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Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803
从蓝藻集胞藻属PCC 6803提取和量化GABA和谷氨酸盐   

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Abstract

GABA (γ-amino butyric acid) is a biologically active four carbon non-protein amino acid found in prokaryotes and eukaryotes. Glutamate is a five carbon α-amino acid which can be converted to GABA catalyzed by the enzyme glutamate decarboxylase. In this protocol, we describe the procedure for extraction and quantification of GABA and glutamate from cells of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Apart from Synechocystis, this protocol has already been successfully tested for the cyanobacterium Nostoc punctiforme and for the green alga Tetraspora sp. CU2551. The protocol can also be used for the analyses of other primary amino acids. We have successfully employed this protocol in our studies of GABA and glutamate analyses in Synechocystis (Kanwal and Incharoensakdi, 2016).

Keywords: GABA assay(GABA法), Glutamate assay(谷氨酸测定), Amino acid analysis(氨基酸分析), Cyanobacteria(蓝藻), Synechocystis(集胞藻)

Background

Amino acid analysis has been done previously by employing a number of methods that include the detection using thin layer chromatography or ion exchange separation coupled with post-column derivatization with ninhydrin method or also using HPLC by fluorescent detection. However, several challenges are encountered in those detection methods such as poor sensitivity, degradation of derivatives, formation of multiple derivatives and longer retention times causing higher solvent consumption. Furthermore, the UV detection methods alone could not be used owing to optically inactive nature of amino acids.

Here, we describe the detailed procedure for extraction and analyses of amino acids such as GABA and glutamate in Synechocystis. The method used for amino acids separation and detection is high performance reversed-phase liquid chromatography followed by UV detection of pre-column OPA (o-phthaldehyde) derivatization of amino acids adopted from Henderson et al. (2006). This method offers several advantages over previous methods by improving the sensitivity of detection. Reversed-phase chromatography allows faster separation of amino acids, whereas UV detection offers improved sensitivity. OPA derivatization of amino acids results in stable derivatives that make the UV detection of amino acids possible. The method also offers the detection and quantification of amino acids within 26 min, which consequently reduces solvent consumption.

Materials and Reagents

  1. 50 ml polypropylene tubes (Corning®, CentristarTM, Mexico)
  2. 1.5 ml Eppendorf tubes (Hycon plastics, catalog number: HCE-1004-MIC )
  3. 200 µl conical inserts with polymer feet (Borosilicate glass) (JG Finneran, USA)
  4. Amber wide-opening vial with screw cap (Borosilicate glass) (JG Finneran, USA)
  5. 0.2 µm Millipore filter (Nylon or cellulose filter membrane) (Sartorius Stedim biotech, Germany)
  6. Synechocystis cells (fresh or frozen at -20 °C)
  7. BG11 growth medium
  8. Deionized water
  9. Methanol, AR grade (RCI Labscan, Thailand)
  10. Ethanol, AR grade (QRec, New Zealand)
  11. Chloroform (RCI Labscan, Thailand)
  12. Hydrochloric acid (HCl) (QRec, New Zealand)
  13. NaH2PO4 (Carlo Erba Reagents, Chaussée du Vexin, Val de Reuil, France)
  14. NaOH (Merck, Germany)
  15. UP water, HPLC grade (TEDIA, OH, USA)
  16. Acetonitrile CAN, HPLC grade (Burdick and Jackson, Korea)
  17. Methanol (MeOH), HPLC grade (Burdick and Jackson, Korea)
  18. Standards of glutamate and GABA, > 99% purity (Sigma-Aldrich)
  19. 0.4 M borate buffer, pH 10.2 (Agilent Technologies, catalog number: 5061-3339 )
  20. O-phthaldehyde reagent (OPA) (Agilent Technologies, catalog number: 5061-3335 )
  21. BG11 growth medium (see Recipes)
  22. HPLC solvent A (see Recipes)
  23. HPLC solvent B (see Recipes)
  24. OPA reagent (see Recipes)

Equipment

  1. Pyrex® 250 ml conical flasks (Corning, Pyrex®, catalog number: 4980 )
  2. Shaker (Eppendorf, model: InnovaTM 2100 )
  3. Tabletop centrifuge (Hettich Lab Technology, model: MIKRO220/220R )
  4. Vortex mixer (Scientific Industries, model: SI-0236 )
  5. Ultrasonic laboratory homogenizer (Sonuplus UW2200)
  6. Centrivap concentrator (Labconco, USA)
  7. Shimadzu prominence ultra-fast liquid chromatography system (Shimadzu Scientific Instruments)
  8. Shimadzu prominence autosampler (Shimadzu Scientific Instruments, model: SIL-21A HT )
  9. Degasser (Shimadzu Scientific Instruments, model: DGU-20A5 )
  10. UV-vis detector (Shimadzu Scientific Instruments, model: SPD-20A )
  11. Reverse phase column (ZORBAX Eclipse AAA analytical column, 4.6 x 150 mm, 5.0 μm) (Agilent Technologies, catalog number: 93400-902 )
  12. Guard column (ZORBAX Eclipse AAA analytical guard column, 4.6 x 12.5 mm, 5.0 μm) (Agilent Technologies, catalog number: 820950-931 )

Procedure

  1. GABA and glutamate extraction
    1. Grow Synechocystis cells, at 30 °C in BG11 medium with a total volume of 50 ml in a cotton-plugged 250 ml conical flask.
    2. Harvest cells at exponential phase of growth (3 to 8 days) by centrifuging at 2,500 x g for 10 min, room temperature. Discard supernatant, resuspend cell pellet in 500 µl of deionized H2O and transfer the cell suspension into a 1.5 ml Eppendorf tube. Spin down the cells at 10,000 x g for 10 min and remove all water by micropipette (at this point, cells can be frozen at -20 °C for up to a month).
    3. Resuspend the cells in 1.5 ml of 80% (v/v) methanol by vigorous mixing. Incubate the mixture for a few hours at room temperature (or incubate overnight) and homogenize using ultrasonic homogenizer for 2 min.
    4. Pellet cell debris by centrifuging at 10,000 x g for 10 min, 4 °C. Collect and transfer the supernatant into a new 1.5 ml Eppendorf tube to obtain the “methanol extract”.
    5. Dry the methanol extract in a vacuum evaporator at 40 °C for 5 h and keep the dried pellet at room temperature (steps A6 and 7 can be omitted, see Note 1 below).
    6. To the cell debris obtained from step A4, add 2 ml of 80% (v/v) ethanol for further re-extraction in the same way to get the “ethanol extract” (see steps A2 and A3).
    7. Add the ethanol extract to previously obtained dried pellet from methanol extract at step A5 and dry in a vacuum evaporator at 40 °C.
    8. Dissolve the pellet obtained after drying with 250 µl of deionized water and 50 µl of chloroform, followed by centrifugation at 10,000 x g for 10 min, 4 °C. Collect the upper aqueous phase (collect sufficient amount of aqueous phase and be careful not to collect the chloroform phase) and transfer into a new 1.5 ml Eppendorf tube.
    9. Add diluted HCl to the aqueous phase to make a final concentration of 0.1 N. Filter the solution through a 0.2 µm Millipore filter before GABA/glutamate detection by HPLC. The filtered sample can be stored at -20 °C until further use.

  2. HPLC analysis of GABA and glutamate
    1. Filter deionized water (or use UP water, HPLC grade), solvent A and solvent B through a 0.2 µm Millipore filter.
    2. The pre-column sample derivatization with OPA reagent is performed as described below.
      1. Transfer 30 µl of filtered sample from step A9 into a 1.5 ml Eppendorf tube and add 5 µl of borate buffer.
      2. Mix thoroughly and incubate for 15 sec at room temperature.
      3. Add 0.5 µl of OPA reagent and mix well (see Note 2).
      4. Transfer the solution into a 200 µl conical insert (Figure 1A). Place the insert in a transparent wide-opening vial (Figure 1B).
      5. Close the vial with screw cap (Figure 1C) and the sample is immediately injected into the HPLC system by an autoinjector.
    3. Separation is performed on a reverse phase column with solvent A and solvent B in 26 min at a flow rate of 2 ml/min at 40 °C (column oven temperature) using a gradient program (Table 1) (see Note 3). The UV-detection wavelength is 338 nm for OPA derivatized samples.
    4. Quantification of GABA and glutamate content is done by comparing the peak areas with those of standards (linearity is obtained for the concentration range of 4.5 to 450 pmol in 0.5 µl of sample). Retention time for glutamate and GABA using these HPLC conditions is 3.0-3.7 min and 10.0-10.5 min respectively (see the Data analysis, Figure 2) (see Note 4).


      Figure 1. A photograph of (A) conical insert, (B) transparent wide-opening vial, and (C) screw cap for GABA and glutamate analyses using the Shimadzu prominence autosampler

      Table 1. HPLC gradient program settings for a single run

Data analysis



Figure 2. Representative chromatograms of separated amino acids extracted from the cyanobacterium Synechocystis sp. PCC 6803. A. Chromatogram of standard glutamate and GABA for the concentration of 2 nmol/ml; B. Chromatogram of amino acid separation from Synechocystis sp. PCC 6803 extract. Data were obtained from Shimadzu LC solution software and were confirmed by three independent experiments.

Notes

  1. The ethanol extraction step can be omitted. However, to ensure the complete quantitative recovery of amino acids, this step is recommended.
  2. The OPA reagent slowly degrades in the presence of oxygen. Inject immediately the freshly derivatized reaction mixture into HPLC.
  3. In case if further cleaning is required for column, the program could be continued by column washing with 100% solvent B for 20-30 min followed by equilibration with buffer (solvent A) for 20-30 min.
  4. To ensure the consistency of HPLC conditions and retention time of standards and sample, it is important to run standards regularly.

Recipes

  1. BG11 growth medium
    Note: Preparation of BG11 growth medium is described in detail in Rippka et al., 1979.
  2. HPLC solvent A
    40 mM NaH2PO4, pH 7.8
    Dissolve 5.5 g NaH2PO4·H2O in 1 L of water
    Adjust pH with 10 N NaOH solution
  3. HPLC solvent B
    ACN:MeOH:water (45:45:10, v/v/v)
  4. OPA reagent
    10 mg/ml o-phthalaldehyde
    10 mg/ml 3-mercaptopropionic acid
    0.4 M borate buffer, pH 10.2

Acknowledgments

The method for GABA and glutamate extraction was adapted from Bieleski and Turner (1966) with some modifications. The HPLC-based method for the detection and quantification of GABA and glutamate is a modification of protocol published by Henderson et al. (2006). This work was supported by research grants from Chulalongkorn University (CU) Ratchadaphiseksomphot Endowment Fund (Food and Water Cluster, CU-58-011-FW) and from the Thailand Research Fund (IRG5780008). Simab Kanwal thanks CU Graduate School for providing post-doctoral fellowship.

References

  1. Bieleski, R. L. and Turner, N. A. (1966). Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal Biochem 17: 278-293.
  2. Henderson, J. W., Ricker, R. D., Bidlingmeyer, B. A. and Woodward, C. (2006). Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. Agilent Application Note 5980-1193EN: 1-10.
  3. Kanwal, S. and Incharoensakdi, A. (2016). Characterization of glutamate decarboxylase from Synechocystis sp. PCC 6803 and its role in nitrogen metabolism. Plant Physiol Biochem 99: 59-65.
  4. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61.

简介

GABA(γ-氨基丁酸)是在原核生物和真核生物中发现的生物活性四碳非蛋白质氨基酸。谷氨酸是一种五碳α-氨基酸,其可以被谷氨酸脱羧酶催化转化为GABA。在该协议中,我们描述了从蓝细菌集胞藻的细胞中提取和定量GABA和谷氨酸的程序。 PCC 6803(以下称为"集胞藻")。除了集胞藻 ,该协议已经成功地测试了蓝细菌点状念珠菌 和绿藻 Tetraspora CU2551。该方案也可用于其他一级氨基酸的分析。我们已经在我们的集胞藻中的GABA和谷氨酸分析研究中成功地使用该方案(Kanwal和Incharoensakdi,2016)。

[背景] 酸分析先前已通过使用许多方法进行,包括使用薄层色谱或离子交换分离偶联的后柱衍生化与茚三酮方法或也使用HPLC通过荧光检测的检测。然而,在那些检测方法中遇到几个挑战,例如差的灵敏度,衍生物的降解,多重衍生物的形成和更长的保留时间,导致更高的溶剂消耗。此外,由于氨基酸的光学非活性,不能单独使用UV检测方法。
  在这里我们描述了提取和分析氨基酸如GABA和谷氨酸在集胞藻中的详细程序。用于氨基酸分离和检测的方法是高性能反相液相色谱,随后是从Henderson等人采用的氨基酸的前柱OPA(邻苯二甲醛)衍生化的UV检测。 (2006)。通过提高检测的灵敏度,该方法提供了优于先前方法的几个优点。反相色谱允许更快地分离氨基酸,而UV检测提供改进的灵敏度。氨基酸的OPA衍生产生稳定的衍生物,其使得氨基酸的UV检测成为可能。该方法还提供在26分钟内氨基酸的检测和定量,因此减少溶剂消耗。

关键字:GABA法, 谷氨酸测定, 氨基酸分析, 蓝藻, 集胞藻

材料和试剂

  1. 50ml聚丙烯管(Corning),,Centristar TM ,墨西哥)
  2. 1.5ml Eppendorf管(Hycon plastics,目录号:HCE-1004-MIC)
  3. 200μl带聚合物脚的锥形插入物(硼硅酸盐玻璃)(JG Finneran,美国)
  4. 带螺帽的琥珀色开口小瓶(Borosilicate glass)(JG Finneran,美国)
  5. 0.2微米Millipore过滤器(尼龙或纤维素过滤膜)(Sartorius Stedim biotech,德国)
  6. 集胞藻细胞(新鲜或冷冻于-20℃)
  7. BG11生长培养基
  8. 去离子水
  9. 甲醇,AR级(RCI Labscan,泰国)
  10. 乙醇,AR级(QRec,新西兰)
  11. 氯仿(RCI Labscan,泰国)
  12. 盐酸(HCl)(QRec,新西兰)
  13. NaH 2 PO 4(Carlo Erba试剂,Chausséedu Vexin,Val de Reuil,法国)
  14. NaOH(Merck,Germany)
  15. UP水,HPLC级(TEDIA,OH,USA)
  16. 乙腈CAN,HPLC级(Burdick和Jackson,Korea)
  17. 甲醇(MeOH),HPLC级(Burdick和Jackson,Korea)
  18. 谷氨酸和GABA的标准, 99%纯度(Sigma-Aldrich)
  19. 0.4M硼酸盐缓冲液,pH10.2(Agilent Technologies,目录号:5061-3339)
  20. O-苯二甲醛试剂(OPA)(Agilent Technologies,目录号:5061-3335)
  21. BG11生长培养基(参见配方)
  22. HPLC溶剂A(参见配方)
  23. HPLC溶剂B(参见配方)
  24. OPA试剂(参见配方)

设备

  1. 250ml锥形瓶(Corning,Pyrex ,目录号:4980)。
  2. Shaker(Eppendorf,型号:Innova TM 2100)
  3. 台式离心机(Hettich Lab Technology,型号:MIKRO220/220R)
  4. 涡旋混合器(Scientific Industries,型号:SI-0236)
  5. 超声实验室匀浆器(Sonuplus UW2200)
  6. Centrivap浓缩器(Labconco,美国)
  7. Shimadzu prominence超高速液相色谱系统(Shimadzu Scientific Instruments)
  8. 岛津突出物自动进样器(Shimadzu Scientific Instruments,型号:SIL-21A HT)
  9. 脱气器(Shimadzu Scientific Instruments,型号:DGU-20A5)
  10. UV-vis检测器(Shimadzu Scientific Instruments,型号:SPD-20A)
  11. 反相柱(ZORBAX Eclipse AAA分析柱,4.6×150mm,5.0μm)(Agilent Technologies,目录号:93400-902)
  12. 保护柱(ZORBAX Eclipse AAA分析保护柱,4.6×12.5mm,5.0μm)(Agilent Technologies,目录号:820950-931)

程序

  1. GABA和谷氨酸提取
    1. 在含有棉塞的250ml锥形瓶中的总体积为50ml的BG11培养基中,在30℃下培养集胞藻细胞。
    2. 通过在2,500×g离心10分钟,室温下,在生长的指数期(3至8天)收获细胞。 弃去上清液,将细胞沉淀重悬在500μl去离子H 2 O中,并将细胞悬浮液转移到1.5ml Eppendorf管中。 将细胞以10,000×g离心10分钟,并通过微量移液管除去所有的水(此时,细胞可以在-20℃下冷冻一个月)。
    3. 通过剧烈混合将细胞重悬于1.5ml的80%(v/v)甲醇中。在室温下孵育混合物几个小时(或孵育过夜),并使用超声均化器均化2分钟。
    4. 通过在10,000×g离心10分钟,4℃,沉淀细胞碎片。收集上清液并将其转移到新的1.5ml Eppendorf管中,得到"甲醇提取物"
    5. 在真空蒸发器中在40℃下干燥甲醇提取物5小时,并将干燥的沉淀物保持在室温下(可以省略步骤A6和7,参见下面的注释1)。
    6. 向从步骤A4获得的细胞碎片中加入2ml的80%(v/v)乙醇,以相同的方式进一步再提取,得到"乙醇提取物"(参见步骤A2和A3)。
    7. 在步骤A5中将乙醇提取物加入到先前从甲醇提取物获得的干燥沉淀中,并在40℃下在真空蒸发器中干燥。
    8. 将干燥后得到的沉淀用250μl去离子水和50μl氯仿溶解,然后在10,000×g下离心10分钟,4℃。收集上层水相(收集足够量的水相,小心不要收集氯仿相),转移到新的1.5 ml Eppendorf管中。
    9. 向水相中加入稀HCl,使最终浓度为0.1N。通过0.2μm微孔过滤器过滤溶液,然后通过HPLC检测GABA /谷氨酸。 过滤的样品可以储存在-20℃下直到进一步使用
  2. GABA和谷氨酸的HPLC分析
    1. 用0.2μmMillipore过滤器过滤去离子水(或使用UP水,HPLC级),溶剂A和溶剂B.
    2. 使用OPA试剂的预柱样品衍生化如下所述进行。
      1. 将30μl步骤A9的过滤样品转移到1.5 ml Eppendorf管中,加入5μl硼酸缓冲液。
      2. 充分混合并在室温下孵育15秒
      3. 加入0.5μlOPA试剂并混匀(见注2)
      4. 将溶液转移到200μl锥形插入物(图1A)。将插入物放置在透明的大开瓶中(图1B)。
      5. 用螺旋盖关闭小瓶(图1C),并通过自动注射器将样品立即注入HPLC系统。
    3. 使用梯度程序(表1)(参见注释3),在反相柱上使用溶剂A和溶剂B在2分钟内以2ml/min的流速在40℃(柱温箱温度)下进行分离。对于OPA衍生的样品,UV检测波长为338nm
    4. 通过将峰面积与标准物的峰面积进行比较来进行GABA和谷氨酸盐含量的定量(在0.5μl样品中对于4.5至450pmol的浓度范围获得线性)。使用这些HPLC条件的谷氨酸和GABA的保留时间分别为3.0-3.7min和10.0-10.5min(参见数据分析,图2)(参见注释4)。


      图1.使用Shimadzu显微自动进样器进行GABA和谷氨酸分析的(A)锥形插入物,(B)透明开口小瓶和(C)螺帽的照片

      表1.单次运行的HPLC梯度程序设置

数据分析



图2.从蓝细菌集胞藻中提取的分离的氨基酸的代表性色谱图。 A.标准谷氨酸和GABA的色谱图,浓度为2nmol/ml; B.从集胞藻中分离氨基酸的色谱图。 PCC 6803提取物。数据获自Shimadzu LC溶液软件,并通过三次独立实验确认。

笔记

  1. 可以省略乙醇提取步骤。 然而,为了确保氨基酸的完全定量回收,推荐这一步骤。
  2. OPA试剂在氧存在下缓慢降解。 立即将新衍生的反应混合物注入HPLC中
  3. 如果柱需要进一步清洗,程序可以通过用100%溶剂B柱洗涤20-30分钟,然后用缓冲液(溶剂A)平衡20-30分钟继续。
  4. 为了确保HPLC条件和标准品和样品的保留时间的一致性,定期运行标准品很重要。

食谱

  1. BG11生长培养基
    注意:BG11生长培养基的制备在Rippka等,1979中有详细描述。
  2. HPLC溶剂A
    40mM NaH 2 PO 4,pH 7.8 在1L水中溶解5.5g NaH 2 PO 4 H 2·H 2 O·
    用10N NaOH溶液调节pH
  3. HPLC溶剂B
    ACN:MeOH:水(45:45:10,v/v/v)洗脱
  4. OPA试剂
    10mg/ml邻苯二甲醛
    10mg/ml 3-巯基丙酸 0.4M硼酸盐缓冲液,pH 10.2

致谢

GABA和谷氨酸盐提取的方法改编自Bieleski和Turner(1966),有一些修改。用于检测和定量GABA和谷氨酸的基于HPLC的方法是Henderson等人公布的方案的修改。 (2006)。这项工作是由朱拉隆功大学(CU)Ratchadaphiseksomphot捐赠基金(食品和水集群,CU-58-011-FW)和泰国研究基金(IRG5780008)的研究拨款支持。 Simab Kanwal感谢CU研究生院提供博士后的奖学金。

参考文献

  1. Bieleski,RL和Turner,NA(1966)。  分离和通过薄层电泳和色谱法估计粗制植物提取物中的氨基酸。生化。 17:278-293。
  2. Henderson,JW,Ricker,RD,Bidlingmeyer,BA和Woodward,C.(2006)。  快速,准确,灵敏,可重复的氨基酸HPLC分析。 5980-1193EN:1-10。
  3. Kanwal,S.和Incharoensakdi,A.(2016)。  表征来自集胞藻的谷氨酸脱羧酶 PCC 6803及其在氮代谢中的作用。 Plant Physiol Biochem 99:59-65。
  4. Rippka,R.,Deruelles,J.,Waterbury,JB,Herdman,M.and Stanier,RY(1979)。  蓝藻菌的纯培养物的通用分配,菌株历史和性质。 J Gen Microbiol :1-61。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kanwal, S. and Incharoensakdi, A. (2016). Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803. Bio-protocol 6(18): e1928. DOI: 10.21769/BioProtoc.1928.
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