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Nov 2019

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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   

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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

  1. Pipette tips: 0.1-10 µl, 1-200 µl and 100-1,000 µl
  2. 1.5 ml microcentrifuge tubes
  3. 50 ml conical centrifuge tubes
  4. Sterile syringes: 1 ml and 10 ml
  5. Sterile acid-washed glass beads (150-212 µm) (Sigma-Aldrich, catalog number: G1145 )
  6. 0.22 µm nylon menbrane filters (e.g., GVS, catalog number: FJ13BNPNY002AD01 )
  7. Synechocystis PCC 6803 liquid cultures
  8. Ice
  9. Liquid nitrogen
  10. Sterile Mili-Q water
  11. Water for HPLC (Sigma-Aldrich, catalog number 900682)
  12. Bond Elut C18 cartridges (Agilent Technologies, catalog number: 12102028 )
  13. TFA (Trifluoroacetic acid) (Carlo Erba, catalog number: 411564 )
  14. DTT (Dithiothreitol) (Sigma-Aldrich, catalog number: D0632 )
  15. Methanol HPLC-grade (Sigma-Adrich, catalog number: 34860
  16. Phosphoric acid for HPLC (85%) (Sigma-Adrich, catalog number: 49685 )
  17. Potassium phosphate dibasic (Sigma-Aldrich, catalog number: P8281 )
  18. Potassium dihydrogen phosphate anhydrous (Sigma-Aldrich, catalog number: 543841 )
  19. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 431788 )
  20. NADPH (β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate ≥93% (HPLC) (Sigma-Aldrich, catalog number: N1630 )
  21. DTNB (5,5′-Dithiobis(2-nitrobenzoic acid)) (Sigma-Aldrich, catalog number: D8130 )
  22. GSSG (L-Glutathione oxidized) (Sigma-Aldrich, catalog number: G4376 )
  23. Glutathione Reductase from baker's yeast (S. cerevisiae) (Sigma-Aldrich, catalog number: G3664 )
  24. 4-Vinylpyridine (Sigma-Aldrich, catalog number: V3204 )
  25. L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  26. L-Ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
  27. Neubauer counting chamber (BrandTM BlaubrandTM, catalog number: 10350141 )
  28. Phosphate buffer 100 mM pH7 (see Recipies)
  29. Phosphate buffer 100 mM pH 7.5 + EDTA 5 mM (see Recipies)

Equipment

  1. Pipettes
  2. Neubauer counting chamber
  3. Refrigerated centrifuge (50 ml conical tubes: Thermo ScientificTM, model: IECTM CL31 ; 1.5 ml tubes: Thermo ScientificTM, model: SorvallTM LegendTM Micro 17R )
  4. Vortex mixer (Scientific Industries, model: VORTEX-GENIE 2 )
  5. Spectrophotometer (Shimadzu, model: UV-160A-Vis )
  6. pH-meter (Milwaukee, model: MI-150, catalogue number: 11861 )
  7. HPLC system (Shimadzu Scientific Instruments) equipped with:
    1. LC pump (Shimadzu Scientific Instruments, model: LC-10 AT )
    2. UV-VIS detector (Shimadzu Scientific Instruments, model: SPD-10AV )
    3. Microsphere C-18 column (Agilent technologies, SS 100 x 4.6 mm, 3 μm; catalog number: CP28076 )

Software

  1. Microsoft Excel (Microsoft Corporation, Available from: https://office.microsoft.com/excel)

Procedure

  1. Cell harvest and antioxidants extraction
    1. Place fifty milliliters of exponentially-growing culture (~2.5 × 107 cells ml-1) in 50 ml conical centrifuge tubes.
    2. Harvest cells by centrifugation (9,300 × g for 10 min, 4 °C).
    3. Discard the supernatant and place the pellet with a sterile spatula in a new and sterile 1.5 ml microcentrifuge tube.
    4. Place the tube on ice. Resuspend cells in 1 ml 3% TFA and add approximately 100 μl of sterile acid-washed glass beads (150-212 µm).
    5. Lyse the cells by mixing vigorously (vortex, 1 min), freeze with liquid nitrogen and thaw in ice.
      Note: Broken cells can be observed under an optical microscope, in order to ensure a correct rupture of the cells.
    6. Repeat Step A5 at least 3 times.
    7. Centrifuge the homogenate (16,000 × g 15 min, 4 °C).
    8. Collect the supernatant in a new 1.5 ml microcentrifuge tube and keep it on ice before use.
    9. Activate Bond Elut C18 columns by applying in the following order:
      1. 1.5 ml methanol.
      2. 1.0 ml water for HPLC.
      3. 1.0 ml phosphate buffer 100 mM pH 7.00.
      Discard the solution from the washing steps.
    10. Pass 500 μl of the supernantant (Step A8) through a previously activated Bond Elut C18 cartridge using a siringe.
    11. Discard flow-through (500 μl) and pass 1.5 ml phosphate buffer 100 mM pH 7.00 to elute (pH of the elution around 2). Collect the filtrate in a new 1.5 ml microcentrifuge tube and mix thoroughly by vortexing for 10-20 s.
      This filtrate is used for ascorbic acid and glutation determination.
      Note: It is highly recommended to perform the measurements inmediately after obtaining the filtrate. In particular, ascorbic acid determination needs to be done after the preparation. Freezing the samples is not recommended because after thawing a drastic reduction in the antioxidant content is observed. For glutathione measurements, the supernatant can be stored at -80 °C.

  2. Reduced ascorbic acid and total ascorbate measurements
    1. To determine reduced ascorbic acid mix in a new 1.5 ml microcentrifuge tube:
      240 μl of the filtrate obtained in Step A10
      240 μl of K2HPO4 100 mM pH 8.5
      50 µl of TFA 10%
      15 µl of water for HPLC
    2. To determine total ascorbate mix in a new 1.5 ml microcentrifuge tube:
      240 μl of the filtrate obtained in Step A10
      240 μl of K2HPO4 100 mM pH 8.5
      15 µl of DTT 100 mM (final concentration: 3 mM)
      Incubate for 5 min at at room temperatur and stop the reaction by adding 50 µl of TFA 10%.

  3. Reduced ascorbic acid and total ascorbate quantification
    1. To quantify the reduced ascorbic acid concentration, filter the solution obtained in Step B1 through a 0.22 µm nylon menbrane filter and inject 10 μl in a HPLC equipped with and a C-18 column (100 × 4.6 mm, 3 μM). As mobile phase use a solution of KH2PO4 100 mM (pH 3.00) in water for HPLC (adjust pH with phosphoric acid 85%) at 0.35 and 0. 5 ml min-1 flow.
    2. Quantify the area under the peak that displays a maximum at 265 nm (Figure 1).
    3. To quantify total ascorbate concentration filter and inject 10 μl of the second solution obtained in step B2 using the same equipment and conditions described in Steps C1 and C2.
    4. Prepare a calibration curve using a series of standard L-ascorbic acid dilutions (e.g., final concentrations of 0.1, 0.5 and 1 μM) and measure the peak area following the same procedure as described in Step C2 (Figure 1).
    5. Adjust the linear function of ascorbic acid concentration vs. peak area and use the slope to calculate the concentration of the unknown samples as described in the formula:



      The x2 is included in the formula because cells are resuspended in 1 ml TFA (Step A4) but only half of the supernatant is passed trought the column (Step A9). The x3 is included because in Step A10 one volume of sample is passed through the column (500 μl) and three volumes are collected (1.5 ml).


      Figure 1. Area under the peak and standard curve to Asa quantification

  4. Measurement of glutathione content
    The quantitative determination of the total amount of glutathione (GSH + GSSG) employs an enzymatic method. In this, the reduction of DTNB to 5-thio-2-Nitrobenzoato (TNB) by NADPH is quantified spectrophotometrically. This reaction is used to measure the reduction of GSSG to GSH. The rate of the reaction is proportional to the GSH and GSSG concentration.
    1. To determine the total glutathione, in a new 1.5 ml microcentrifuge tube add:
      650 µl phosphate buffer 100 mM pH 7.5 + EDTA 5 mM
      25 µl DNTB (5 mg/ml) (final concentration 0.2 mM)
      5 µl NADPH (1 mg/100 µl)
      5 µl l glutathione reductase 1:30 (0.5 U)
      300 µl of filtrate obtained in Step A10
      Note: The blank solution contained all reagents except the filtrate obtained in Step A10.
    2. Mix gently and read the absorbance at 412 nm using a spectrophotometer. 
    3. To extract the oxidized gluthathion (GSSG), add 3 µl of Vinylpyridine (95%) to 300 µl of the filtrate obtained in Step A10, gently mix by vortexing and incubate at room temperature for 25 min.
    4. Centrifuge (16,000 × g 10 min, 4 °C) and place the tube on ice
    5. In a new 1.5 ml microcentrifuge tube add
      365 µl phosphate buffer 100 mM pH 7.5 + EDTA 5 mM.
      25 µl DNTB (5 mg/ml).
      5 µl NADPH (1 mg/100 μl)
      5 µl l glutathione reductase 1:30 (0.5 U)
      100 µl of the supernatant obtained in Step D4
    6. Mix gently and read the absorbance at 412 nm.
    7. Quantification of glutathione levels is based on the oxidized form of glutathione (GSSG). To quantify the glutathione levels, prepare a calibration curve with 0-100 µM standard L-Glutathione oxidized solution.

    Note: The content of the antioxidant can be expressed either as per mg of fresh or dry weight or as pmol per cell (in the case of of ascorbic acid) and nmol per cell (in the case of glutathione). The content of antioxidants can be correlated with cell number determined with a Neubauer counting chamber.

Recipes

  1. Phosphate buffer 100 mM pH 7
    Add 1.071g K2HPO4 and 0.524 g KH2PO4 in 100 ml Sterile Mili-Q water
  2. Phosphate buffer 100 mM pH 7.5 + EDTA 5 mM
    Solution 1: mix 0.681 g of KH2PO4 and 0.093 g of EDTA in 50 ml sterile Mili-Q water
    Solution 2: mix 0.871 g of K2HPO4 and 0.093 g of EDTA in 50ml sterile Mili-Q water
    Adjuts the pH of solution 2 by adding solution 1 till reach pH 7.5

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

  1. Aguilera, A., Berdun, F., Bartoli, C., Steelheart, C., Alegre, M., Salerno, G., Pagnussat, G. and Martin, M. V. (2019). Heat stress induces ferroptosis in a photosynthetic prokaryote. bioRxiv: 828293.
  2. Bartoli, C. G., Yu, J., Gómez, F., Fernández, L., McIntosh, L. and Foyer, C. H. (2006). Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves. J Exp Bot 57(8): 1621-1631. 
  3. Distéfano, A. M., Martin, M. V., Córdoba, J. P., Bellido, A. M., D'Ippólito, S., Colman, S. L., Soto, D., Roldán, J. A., Bartoli, C. G., Zabaleta, E. J., Fiol, D. F., Stockwell, B. R., Dixon, S. J. and Pagnussat, G. C. (2017). Heat stress induces ferroptosis-like cell death in plants. J Cell Biol 216(2): 463-476. 
  4. Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106(1): 207-212. 
  5. Narainsamy, K., Farci, S., Braun, E., Junot, C., Cassier-Chauvat, C. and Chauvat, F. (2016). Oxidative-stress detoxification and signalling in cyanobacteria: the crucial glutathione synthesis pathway supports the production of ergothioneine and ophthalmate. Mol Microbiol 100(1): 15-24.

简介

[摘要] 抗坏血酸(AsA)和明胶硫(GSH)是真核细胞和原核细胞抗氧化机制的两个重要组成部分。蓝藻Synechocystis sp.pcc6803以不同浓度(AsA,20-100μM和GSH,2-5mm)呈现这两种化合物。因此,有精确和敏感的方法来确定细胞中的氧化还原状态和检测这种抗氧化剂的变化是很重要的。在该方案中,我们描述了一种改进的方法,用以从Synechocystis sp.pcc6803的液体培养中获得的同一样品中估算两种抗氧化剂(以还原形式和氧化形式)的含量。

[背景] 细胞中的氧化还原状态可以被多种因素改变,产生氧化应激。我们使用该方案来量化暴露于50℃高温下的Synechocystis sp. PCC 6803中的GSH和AsA含量。如拟南芥所述,热胁迫导致抗氧化剂含量下降,并通过铁作用引起细胞死亡(Distefano et al., 2017;Aguilera等人,2019年预印本)。虽然本方案是针对Synechocystis sp. PCC 6803制定的,但也可用于测定其他蓝藻中GSH和AsA的含量。蓝藻中AsA含量很低,正常条件下uM含量在一定范围内,某些处理下pM含量在一定范围内。因此,我们提出了这个敏感的方法与一个改进的细胞裂解程序。

关键字:蓝藻, 抗氧化剂, 谷胱甘肽, 抗坏血酸, 氧化应激

材料和试剂
 
1.      移液管尖端:0.1-10µl、1-200µl和100-1000µl
2.     1.5 ml微量离心管
3.     50毫升锥形离心管
4.     无菌注射器:1ml和10ml
5.     无菌酸洗玻璃珠(150-212µm)(Sigma-Aldrich,目录号:G1145)
6.     0.22µm尼龙薄膜过滤器(例如,GVS,目录号:FJ13BNPNY002AD01)
7.     联合囊肿PCC 6803液体培养
8.     冰
9.     液氮
10.  无菌米利-Q水
11.  HPLC用水(Sigma-Aldrich,目录号900682)
12.  键洗脱C18卡式瓶(安捷伦技术公司,目录号:12102028)
13.  三氟乙酸(三氟乙酸)(Carlo Erba,目录号:411564)
14.  DTT(二硫苏糖醇)(Sigma-Aldrich,目录号:D0632)
15.  甲醇HPLC等级(Sigma Adrich,目录号:34860)
16.  HPLC用磷酸(85%)(Sigma Adrich,目录号:49685)
17.  磷酸二氢钾(Sigma-Aldrich,目录号:P8281)
18.  无水磷酸二氢钾(Sigma-Aldrich,目录号:543841)
19.  乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:431788)
20.  NADPH(β-烟酰胺腺嘌呤二核苷酸2′-磷酸还原四钠盐水合物≥93%(HPLC)(Sigma-Aldrich,目录号:N1630)
21.  DTNB(5,5′-二硫代(2-硝基苯甲酸))(Sigma-Aldrich,目录号:D8130)
22.  GSSG(L-谷胱甘肽氧化)(Sigma-Aldrich,目录号:G4376)
23.  来自面包酵母(酿酒酵母)的谷胱甘肽还原酶(Sigma-Aldrich,目录号:G3664)
24.  4-乙烯基吡啶(Sigma-Aldrich,目录号:V3204)
25.  还原型L-谷胱甘肽(Sigma-Aldrich,目录号:G4251)
26.  L-抗坏血酸(Sigma-Aldrich,目录号:A5960)
27.  Neubauer计数室(BrandTM-BlaubrandTM,目录号:10350141)
28.  磷酸盐缓冲液100 mM pH7(见回收物)
29.  磷酸盐缓冲液100 mM pH值7.5+EDTA 5 mM(见配方)
 
设备
 
1.     微量加样器
2.     纽鲍尔计数室
3.     冷冻离心机(50毫升锥形管:Thermo ScientificTM,型号:IECTM CL31;1.5毫升试管:Thermo ScientificTM,型号:SorvallTM LegendTM Micro 17R)
4.     涡流混合器(科学工业,型号:Vortex-GENIE 2)
5.     分光光度计(岛津,型号:UV-160A-Vis)
6.     型号:118PH,密尔沃基,型号:118PH
7.     HPLC系统(岛津科学仪器)配备:
a、 LC泵(岛津科学仪器,型号:LC-10AT)
b、 紫外可见探测器(岛津科学仪器,型号:SPD-10AV)
c、 微球c-18柱(安捷伦科技,SS 100 x 4.6 mm,3 m;目录号:CP28076)μ
 
软件
 
1.     Microsoft Excel(微软公司,可从:https://office.microsoft.com/excel)
 
程序
 
A、 细胞收获和抗氧化剂提取
1.     将50毫升指数生长培养物(~2.5×107细胞ml-1)放入50 ml锥形离心管中。
2.     通过离心法(9300×g,4°C)收获细胞。
3.     弃去上清液,用无菌抹刀将小球放入新的无菌1.5毫升微型离心管中。
4.     把管子放在冰上。在1 ml 3%TFA中再悬浮细胞,并添加约100μl无菌酸洗玻璃珠(150-212µm)。
5.     通过剧烈混合(漩涡,1分钟)溶解细胞,用液氮冷冻并在冰中解冻。
注意:可以在光学显微镜下观察破裂的细胞,以确保细胞正确破裂。
6.     重复步骤A5至少3次。
7.     离心匀浆(16000×g 15min,4°C)。
8.     将上清液收集在一个新的1.5毫升微型离心管中,并在使用前将其保存在冰上。
9.     按以下顺序应用,激活键洗脱C18柱:
a、 1.5毫升甲醇。
b、 1.0 ml水用于HPLC。
c、 1.0毫升磷酸盐缓冲液100毫米pH 7.00。
丢弃洗涤步骤中的溶液。
10.  使用siringe将500μl助溶剂(步骤A8)通过先前激活的键洗脱C18盒。
11.  丢弃流动液(500μl),通过1.5 ml磷酸盐缓冲液100 mM pH7.00洗脱(洗脱液的pH值约为2)。将滤液收集在一个新的1.5毫升微型离心管中,通过旋转10-20秒使其充分混合。
该滤液用于抗坏血酸和谷胱甘肽的测定。
注:强烈建议在获得过滤。尤其是抗坏血酸的测定需在制剂完成后进行。不建议冷冻样品,因为解冻后可观察到抗氧化剂含量急剧下降。为谷胱甘肽测量,上清液可储存在-80°C。
 
B、 还原抗坏血酸和总抗坏血酸测量
1.     在新的1.5 ml微型离心管中测定还原抗坏血酸混合物:
240 步骤A10中获得的滤液μl
240 μl K2HPO4 100 mM pH 8.5
50µl TFA 10%
15µl水用于HPLC
2.     在新的1.5 ml微型离心管中测定总抗坏血酸混合物:
240 步骤得到的滤液为10μl
240 μl K2HPO4 100 mM pH 8.5
15µl DTT 100 mM(最终浓度:3 mM)
在室温下孵育5分钟,加入50µl TFA 10%停止反应。
 
C、 还原抗坏血酸和总抗坏血酸定量
1.     为了定量降低的抗坏血酸浓度,0.22µm尼龙膜过滤并在配备有C-18柱(100×4.6 mm,3μm)的HPLC中注入10μl。作为流动相,使用KH2PO4 100 mM(pH 3.00)的水溶液进行HPLC(用85%的磷酸调节pH值)为0.35和0。5ml min-1流量。过滤步骤B1到a中获得的溶液
2.     量化在265nm处显示最大值的峰下面积(图1)。
3.     用步骤C1和C2中描述的相同设备和条件,量化总抗坏血酸浓度过滤器并注入10μl步骤B2中获得的第二种溶液。
4.     使用一系列标准L-抗坏血酸稀释液(例如,最终浓度为0.1、0.5和1μM)制备校准曲线,并按照步骤C2(图1)中所述的相同程序测量峰面积。
5.     调整抗坏血酸浓度与峰面积的线性函数,用斜率计算未知样品的浓度,公式如下:
 
 
 
x2包含在公式中,因为细胞在1mL TFA中重新悬浮(步骤A4),但只有一半的上清液通过柱(步骤A9)。包括x3,因为在步骤A10中,一个体积的样品通过色谱柱(500μl),收集三个体积的样品(1.5 ml)。
 
 
图1。Asa定量的峰下面积和标准曲线下面积
 
D、 谷胱甘肽含量的测定
谷胱甘肽总量(GSH+GSSG)的定量测定采用酶法。在这篇文章中,用分光光度法定量地研究了用NADPH还原DTNB到5-硫代-2-硝基苯甲酸(TNB)的过程。该反应用于测量GSSG还原为GSH。反应速率与GSH和GSSG浓度成正比。
1.     为了测定总谷胱甘肽,在一个新的1.5毫升微型离心管中加入:
650 µl磷酸盐缓冲液100 mM pH 7.5+EDTA 5 mM
25µl DNTB(5毫克/毫升)(最终浓度0.2毫米)
5µl NADPH(1毫克/100微升)
5微升谷胱甘肽还原酶1:30(0.5 U)
300 步骤A10中获得的滤液微升
注:空白溶液含有除步骤A10中获得的滤液外的所有试剂。
2.     轻轻混合,用分光光度计读取412nm处的吸光度。
3.     为了提取氧化明胶硫磷(GSSG),向步骤A10中获得的300µl滤液中添加3µl乙烯基吡啶(95%),通过涡流轻轻混合并在室温下培养25 min。
4.     离心(16000×g 10min,4°C)并将试管放在冰上
5.     在新的1.5 ml微型离心管中添加
365µl磷酸盐缓冲液100 mM pH 7.5+EDTA 5 mM。
25µl DNTB(5毫克/毫升)。
5µl NADPH(1毫克/100微升)
5微升谷胱甘肽还原酶1:30(0.5 U)
100 µl步骤D4中获得的上清液
6.     轻轻搅拌,读取412nm处的吸光度。
7.     谷胱甘肽水平的量化是基于谷胱甘肽(GSSG)的氧化形式。为了量化谷胱甘肽水平,用0-100µM标准L-谷胱甘肽氧化溶液制备校准曲线。
 
注:抗氧化剂的含量可以表示为每毫克新鲜或干重或作为每细胞pmol(抗坏血酸)和nmol/cell(谷胱甘肽)。抗氧剂的含量与用a纽鲍尔计数室。
 
食谱
 
1磷酸盐缓冲液1磷酸盐缓冲液100毫米pH值7
在100 ml无菌Mili-Q水中添加1.071g K2HPO4和0.524g KH2PO4
2.     磷酸盐缓冲液100 mM pH 7.5+EDTA 5 mM
溶液1:将0.681 g KH2PO4和0.093 g EDTA混合在50 ml无菌Mili-Q水中
溶液2:将0.871g K2HPO4和0.093g EDTA混合在50ml无菌Mili-Q水中
通过添加溶液1来调整溶液2的pH值,直到pH值达到7.5
 
致谢
 
这项研究由阿根廷国家促进发展署(Agencia Nacional de Promoción Cient y Técnica Argentina)(PICT 1956和PICT 0173)向M.V.Martin和阿根廷国家促进发展署(Agencia Nacional de Promoción Cient y Técnica Argentina)(PICT-2017-0201)向G.C.Pagnusat提供了资助。
本方案改编自先前发表的研究(Griffith,1980;Bartoli等人,2006;Narainsamy等人,2016)。
 
相互竞争的利益
 
作者声明没有竞争性的经济利益。
 
工具书类
 
1.     Aguilera,A.,Berdun,F.,Bartoli,C.,Steelheart,C.,Alegre,M.,Salerno,G.,Pagnusat,G.和Martin,M.V.(2019年)。热胁迫诱导光合原核生物的铁下垂。生物XIV:828293。
2.     Bartoli,C.G.,Yu,J.,Gómez,F.,Fernández,L.,McIntosh,L.和Foyer,C.H.(2006年)。拟南芥叶片抗坏血酸合成与积累调控中光与呼吸的相互关系。《出口管理条例》57(8):1621-1631。
3.     Distéfano,A.M.,Martin,M.V.,Córdoba,J.P.,Bellido,A.M.,D'Ippólito,S.,Colman,S.L.,Soto,D.,Roldán,J.A.,Bartoli,C.G.,Zabaleta,E.J.,Fiol,D.F.,Stockwell,B.R.,Dixon,S.J.和Pagnussat,G.C.(2017年)。热胁迫诱导植物铁下垂样细胞死亡。细胞生物学杂志216(2):463-476。
4.     格里菲斯,O.W.(1980年)。用谷胱甘肽还原酶和2-乙烯基吡啶测定谷胱甘肽和谷胱甘肽二硫化物。肛生物化学106(1):207-212。
5.     Narainsamy,K.,Farci,S.,Braun,E.,Junot,C.,Cassier Chauvat,C.和Chauvat,F.(2016年)。蓝藻氧化应激解毒和信号转导:谷胱甘肽合成的关键途径支持麦角碱和眼酸盐的产生。分子微生物学100(1):15-24。
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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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