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Peer-reviewed

Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

采用HPLC法定量测定绿藻莱茵衣藻中的抗坏血酸

László Kovács László Kovács
André Vidal-Meireles André Vidal-Meireles
Valéria Nagy Valéria Nagy
Szilvia Z. Tóth Szilvia Z. Tóth

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2067 浏览次数: 8309

评审: Maria SinetovaClaudia CatalanottiAnonymous reviewer(s)

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Cover of Plant, Cell & Environment, featuring study using the protocol.

Jul 2016

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Abstract

Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the quantitative determination of cellular Asc content in the green alga Chlamydomonas reinhardtii.

Keywords: Ascorbate (抗坏血酸) search Cell counter (细胞计数器) search Chlamydomonas reinhardtii (莱茵衣藻) search HPLC (HPLC) search Dehydroascorbate (脱氢抗坏血酸盐) search

Background

Previous protocols for the determination of cellular Asc content were developed for higher plants, of which the amounts of cellular Asc are high enough for the analysis. Those protocols are not suitable for green algae, since both the amounts of the plant material and the levels of the cellular Asc are usually limited. Therefore, there was a need to develop a novel method with sensitivity improved to the µM range to measure the cellular Asc in green algae.

Materials and Reagents

  1. 15-ml conical centrifuge tubes (Corning, Falcon®)
  2. Polypropylene microcentrifuge tubes of 1.5 ml (Eppendorf)
  3. 0.3 ml polypropylene chromatographic vials (Focus Technology, model: VP91 ) with polypropylene cap and PTFE/silicone septa (Focus Technology, model: SC9291 )
  4. 4 mm hydrophilic PTFE syringe filter with a 0.22 µm pore size (Nantong Filterbio Membrane, catalog number: FBS4PTFE022L )
  5. 47 mm hydrophilic PTFE membrane with a 0.45 µm pore size (Nantong Filterbio Membrane, catalog number: FBM047PTFE045L )
  6. Glass beads 212-300 µm (Sigma-Aldrich, catalog number: G9143-250G )
  7. 1 ml disposable polypropylene syringe (B. Braun Medical, catalog number: 9166017V )
  8. Millex-GS 33 mm sterile syringe filter with a 0.22 µm pore size (EMD Millipore, catalog number: SLGS033SS )
  9. Milli-Q water
  10. Liquid nitrogen
  11. Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (Carl Roth, catalog number: HN95.2 )
  12. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: 3904.1 )
  13. Orthophosphoric acid (H3PO4) (Sigma-Aldrich, catalog number: W290017 )
  14. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
  15. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520.1000 )
  16. Potassium chloride (KCl) (Duchefa Biochemie, catalog number: P0515.1000 )
  17. Di-sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: P030.2 )
  18. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2393-100g )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Duchefa Biochemie, catalog number: C0504.1000 )
  20. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148-2.5L-D )
  21. Sodium L-Asc (Sigma-Aldrich, catalog number: 4034-100G )
  22. Acetonitrile HPLC gradient grade (VWR, HiPerSolv Chromanorm®, catalog number: 83639.320 )
  23. Phosphate-buffered saline (PBS) (see Recipes)
  24. Extraction buffer (see Recipes)
  25. HPLC mobile phase A (see Recipes)
  26. HPLC mobile phase B (see Recipes)
  27. Sodium L-Asc standard solutions (see Recipes)

Equipment

  1. ScepterTM handheld automated cell counter (EMD Millipore, catalog number: PHCC00000 )
  2. ScepterTM Sensors – 40 µm (EMD Millipore, catalog number: PHCC40050 )
  3. Refrigerated table centrifuge and microcentrifuge
  4. HPLC system (Shimadzu Scientific Instruments) equipped with:
    1. LC pump (Shimadzu Scientific Instruments, model: LC-20AD )
    2. Thermostated autosampler (Shimadzu Scientific Instruments, model: SIL-20AC )
    3. Photo Diode Array detector (Shimadzu Scientific Instruments, model: SPD-M30A )
    4. Column oven (Shimadzu Scientific Instruments, model: CTO-20AC )
    5. Synergy hydro 4u Hydro – RP 80 Å 250 x 4.6 mm (Phenomenex, catalog number: 00G-4375-E0 ) directly mounted with a guard column AQ C18 4.0 x 3.0 mm (Phenomenex, catalog number: KJ0-4282 )

Software

  1. LabSolution (Shimadzu Scientific Instruments)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Kovács, L., Vidal-Meireles, A., Nagy, V. and Tóth, S. Z. (2016). Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC. Bio-protocol 6(24): e2067. DOI: 10.21769/BioProtoc.2067.

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分类

植物科学 > 植物生物化学 > 其它化合物

生物化学 > 其它化合物 > 抗坏血酸盐

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