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Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea
极端环境微藻Galdieria phlegrea中还原型和总谷胱甘肽含量的测定   

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Abstract

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Keywords: Microalgae(微藻), Galdieria phlegrea(Galdieria phlegrea), Glutathione(谷胱甘肽), Glutathione reductase(谷胱甘肽还原酶), Redox state(氧化还原状态)

Background

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is an essential tripeptide existing in all known organism, ranging from bacteria to humans (Frendo et al., 2013). It is involved in several different cell protective roles (Figure 1). Glutathione is considered an excellent antioxidant molecule having redox signalling function. It is also involved in response to abiotic and biotic stress, and implicated in the primary metabolism (C, N, S metabolism) of the cell (Noctor et al., 2012; Hernández et al., 2015; Salbitani et al., 2015).

In plant cells, the ratio between the reduced and oxidized forms of glutathione (GSH/GSSG) plays a significant part in signalling and in the activation of numerous defence mechanisms (Foyer and Noctor, 2012; Salbitani et al., 2015). The GSH/GSSG redox state, which is normally tightly controlled, may transiently shift towards a slightly more oxidized value during a stress condition (Tausz et al., 2004).

In the past, some researchers have developed and modified spectrophotometric methods to determine the GSH and GSSG in several organism (Anderson, 1985; Bashir et al., 2013). Here we describe a protocol for a simple glutathione determination, optimized on the extremophilic microalga Galdieria phlegrea. The method illustrated is based on the reaction of GSH with the thiol reagent DTNB (5,5-dithiobis(2-nitrobenzoic acid)) to form GSSG and TNB (5-thionitrobenzoic acid), which is detected spectrophotometrically at 412 nm (Giustarini et al., 2013).


Figure 1. Glutathione redox cycle. Reduced glutathione (GSH) is a tripeptide composed of cysteine, glutamic acid and glycine, which plays a key role in the control of signalling processes, detoxification and various other cell processes. Glutathione disulfide (GSSG) is the oxidized form of glutathione. It is reduced to GSH in presence of NADPH by the glutathione reductase (GR). The glutathione peroxidase (GP) converts hydrogen peroxide to water.

Materials and Reagents

  1. Disposable plastic cuvettes (1.5 ml) (BRAND, catalog number: 759150 )
  2. 5-sulfosalicylic acid hydrate (Sigma-Aldrich, catalog number: 390275 )
  3. L-glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G6013 )
  4. 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) (Sigma-Aldrich, catalog number: D8130 )
  5. β-Nicotinamide adenine dinucleotide phosphate, reduced tetra(cyclohexylammonium) salt (NADPH) (Sigma-Aldrich, catalog number: N5130 )
  6. Glutathione reductase (GR) (Sigma-Aldrich, catalog number: G3664 )
  7. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  8. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  9. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  10. Reaction buffer (see Recipes)

Equipment

  1. Bench centrifuge (Thermo Electron Corporation, model: IEC CL30 )
  2. French pressure cell press (Aminco Resources, model: FA-078 )
  3. Superspeed centrifuge (Thermo Fisher Scientific, model: SorvallTM RC-5C Plus )
  4. Vortex mixer (Troemner, catalog number: TY-LP-945302 )
  5. Spectrophotometer (Cole-Parmer, Jenway, model: 7315 )
  6. pH-meter (Mettler-Toledo International, model: FE20 )
  7. Optical microsccope (leitz laborlux K)
  8. Bϋrker chamber (BRAND, catalog number: 719520 )

Procedure

  1. Preparation of microalgae extracts
    1. Collect 100 ml of culture during the exponential growth phase (OD550 between 0.8 and 2.5) by low speed centrifugation (4,500 x g for 10 min) by Bench centrifuge from 100 ml of algal culture. Discard the supernatant, re-suspend the pellet in 3 ml of 5% (w/v) sulfosalicylic acid and mix by vortexing.
      Note: Sulfosalicilyc acid is added to the pellet for its protective effect on stability of GSH (Stempak et al., 2001) but also for the removal of proteins from samples.
    2. Lyse the cells by passing twice through a French pressure cell (1,100 psi). Centrifuge at 11,000 x g for 20 min at 4 °C by Superspeed centrifuge. Use the resulting supernatant as crude extract (CE) and assay it for the glutathione content.
      Note: Other methods and procedures can be used to lyse Galdieria cells; among the most common methods, there are the use of a magnetic stirrer, microwave radiation, ultrasonication and enzyme treatment (Dvoretskyet al., 2016; Farooq et al., 2016; Huang et al., 2016). The breaking of the cells can be observed with an optical microscope.
    3. Keep the crude extract on ice before use.
      Note: The CE for glutathione determination needs to be used within 2 h after the preparation; in fact, after 2 h and after freezing a drastic reduction in GSH content was observed.
  2. Preparation of the calibration curve
    Quantification of glutathione levels is based on the reduced form of glutathione (GSH). Calibration curve is performed with 0-500 μM standard GSH solution. The molar extinction coefficient (ε), calculated at 412 nm, is estimated to be 0.017 mM-1 cm-1.
  3. Determination of reduced and total glutathione
    1. To determine the reduced glutathione (GSH) content add to a 1.5 ml cuvette 600 µl of reaction buffer, 40 µl of 0.4% (w/v) DTNB, 10-100 µl of CE and Milli-Q water to a final volume of 1,140 µl, and mix gently. The blank solution contained all reagents except CE.
      Note: The DTNB solution can be prepared, aliquoted and stored at -20 °C for six months.
    2. Incubate the cuvettes at room temperature for 5 min and measure the absorbance at 412 nm using a spectrophotometer.
    3. To determine the total glutathione, add to the reaction mix already present in the cuvettes, 50 µl of 0.4% NADPH and 1 µl of GR (0.5 U).
    4. Incubate the cuvettes at room temperature for 30 min. Mix gently and read the absorbance by a spectrophotometer at 412 nm.
      Note: This method has been improved for a cuvette assay but adaption to microplates assays is possible.

Data analysis

  1. The GSSG content was calculated as the difference between the total glutathione and GSH contents.
  2. The thiol levels were expressed as nmol ml-1 extract. The glutathione content in each sample was correlated with the cell number that was determined by Bürker chamber.
  3. The approximate concentrations of GSH, expected in a control culture, should be around 0.8 pmol 10-5 cell-1; in addition, the concentration of total glutathione, in the same extracts, should be around 3.3 pmol 10-5 cell-1.

Recipes

  1. Reaction buffer
    0.1 M Na-phosphate buffer pH 7.00
    1 mM EDTA

Acknowledgments

This protocol was adapted from previously published studies (Anderson, 1985; Bashir et al., 2013). This study was financed by Regione Campania (PON-Smart Generation and LR 5/2002).

References

  1. Anderson, M. E. (1985). Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113: 548-555.
  2. Bashir, H., Ahmad, J., Bagheri, R., Nauman, M. and Irfan Quereshi, M. (2013). Limited sulphur resource forces Arabidopsis thaliana to shift towards non-sulfur tolerance under cadmium stress. Environ Exp Bot 94:19-32.
  3. Dvoretsky, D., Dvoretsky, S., Temnov, M., Akulinin, E., and Peshkova, E. (2016). Enhanced lipid extraction from microalgae Chlorella vulgaris biomass: experiments, modelling, optimization. Chem Eng Trans 49: 175-180.
  4. Farooq, W., Mishra, S. K., Moon, M., Suh, W. I., Shrivastav, A., Kumar, K., Kwon, J. H., Park, M. S., and Mu, Y. (2016). Energy efficient process for microalgae cell disruption for oil recovery using triiodide resin. Algal Res 13: 102-108.
  5. Foyer, C. H. and Noctor, G. (2012). Managing the cellular redox hub in photosynthetic organisms. Plant Cell Environ 35(2): 199-201.
  6. Frendo, P., Baldacci-Cresp, F., Benyamina, S. M. and Puppo, A. (2013). Glutathione and plant response to the biotic environment. Free Radic Biol Med 65: 724-730.
  7. Giustarini, D., Dalle Donne, I., Milzani, A., Fanti, P., Rossi, R. (2013). Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc 8(9): 1660-1669.
  8. Hernández, L. E., Sobrino-Plata, J., Montero-Palmero, M. B., Carrasco-Gil, S., Flores-Cáceres, M. L., Ortega-Villasante, C. and Escobar, C. (2015). Contribution of glutathione to the control of cellular redox homeostasis under toxic metal and metalloid stress. J Exp Bot 66(10): 2901-2911.
  9. Huang, Y., Qin, S., Zhang, D., Li, L., and Mu, Y. (2016). Evaluation of cell disruption of Chlorella vulgaris by pressure-assisted ozonation and ultrasonication. Energies 13: 173-184.
  10. Noctor, G., Mhamdi, A., Chaouch, S., Han, Y., Neukermans, J., Marquez-Garcia, B., Queval, G. and Foyer, C. H. (2012). Glutathione in plants: an integrated overview. Plant Cell Environ 35(2): 454-484.
  11. Salbitani, G., Vona, V., Bottone, C., Petriccione, M. and Carfagna, S. (2015). Sulfur deprivation results in oxidative perturbation in chlorella sorokiniana (211/8k). Plant Cell Physiol 56(5): 897-905.
  12. Stempak, D., Dallas, S., Klein, J., Bendayan, R., Koren, G. and Baruchel, S. (2001). Glutathione stability in whole blood: effect of various deproteinizing acids. Ther Drug Monit 23(5): 542-549.
  13. Tausz, M., Sircelj, H. and Grill, D. (2004). The glutathione system as a stress marker in plant ecophysiology: is a stress-response concept valid? J Exp Bot 55(404): 1955-1962.

简介

谷胱甘肽是涉及所有生物体的初级和次级代谢的重要分子。 谷胱甘肽氧化还原状态是细胞氧化还原状态的指标。 因此,重要的是要有精确的方法来确定细胞中的谷胱甘肽氧化还原状态。 在本协议中,我们描述了一种改进的分光光度法,用于估计极地微藻Galdieria phlegrea中还原(GSH)和氧化(GSSG)形式的谷胱甘肽的含量。
【背景】谷胱甘肽(γ-L-谷氨酰基-L-半胱氨酰 - 甘氨酸)是存在于所有已知生物体中的必需三肽,其范围从细菌到人类(Frendo等人,2013)。它涉及到几种不同的细胞保护作用(图1)。谷胱甘肽被认为是具有氧化还原信号功能的优异的抗氧化分子。它还参与对非生物和生物胁迫的反应,并涉及细胞的初级代谢(C,N,S代谢)(Noctor et al。,2012;Hernándezet al。 ,2015; Salbitani等人,2015)。
 在植物细胞中,还原型和氧化形式的谷胱甘肽(GSH / GSSG)之间的比例在信号传导和许多防御机制的激活中起重要作用(Foyer和Noctor,2012; Salbitani等人, ,2015)。通常严格控制的GSH / GSSG氧化还原状态可能在应力条件下瞬时转向稍微氧化的值(Tausz等人,2004)。
 在过去,一些研究人员已经开发和修改了分光光度法来确定几种生物体中的GSH和GSSG(Anderson,1985; Bashir等人,2013)。在这里,我们描述了一种简单的谷胱甘肽测定方案,优化极值微藻Galdieria phlegrea。所述方法基于GSH与硫醇试剂DTNB(5,5-二硫代双(2-硝基苯甲酸))的反应,形成GSSG和TNB(5-硫代苯甲酸),其在412nm分光光度法检测(Giustarini et al。,2013)。


图1.谷胱甘肽氧化还原循环谷胱甘肽(GSH)是由半胱氨酸,谷氨酸和甘氨酸组成的三肽,其在信号转导,解毒和各种其他细胞过程的控制中起关键作用。谷胱甘肽二硫化物(GSSG)是氧化形式的谷胱甘肽。在谷胱甘肽还原酶(GR)的NADPH存在下被还原成GSH。谷胱甘肽过氧化物酶(GP)将过氧化氢转化为水。

关键字:微藻, Galdieria phlegrea, 谷胱甘肽, 谷胱甘肽还原酶, 氧化还原状态

材料和试剂

  1. 一次性塑料比色杯(1.5ml)(BRAND,目录号:759150)
  2. 5-磺基水杨酸水合物(Sigma-Aldrich,目录号:390275)
  3. L-谷胱甘肽还原(GSH)(Sigma-Aldrich,目录号:G6013)
  4. 5,5-dithiobis(2-硝基苯甲酸)(DTNB,Ellman's试剂)(Sigma-Aldrich,目录号:D8130)
  5. β-烟酰胺腺嘌呤二核苷酸磷酸酯,还原四(环己基铵)盐(NADPH)(Sigma-Aldrich,目录号:N5130)
  6. 谷胱甘肽还原酶(GR)(Sigma-Aldrich,目录号:G3664)
  7. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  8. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  9. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
  10. 反应缓冲液(见配方)

设备

  1. 台式离心机(Thermo Electron Corporation,型号:IEC CL30)
  2. 法国压力机(Aminco Resources,型号:FA-078)
  3. 超速离心机(Thermo Fisher Scientific,型号:Sorvall TM RC / 5C Plus)
  4. 涡街搅拌机(Troemner,目录号:TY-LP-945302)
  5. 分光光度计(Cole-Parmer,Jenway,型号:7315)
  6. pH计(Mettler-Toledo International,型号:FE20)
  7. 光学显微镜(leitz laborlux K)
  8. Bϋ克室(BRAND,目录号:719520)

程序

  1. 微藻提取物的制备
    1. 通过Bench离心机通过低速离心(4,500×g×10分钟)在指数生长期(OD值550和0.8之间)收集100ml培养物,从100ml的藻类文化。丢弃上清液,将沉淀重新悬浮在3ml 5%(w / v)磺基水杨酸中,并通过涡旋混合。
      注意:将磺基水杨酸添加到颗粒中以对GSH的稳定性(Stempak等人,2001)的保护作用,还用于从样品中去除蛋白质。
    2. 通过两次通过法国压力池(1,100psi)使细胞裂解。通过Superspeed离心机在4℃下以11,000 x g离心20分钟。使用所得上清液作为粗提物(CE),并测定其为谷胱甘肽含量。
      注意:其他方法和程序可用于裂解Galdieria细胞;在最常见的方法中,使用磁力搅拌器,微波辐射,超声波和酶处理(Dvoretskyet等,2016; Farooq等,2016; Huang等,2016)。可以用光学显微镜观察细胞的断裂。
    3. 使用前将粗提物放在冰上。
      注意:需要在准备2小时内使用谷胱甘肽测定的CE;事实上,在2小时后,在冷冻后,观察到GSH含量急剧下降。
  2. 校准曲线的准备
    谷胱甘肽水平的定量是基于还原形式的谷胱甘肽(GSH)。校准曲线用0-500μM标准GSH溶液进行。在412nm处计算出的摩尔消光系数(ε)估计为0.017mM -1 -1.0
  3. 还原和总谷胱甘肽的测定
    1. 为了测定还原型谷胱甘肽(GSH)含量,加入1.5ml比色皿600μl反应缓冲液,40μl0.4%(w / v)DTNB,10-100μlCE和Milli-Q水,终体积为1,140 μl,轻轻混匀。空白溶液中含有除CE以外的所有试剂。
      注意:DTNB溶液可以制备,等分并储存在-20°C六个月。
    2. 在室温下孵育反应杯5分钟,并使用分光光度计测量412nm处的吸光度
    3. 为了确定总谷胱甘肽,加入已经存在于比色皿中的反应混合物,50μl的0.4%NADPH和1μl的GR(0.5U)。
    4. 在室温下孵育反应杯30分钟。轻轻混合,用分光光度计在412 nm读取吸光度 注意:对于比色皿测定法已经改进了该方法,但是适应微孔板测定是可能的。

数据分析

  1. GSSG含量计算为总谷胱甘肽与GSH含量之间的差异。
  2. 硫醇水平用nmol ml -1提取物表达。每个样品中的谷胱甘肽含量与通过Bürker腔确定的细胞数相关
  3. 在对照培养物中预期的GSH的近似浓度应为约0.8pmol 10℃-5细胞 -1。此外,同一提取物中的总谷胱甘肽的浓度应为约3.3 pmol 10至-5细胞 -1

食谱

  1. 反应缓冲液
    0.1M磷酸钠缓冲液pH 7.00
    1 mM EDTA

致谢

该协议是从先前发表的研究(Anderson,1985; Bashir等人,2013)中改编的。本研究由Regione Campania(PON-Smart Generation和LR 5/2002)资助。

参考

  1. Anderson,ME(1985)。  测定谷胱甘肽和谷胱甘肽二硫化物在生物样品中。方法Enzymol 113:548-555。
  2. Bashir,H.,Ahmad,J.,Bagheri,R.,Nauman,M.和Irfan Quereshi,M.(2013)。&nbsp; Environ Exp Bot < / em> 94:19-32。
  3. Dvoretsky,D.,Dvoretsky,S.,Temnov,M.,Akulinin,E.和Peshkova,E。(2016)。从微藻增强的脂质提取生物小球藻生物质:实验,建模,优化。 Trans 49:175-180。
  4. Farooq,W.,Mishra,SK,Moon,M.,Suh,WI,Shrivastav,A.,Kumar,K.,Kwon,JH,Park,MS和Mu,Y。(2016)。使用三碘化物树脂进行油回收的微藻细胞破坏的节能过程。 > Algal Res 13:102-108。
  5. Foyer,CH and Noctor,G.(2012)。&nbsp; 管理光合生物体内的细胞氧化还原途径。植物细胞环境35(2):199-201。
  6. Frendo,P.,Baldacci-Cresp,F.,Benyamina,SM and Puppo,A.(2013)。&nbsp; 谷胱甘肽和植物对生物环境的反应。免费Radic Biol Med 65:724-730。
  7. Giustarini,D.,Dalle Donne,I.,Milzani,A.,Fanti,P.,Rossi,R。(2013)。&nbsp; 使用N-乙基马来酰亚胺衍生化后的GSH和GSSG分析。Nat Protoc 8(9):1660-1669 。
  8. Hernández,LE,Sobrino-Plata,J.,Montero-Palmero,MB,Carrasco-Gil,S.,Flores-Cáceres,ML,Ortega-Villasante,C。和Escobar,C。(2015)。谷胱甘肽在有毒金属和准金属应激下控制细胞氧化还原稳态的贡献。 J Exp Bot 66(10):2901-2911。
  9. Huang,Y.,Qin,S.,Zhang,D.,Li,L.,and Mu,Y.(2016)。&nbsp; 细胞的细胞破坏。 / em> 13:173-184。
  10. No。J,Mhamdi,A.,Chaouch,S.,Han,Y.,Neukermans,J.,Marquez-Garcia,B.,Queval,G。和Foyer,CH(2012) “ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/21777251”target =“_ blank”>植物中的谷胱甘肽:综合概述植物细胞环境 35(2):454-484。
  11. Salbitani,G.,Vona,V.,Bottone,C.,Petriccione,M.和Carfagna,S。(2015)。硫剥夺导致小球藻sorokiniana(211 / 8k)中的氧化微扰植物细胞生理学56(5): 897-905。
  12. Stempak,D.,Dallas,S.,Klein,J.,Bendayan,R.,Koren,G.and Baruchel,S。(2001)。全血中的谷胱甘肽稳定性:各种脱蛋白酸的作用。 Ther Drug Monit 23(5):542-549。
  13. Tausz,M.,Sircelj,H.和Grill,D。(2004)。谷胱甘肽系统作为植物生态生理学中的应激标记:是一种应激反应概念有效的? 55(404):1955-1962。 />
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引用:Salbitani, G., Bottone, C. and Carfagna, S. (2017). Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea. Bio-protocol 7(13): e2372. DOI: 10.21769/BioProtoc.2372.
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