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Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.

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Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors
利用双荧光探测器的高效液相色谱(HPLC)检测和定量血红素和叶绿素前体

植物科学 > 植物生理学 > 光合作用
作者: Jan Pilný
Jan PilnýAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1970
Jana Kopečná
Jana KopečnáAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a86
Judith Noda
Judith NodaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1971
 and Roman Sobotka
Roman SobotkaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
For correspondence: sobotka@alga.cz
Bio-protocol author page: a84
Vol 5, Iss 3, 2/5/2015, 3313 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1390

[Abstract] Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.
Keywords: Chlorophyll biosynthesis(叶绿素的生物合成), Tetrapyrroles(四吡咯), Flourescence detector(荧光检测器), High Performance Liquid Chromatography(高效液相色谱法), Synechocystis 6803(集胞藻6803)

[Abstract] 四吡咯生物合成途径的中间体是低丰度化合物,它们的定量通常是困难的,耗时的并且需要大量的输入材料。 在这里,我们描述了一种方法,允许快速和准确的量化几乎所有的血红素和叶绿素生物合成的中间体,包括单乙烯基和二乙烯基形式的(原)叶绿素,只使用几毫升的蓝藻培养。 提取的前体通过高效液相色谱系统(HPLC)分离,并通过设置为不同波长的两个超灵敏荧光检测器检测。

Materials and Reagents

  1. Methanol (HPLC grade)
  2. Pyridine reagent Plus >99% (Sigma-Aldrich, catalog number: P57506 )
  3. H2O (HPLC grade) (Merck KGaA)
  4. Acetonitrile (HPLC grade)
  5. HPLC solvent A (see Recipes)
  6. HPLC solvent B (see Recipes)

Equipment

  1. 0.5 ml polypropylene tubes
  2. HPLC 2 ml glass vials and 200 μl glass inserts
  3. HPLC system with an autosampler (Agilent, model: 1200 ) (Figure 1)


    Figure 1. The Agilent 1200 HPLC system equipped with an autosampler and two fluorescence detectors marked as FLD #1 and FLD #2

  4. Two HPLC fluorescence detectors (Agilent, model: 1200) (see Note 1)
    The original Agilent 1200 detectors offer adequate sensitivity for emission wavelengths ranging 300-600 nm, however for longer wavelengths the sensitivity quickly falls almost to zero. This issue also applies to the newer 1260 version of the detector. As sensitivity to longer wavelengths is essential for detection of several heme/chlorophyll precursors, the original Agilent photomultiplier was replaced with the R928 photomultiplier tube (Hamamatsu, catalog number: R928) in both detectors. This modification significantly improved sensitivity within the entire wavelength range, and dramatically improved the sensitivity to wavelengths >600 nm. The replacement for the photomultiplier was provided by Agilent’s technical service.
  5. Tabletop centrifuge (Eppendorf, MiniSpin plus)
  6. Reverse phase column (ReproSil 100 C8-AB, 5-µm particle size, 4 x 250 mm) (Dr. Maisch, catalog number: r15.8b.s2504 )

Procedure

  1. The protocol requires 2 ml of a culture of cyanobacterial cells at the exponential phase of growth. The protocol works very well for the cyanobacterium Synechocystis PCC 6803 grown at 30 °C in the liquid medium BG11 (Rippka et al., 1979) with optical density at 750 nm ~ 0.4 (see Note 2). The BG11 medium is used frequently for various cyanobacterial strains, it contains a balanced mix of macro- and microelements required for photoautotrophic grow and its preparation is described in detail in Rippka et al. (1979).
  2. All further steps should be carried out under dim or green light to prevent degradation of photo labile tetrapyrroles. Alternatively, the tubes can be covered by aluminum foil.
  3. Harvest cells by centrifuging at room temperature and maximal speed (≥ 12 000 x g) for 5 min. Remove the supernatant and resuspend cells in 200 µl of ultrapure H2O. Transfer the cell solution into a 500 µl tube, spin cells down again and carefully remove all water by pipette. At this point, cells can be frozen in liquid nitrogen and stored in -70 °C for up to a month.
  4. Resuspend the pellet from the end of step 3 in 25 µl of ultrapure water, then add 75 µl of methanol, mix thoroughly and incubate in dark and at room temperature for 15 min to extract tetrapyrroles. Pellet cell debris by centrifuging for 5 min at maximal speed.
  5. Transfer the supernatant (~ 90 µl) to a new 500 µl tube.
  6. Resuspend the pellet from the end of step 3 in 20 µl of ultrapure water, add 80 µl of methanol, mix thoroughly and incubate again in dark and at room temperature for 15 min. Pellet cell debris by centrifuging for 5 min at maximal speed.
  7. Pool both supernatants (~ 180 µl) in the 500 µl tube, mix thoroughly and centrifuge again for 5 min at maximal speed to remove any traces of cell debris. Transfer the supernatant to the 200 µl insert and inject it immediately into the HPLC system.
  8. Separation is performed on a reverse phase column with 35% methanol and 15% acetonitrile in 0.25 M pyridine (solvent A) and 50% methanol in acetonitrile as solvent B. Tetrapyrroles are eluted with a gradient of solvent B (35 to 50% in 5 min) followed by 50 to 64% of solvent B in 30 min at a flow rate of 0.8 ml/min at 40 °C (Table 1). The program continues by column washing (36-45 min) and equilibration (36-45 min).

    Table 1. Setting of HPLC pumps for a single run
    Time (min)
    % of the solvent B
    0
    35
    5
    50
    35
    64
    36
    100
    45
    100
    46
    35
    51
    35

  9. Each run is finished using 100% of solvent B for 10 min to wash out more hydrophobic and abundant pigments like chlorophylls of carotenoids. The column is finally equilibrated for 5 min by 35 % of the solvent B (Table 1 for the complete pump program).
  10. Eluted pigments are detected by their specific fluorescence emission maxima. Settings of fluorescence detectors, the elution times and the excitation/emission maxima of individual precursors are summarized in Table 1 and also indicated graphically in Figure 2.

    Table 2. Settings of fluorescent detectors and the list of detectable heme/chlorophyll precursors, including their excitation and emission maxima and approximate elution times
    Detector
    Detector Setting Ex/Em (nm), Time
    Precursor, Ex/Em maxima (nm)
    Elution time
    FLD #1
    400/620, 0-10 min
    Coproporphyrin IX 400/420
    6 min
    440/660, 11-22 min
    DVa-chlorophylide 440/670
    17 min

    MVb-chlorophylide 440/670
    18 min

    DV -protochlorophylide 440/640
    20 min

    MV-protochlorophylide 440/640
    21 min
    400/630, 13-30 min
    Protoporphyrin IX 400/630
    26 min
    FLD #2
    416/595, 0-30 min
    Mg-protoporphyrin IX 416/595
    18 min

    Mg-protoporphyrin IX MEc 416/595
    24 min
    a-divinyl
    b-monovinyl
    c-monomethyl-ester

Representative data



Figure 2. Representative chromatograms of separated precursors extracted from the cyanobacterium Synechocystis PCC 6803. A. Chromatogram recorded by the first fluorescent detector (FLD). B. Chromatogram recorded simultaneously by the second FLD. Settings of excitation and emission wavelengths for FLD #1 and #2 are shown above the each chromatogram. The number in round brackets indicates the FLD sensitivity (gain; the maximum = 16) set for the given wavelengths (see Note 3). Copro III = Coproporphyrin IX; DV-PChlide = divinyl-protochlorophyllide; MV-PChlide = monovinyl-protochlorophyllide; DV-Chlide = divinyl-chlorophyllide; MV-Chlide = monovinyl-chlorophyllide; Proto IX - Protoporphyrin IX; Mg-Proto = Mg-protoporphyrin IX; Mg-Proto ME = Mg-protoporphyrin IX monomethylester.

Notes

  1. The protocol should work using any HPLC system with binary or quaternary pump system and with at least one fluorescence detector sensitive enough for the longer wavelengths (up to 680 nm). To detect the whole spectrum of precursors using a system with one fluorescence detector, each sample needs to be measured twice with different setting of excitation/emission wavelengths.
  2. Apart from the cyanobacterium Synechocystis PCC 6803 the described protocol has already been successfully tested for the green alga Chlamydomonas reinhartii, the moss Physcomitrella patens, the plant Arabidopsis thaliana, and for Escherichia coli. Coproporphyrin IX and Protoporphyrin IX should be detectable virtually in any organism.
  3. Sensitivity of the Agilent fluorescence detector can be set from 1-16 (max). This particular setting (numbers) is specific for the Agilent detectors but a similar increasing/decreasing of the sensitivity should be possible to achieve with virtually any HPLC fluorescence detector.

Recipes

  1. HPLC solvent A
    35% methanol and 15% acetonitrile in 0.25 M pyridine
  2. HPLC solvent B
    50% methanol in acetonitrile

Acknowledgments

This research project was supported by projects Algatech (CZ.1.05/2.1.00/03.0110) and Algain (EE2.3.30.0059) of the Czech Ministry of Education, Youth and Sports.

References

  1. Knoppova, J., Sobotka, R., Tichy, M., Yu, J., Konik, P., Halada, P., Nixon, P. J. and Komenda, J. (2014). Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis. Plant Cell 26(3): 1200-1212.
  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): 1-61.

材料和试剂

  1. 甲醇(HPLC级)
  2. 吡啶试剂Plus> 99%(Sigma-Aldrich,目录号:P57506)
  3. H 2 O(HPLC级)(Merck KGaA)
  4. 乙腈(HPLC级)
  5. HPLC溶剂A(参见配方)
  6. HPLC溶剂B(参见配方)

设备

  1. 0.5 ml聚丙烯管
  2. HPLC 2ml玻璃小瓶和200μl玻璃插入物
  3. HPLC系统,带有自动进样器(Agilent,型号:1200)(图1)

    图1.配有自动进样器和标记为FLD#1和FLD#2的两个荧光检测器的Agilent 1200 HPLC系统

  4. 两个HPLC荧光检测器(Agilent,型号:1200)(见注1)
    原始的Agilent 1200检测器对于300-600nm的发射波长提供足够的灵敏度,然而对于较长波长,灵敏度快速下降到零。此问题也适用于较新的1260版本的检测器。由于对较长波长的灵敏度对于检测几种血红素/叶绿素前体是必需的,所以在两个检测器中用R928光电倍增管(Hamamatsu,目录号:R928)替换原始的Agilent光电倍增管。该修改显着地改善了在整个波长范围内的灵敏度,并且显着提高了对> 600nm的波长的灵敏度。光电倍增管的更换由Agilent技术服务提供。
  5. 台式离心机(Eppendorf,MiniSpin plus)
  6. 反相柱(ReproSil 100 C8-AB,5μm粒径,4×250mm)(Dr.Maisch,目录号:r15.8b.s2504)

程序

  1. 该方案需要在指数生长期的2ml蓝细菌细胞培养物。该方案对于在液体培养基BG11(Rippka等人,1979)中在30℃下生长的蓝细菌集胞藻PCC 6803非常好,其光密度为750nm〜 0.4(见注2)。 BG11培养基经常用于各种蓝细菌菌株,其包含光自养生长所需的大分子和微量元素的平衡混合物,其制备在Rippka等人(1979)中有详细描述。
  2. 所有进一步的步骤应在暗光或绿光下进行,以防止光不稳定四吡咯的降解。或者,管可以由铝箔覆盖。
  3. 通过在室温和最大速度(≥12000xg/g)离心5分钟收获细胞。除去上清液并将细胞重悬在200μl的超纯H 2 O中。转移细胞溶液到500微升管,再次旋转细胞,小心地通过移液器移除所有的水。在这一点上,细胞可以在液氮中冷冻并在-70℃下储存长达一个月
  4. 重悬从步骤3结束的沉淀在25微升的超纯水,然后加入75微升的甲醇,彻底混合和孵育在黑暗和室温下15分钟提取四吡咯。通过以最大速度离心5分钟来沉淀细胞碎片
  5. 转移上清(〜90微升)到一个新的500微升管
  6. 从步骤3的末端重悬在20μl的超纯水中的沉淀,加入80μl的甲醇,充分混合并在黑暗中和在室温下再次孵育15分钟。通过以最大速度离心5分钟来沉淀细胞碎片
  7. 池上清在500μl管中的上清液(〜180μl),充分混合并再次离心5分钟,以最大速度,以清除任何痕迹的细胞碎片。将上清液转移到200μl插入片段,并立即注入HPLC系统
  8. 在反相柱上用在0.25M吡啶(溶剂A)中的35%甲醇和15%乙腈和在乙腈中的50%甲醇作为溶剂B进行分离。用溶剂B的梯度(35至50%,在5分钟内)洗脱四min),然后在40℃下以0.8ml/min的流速在30分钟内加入50至64%的溶剂B(表1)。程序通过柱洗涤(36-45分钟)和平衡(36-45分钟)继续。

    表1.设置单次运行的HPLC泵
    时间(分钟)
    溶剂B的%
    0
    35
    5
    50
    35
    64
    36
    100
    45
    100
    46
    35
    51
    35

  9. 每次运行使用100%的溶剂B完成10分钟以洗出更疏水和丰富的颜料如类胡萝卜素的叶绿素。 该柱最终用35%的溶剂B平衡5分钟(对于完整的泵程序,表1)。
  10. 通过其特异性荧光发射最大值检测洗脱的颜料。 荧光检测器的设置,洗脱时间和单个前体的激发/发射最大值总结在表1中,并且还在图2中图示。

    表2.荧光检测器的设置和可检测的血红素/叶绿素前体的列表,包括它们的激发和发射最大值和近似洗脱时间
    检测器
    检测器设置Ex/Em(nm),时间
    前体,Ex/Em最大值(nm)
    洗脱时间
    FLD#1
    400/620,0-10分钟
    粪卟啉IX 400/420
    6分钟
    440/660,11-22分钟
    DV a -chlorophylide 440/670
    17分钟

    MV b - 叶绿素          440/670
    18分钟

    DV-原叶绿素440/640
    20分钟

    MV-原叶绿素440/640
    21分钟
    400/630,13-30分钟
    原卟啉IX 400/630
    26分钟
    FLD#2
    416/595,0-30分钟
    Mg原卟啉IX 416/595
    18分钟

    Mg原卟啉IX ME c 416/595
    24分钟
    - -
    b -monovinyl
    c-单甲基酯

代表数据



图2.从蓝细菌集胞藻PCC 6803提取的分离的前体的代表性色谱图。 A.由第一荧光检测器(FLD)记录的色谱图。 B.由第二FLD同时记录的色谱图。 FLD#1和#2的激发和发射波长的设置显示在每个色谱图的上方。圆括号中的数字表示为给定波长设置的FLD灵敏度(增益;最大值= 16)(见注3)。 Copro III =粪卟啉IX; DV-PChlide =二乙烯基 - 原叶绿素; MV-PClide =单乙烯基 - 原叶绿素; DV-Chlide =二乙烯基 - 叶绿素; MV-Chlide =单乙烯基 - 叶绿素;原Ⅸ-原卟啉IX; Mg-Proto = Mg-原卟啉IX; Mg-Proto ME = Mg-原卟啉IX单甲酯。

笔记

  1. 该协议应使用任何HPLC系统与二元或四元泵系统和至少一个荧光检测器足够敏感的较长波长(高达680 nm)。检测整个光谱 前体使用具有一个荧光检测器的系统,每个样品需要测量两次,具有不同的激发/发射波长设置
  2. 除了蓝细菌集胞藻PCC 6803之外,所描述的方案已经被成功地测试用于绿藻Chlamydomonas reinhartii ,Physomitrella ,植物拟南芥和大肠杆菌 。鬼卟啉IX和原卟啉IX实际上在任何生物体中都是可检测的
  3. 安捷伦荧光检测器的灵敏度可设置为1-16(最大)。这个特定的设置(数字)是安捷伦探测器的特定设置,但是几乎任何HPLC荧光检测器都可以实现类似的灵敏度增加/减少。

食谱

  1. HPLC溶剂A
    35%甲醇和15%乙腈的0.25M吡啶溶液
  2. HPLC溶剂B
    50%甲醇/乙腈

致谢

这个研究项目得到捷克教育,青年和体育部Algatech(CZ.1.05/2.1.00/03.0110)和Algain(EE2.3.30.0059)项目的支持。

参考文献

  1. Knoppova,J.,Sobotka,R.,Tichy,M.,Yu,J.,Konik,P.,Halada,P.,Nixon,P.J.and Komenda,J.(2014)。 发现参与光合系统II中的光系统II装配的早期步骤的叶绿素结合蛋白复合物 。 植物细胞 26(3):1200-1212
  2. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M。和Stanier,R.Y。(1979)。 蓝藻菌纯培养物的通用分配,菌株历史和特性 J Gen Microbiol 111(1):1-61。
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How to cite this protocol: Pilný, J., Kopečná, J., Noda, J. and Sobotka, R. (2015). Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors. Bio-protocol 5(3): e1390. DOI: 10.21769/BioProtoc.1390; Full Text



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