630 )
- DTNB (5,5′-Dithiobis(2-nitrobenzoic acid)) (Sigma-Aldrich, catalog number: D8130 )
- GSSG (L-Glutathione oxidized) (Sigma-Aldrich, catalog number: G4376 )
- Glutathione Reductase from baker's yeast (S. cerevisiae) (Sigma-Aldrich, catalog number: G3664 )
- 4-Vinylpyridine (Sigma-Aldrich, catalog number: V3204 )
- L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
- L-Ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
- Neubauer counting chamber (BrandTM BlaubrandTM, catalog number: 10350141 )
- Phosphate buffer 100 mM pH7 (see Recipies)
- Phosphate buffer 100 mM pH 7.5 + EDTA 5 mM (see Recipies)
Equipment
- Pipettes
- Neubauer counting chamber
- Refrigerated centrifuge (50 ml conical tubes: Thermo ScientificTM, model: IECTM CL31 ; 1.5 ml tubes: Thermo ScientificTM, model: SorvallTM LegendTM Micro 17R )
- Vortex mixer (Scientific Industries, model: VORTEX-GENIE 2 )
- Spectrophotometer (Shimadzu, model: UV-160A-Vis )
- pH-meter (Milwaukee, model: MI-150, catalogue number: 11861 )
- HPLC system (Shimadzu Scientific Instruments) equipped with:
Software
- Microsoft Excel (Microsoft Corporation, Available from: https://office.microsoft.com/excel)
Procedure
文章信息
版权信息
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Aguillera, A., Steelheart, C., Alegre, M., Berdun, F., Salerno, G., Bartoli, C., Pagnussat, G. C. and Martin, M. V. (2020). Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803. Bio-protocol 10(20): e3800. DOI: 10.21769/BioProtoc.3800.
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分类
微生物学 > 微生物信号传导 > 第二信使
生物化学 > 蛋白质 > 定量
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