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Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)
用[14C]谷氨酸进行体内叶绿素放射性标记并采用薄层色谱分析法(TLC)对标记的叶绿素相对量化测定   

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Abstract

This is an accurate method to assess the rate of chlorophyll biosynthesis in vivo in cyanobacteria. Given that labeled glutamate is used as the very early precursor of chlorophyll together with a short pulse of labeling (30 min), this method provides information about the metabolic flow through the whole chlorophyll biosynthetic pathway on a short timescale.

Keywords: Chlorophyll biosynthesis(叶绿素的生物合成), 14C radiolabeling(14c标记), Cyanobacteria(蓝藻), Synechocystis(集胞藻), Thin Layer Chromatography(薄层色谱法)

Materials and Reagents

  1. Synechocystis PCC 6803
  2. Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([14C]Glu)
  3. Methanol
  4. 25% ammonia solution
  5. 1 M NaCl
  6. Hexane
  7. 10% KOH
  8. Petroleum ether
  9. Chloroform
  10. 1 M Na2HPO4
  11. 1 M NaH2PO4
  12. X-ray film (Eastman Kodak Company)
  13. NH4OH
  14. 1 M TES [2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid]/NaOH (pH 8.2)
  15. Growth medium BG11

Equipment

  1. 10 ml Headspace vials (Sigma-Aldrich)
  2. Water bath shaker
  3. 2 ml o-ring cap tubes
  4. Glass beads (100-200 μm)
  5. Vortex
  6. Tabletop centrifuge (MiniSpin plus, Eppendorf)
  7. Speedvac Concentrator plus (Eppendorf)
  8. Silica gel TLC plate (SIL G-25, MACHEREY-NAGEL)
  9. Rectangular TLC developing tank (Sigma-Aldrich)
  10. Mikro 22R centrifuge (Hettich)
  11. MiniSpin centrifuge (Eppendorf)

Procedure

  1. Labeling requires a 25-ml culture of cyanobacterial cells at the exponential phase of growth. Protocol works very well for the cyanobacterium Synechocystis PCC 6803 grown at 30 °C in growth medium BG11 (Rippka et al., 1979) to optical density at 750 nm~0.4. Harvest cells by centrifuging at 3,500 x g for 5 min at room temperature using brake 5 (Mikro 22R centrifuge).
  2. Discard supernatant and resuspend cell pellet in 2 ml of growth medium BG11, supplemented by 20 mM TES (pH 8.2). Transfer cells into a 2 ml tube and centrifuge at 3,500 x g for 2 min at room temperature (MiniSpin centrifuge).
  3. Discard supernatant and resuspend cell pellet in 450 μl of growth medium/20 mM TES. Transfer cells into a glass vial.
  4. Incubate cells in a water bath shaker for 30 min at 100 rpm under light and temperature conditions you prefer for your experiment.
  5. Add 180 μM of [14C]Glu dissolved in growth medium. Incubate under the same conditions for another 30 min.
  6. Transfer the labeled culture into a 2 ml o-ring cap tube. Spin down the cells at 7,000 x g for 2 min at room temperature and discard supernatant. At this point, the cell pellet can be frozen in liquid nitrogen and stored at -70 °C or used immediately for following pigment extraction.
  7. Resuspend cells in 1 ml of H2O and pellet cells at 7,000 x g for 2 min at room temperature. Discard water and repeat wash twice by 1 ml of H2O to remove all traced of labeled [14C]Glu. Resuspend cells in 1 ml of methanol/0.2% NH4OH. Add 50 μl of glass beads and vortex for 1 min to facilitate pigment extraction. Spin down 4 min at max rpm. Work under a dim light for all following steps
  8. Transfer supernatant (~1 ml) into 2 ml tube and add another 300 μl of methanol/0.2% NH4OH to cells, vortex and spin down again. Combine supernatants and add 140 μl of 1 M NaCl.
  9. Add 400 μl of hexane, vortex and spin down 30 sec at max rpm to accelerate phase separation. Collect upper phase containing chlorophyll. Repeat step 9 three times and combine all hexane into a 2 ml tube.
  10. Evaporate hexane using SpeedVac concentrator set to V-AL and 30 °C for 5 min.
  11. Resuspend the pellet in 190 μl of methanol and add 10 μl of 10% KOH. Incubate at room temperature for 15 min to convert chlorophyll into phytol-less Mg-chlorin.
  12. Extract this solution by 200 μl of hexane and discard upper phase, repeat 4-times.
  13. Transfer remaining ~150 μl of the methanol phase into a new 0.5 ml tube. Evaporate this solution using a SpeedVac concentrator to final volume of 30-50 μl.
  14. Extract this solution 5 times by 150 μl of petroleum ether and discard upper phase containing carotenoids.
  15. Evaporate completely at 30 °C for 30 min and resuspend pellet in 30 μl of methanol:chloroform (1:1). Load 10 μl on the TLC plate. 5 μl can be used for measurement using scintillation counter (see next step).
  16. Load 10 μl of pigment solution on a silica gel TLC plate. Place the plate in a TLC developing tank with 300 ml of mobile phase – methanol: 10 mM NaXHYPO4, pH 6.8 (3:1, v/v). Remove the plate from the tank after 30 min of incubation and dry it at 37 °C for 10 min. Expose the plate to X-ray film for 3-5 days and develop. After development only a one signal should be detected corresponding to Mg-chlorin ‘green’ band on TLC.
    Note: An alternative to TLC followed by detection on an X-ray film is to use a scintillation counter and measure directly radioactivity in the final pigment fraction (5 μl). The later method is faster; however it is less sensitive and also accurate due to presence of 14C labeled impurities.

Acknowledgments

We acknowledge the financial support from the project Algatech (CZ.1.05/2.1.00/03.0110) and Czech Science Foundation, project no. P506/12/1522.

References

  1. Koreny, L., Sobotka, R., Janouskovec, J., Keeling, P. J. and Obornik, M. (2011). Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites. Plant Cell 23(9): 3454-3462.
  2. 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): 51-61.

简介

这是一种精确的方法来评估蓝细菌中体内叶绿素生物合成的速度。 鉴于标记的谷氨酸被用作叶绿素的非常早期的前体以及短的标记脉冲(30分钟),该方法提供关于在短时间尺度上通过整个叶绿素生物合成途径的代谢流的信息。

关键字:叶绿素的生物合成, 14c标记, 蓝藻, 集胞藻, 薄层色谱法

Materials and Reagents

  1. Synechocystis PCC 6803
  2. Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([14C]Glu)
  3. Methanol
  4. 25% ammonia solution
  5. 1 M NaCl
  6. 己烷
  7. 10%KOH
  8. 石油醚
  9. 氯仿
  10. 1 M Na 2 HPO 4
  11. 1 M NaH 2 PO 4
  12. X射线胶片(Eastman Kodak Company)
  13. NH 4 OH
  14. 1 M TES [2 - [[1,3-二羟基-2-(羟甲基)丙-2-基]氨基]乙磺酸]/NaOH(pH8.2)
  15. 生长培养基BG11

设备

  1. 10ml顶空瓶(Sigma-Aldrich)
  2. 水浴摇床
  3. 2 ml O形圈帽管
  4. 玻璃珠(100-200μm)
  5. 涡流
  6. 台式离心机(MiniSpin plus,Eppendorf)
  7. Speedvac集中器加(Eppendorf)
  8. 硅胶TLC板(SIL G-25,MACHEREY-NAGEL)
  9. 矩形TLC显影槽(Sigma-Aldrich)
  10. Mikro 22R离心机(Hettich)
  11. MiniSpin离心机(Eppendorf)

程序

  1. 标记需要在指数生长期的25ml蓝藻细胞培养物。 方案对于在30℃下在生长培养基BG11中生长的蓝细菌集胞藻PCC 6803非常有效(Rippka ,1979),在750nm〜0.4的光密度。收获细胞通过在室温下使用制动器5(Mikro 22R离心机)在3500xg离心5分钟。
  2. 弃去上清液并将细胞沉淀重悬于补充有20mM TES(pH 8.2)的2ml生长培养基BG11中。将细胞转移到2ml管中,并在室温下在3500xg离心2分钟(MiniSpin离心机)。
  3. 弃去上清液并将细胞沉淀重悬在450μl生长培养基/20mM TES中。将细胞转移到玻璃小瓶。
  4. 孵育细胞在水浴摇动器30分钟,在100 rpm在光和温度条件下,你更喜欢你的实验。
  5. 加入溶解在生长培养基中的180μM的[14 C] Glu。在相同条件下再孵育30分钟。
  6. 将标记的培养物转移到2ml的O形环帽管。在室温下以7,000×g离心细胞2分钟,弃去上清液。在这一点上,细胞沉淀可以在液氮中冷冻并储存在-70℃或立即用于随后的颜料提取。
  7. 在室温下将细胞重悬于1ml H 2 O中,并将细胞以7000×g离心2分钟。弃去水,并用1ml H 2 O重复洗涤两次,以除去所有痕量的标记的[14 C] Glu。将细胞重悬于1ml甲醇/0.2%NH 4 OH中。加入50微升玻璃珠,涡旋1分钟,以促进颜料提取。在最大rpm下旋转4分钟。在昏暗的灯光下工作所有以下步骤
  8. 将上清液(〜1ml)转移到2ml管中,并向细胞中再加入300μl甲醇/0.2%NH 4 OH,涡旋并再次旋转。合并上清液并加入140μl的1M NaCl。
  9. 加入400μl己烷,涡旋并以最大转速旋转30秒以加速相分离。收集含有叶绿素的上层相。重复步骤9三次,并将所有己烷结合到2ml管中。
  10. 使用设置为V-AL的SpeedVac浓缩器和30℃蒸发己烷5分钟。
  11. 将沉淀重悬于190μl甲醇中,加入10μl10%KOH。在室温下孵育15分钟,将叶绿素转化为无叶绿素Mg-二氢卟酚。
  12. 用200μl己烷提取该溶液,弃去上层相,重复4次。
  13. 将剩余的〜150μl甲醇相转移到新的0.5ml管中。使用SpeedVac浓缩器蒸发此溶液,终体积为30-50μl。
  14. 用150μl石油醚萃取该溶液5次,弃去含有类胡萝卜素的上层相。
  15. 在30℃下完全蒸发30分钟,并将沉淀重悬在30μl甲醇:氯仿(1:1)中。加载10微升在TLC板上。 5μl可用于使用闪烁计数器测量(见下一步)。
  16. 加载10μl的颜料溶液在硅胶TLC板上。将板置于具有300ml流动相 - 甲醇:10mM Na H X H Y PO 4,pH 6.8的TLC显影槽中3:1,v/v)。在孵育30分钟后从培养皿中取出培养板,并在37℃下干燥10分钟。将板暴露于X射线胶片3-5天并显影。显影后,应检测到一个信号,对应于TLC上的Mg-氯化物"绿"带 注意: >替代TLC,随后在X射线胶片上检测是使用闪烁计数器并直接测量最终颜料级分(5μl)中的放射性。后来的方法更快;然而由于 14 C标记的杂质的存在,敏感度和准确度都会降低。

致谢

我们承认Algatech项目(CZ.1.05/2.1.00/03.0110)和捷克科学基金会项目的资金支持。 P506/12/1522。

参考文献

  1. Koreny,L.,Sobotka,R.,Janouskovec,J.,Keeling,P.J.and Obornik,M。(2011)。 四吡咯合成光合色素可能与apicomplexan寄生虫的不寻常途径同源。 Plant Cell 23(9):3454-3462
  2. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M。和Stanier,R.Y。(1979)。 蓝藻的纯培养物的通用分配,菌株历史和特性。 J Gen Microbiol 111(1):51-61。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sobotka, R., Kořený, L., Kopečná, J. and Oborník, M. (2012). Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC). Bio-protocol 2(16): e249. DOI: 10.21769/BioProtoc.249.
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