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Radioactive Tracer Feeding Experiments and Product Analysis to Determine the Biosynthetic Capability of Comfrey (Symphytum officinale) Leaves for Pyrrolizidine Alkaloids
放射性示踪剂喂养实验测定紫草(聚合草)叶的吡咯里西啶生物碱生物合成能力及其合成产物的分析   

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本实验方案简略版
Plant Physiology
May 2017

Abstract

This protocol delivers a method to determine the biosynthetic capability of comfrey leaves for pyrrolizidine alkaloids independently from other organs like roots or flowers.

The protocol applies and combines radioactive tracer experiments with standard and modern techniques like thin layer chromatography (TLC), solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).

Keywords: Tracer (示踪剂), Radio-HPLC (放射性HPLC), Radio-TLC (放射性TLC), SPE (SPE), GC-MS (GC-MS), Alkaloid (生物碱), Plant-organ specificity (植物器官特异性)

Background

Comfrey roots are known to be able to synthesize pyrrolizidine alkaloids (Frölich et al., 2007) and the key enzyme for biosynthesis, homospermidine synthase (HSS), was localized in the endodermis cells. In addition to this site of synthesis, there have been hints that also leaves of a certain developmental stage might be able to produce pyrrolizidine alkaloids (Niemüller et al., 2012). Therefore, a protocol was developed to determine the biosynthetic capability of comfrey leaves to synthesize pyrrolizidine alkaloids independently from other plant organs.

Materials and Reagents

  1. epT.I.P.S® pipette tips (Eppendorf, catalog number: 0030000730 )
  2. Strata SCX 500 mg/6 ml tubes (Phenomenex, catalog number: 8B-S010-HCH )
  3. Scintillation vial 4 ml (Carl Roth, catalog number: HEE8.1 )
  4. 1.5 ml microcentrifuge tube (SARSTEDT, catalog number: 72.706.700 )
  5. 2 ml microcentrifuge tube (SARSTEDT, catalog number: 72.695.500 )
  6. Scalpel (Carl Roth, catalog number: AH88.1 )
  7. Microcapillary pipette, volume 1-5 µl (Sigma-Aldrich, catalog number: Z611239 )
  8. Silica gel G-25 TLC plates 20 x 20 cm (Merck, catalog number: 1003900001 )
  9. Verex Vial Kit 9 mm, µVial i3 (Phenomenex, catalog number: AR0-9974-12 )
  10. Hamilton syringe 25 µl (Hamilton, catalog number: 80400 )
  11. Fresh comfrey leaves from a flowering plant (size about 3.5 cm in length)
  12. Liquid nitrogen
  13. [1,4-14C]Putrescine (3.95 GBq/mmol, Amersham Int., catalog number: CFA.301, since the end of Amersham Int., [1,4-14C]Putrescine is still available at PerkinElmer, catalog number: NEC150000MC )
  14. [12C]Putrescine (Carl Roth, catalog number: 4141.2 )
  15. Scintillation cocktail, Rotiszint eco plus (Carl Roth, catalog number: 0016.3 )
  16. Acetonitrile (Carl Roth, catalog number: 7330.1 )
  17. Methanol (Carl Roth, catalog number: P717.1 )
  18. Ammonia solution 30% (Carl Roth, catalog number: CP17.1 )
  19. Sulphuric acid solution volumetric, 0.05 M (VWR, catalog number: 319589-500ML )
  20. Zinc dust (Carl Roth, catalog number: 9524.2 )
  21. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.1 )
  22. Potassium phosphate dibasic (K2HPO4) (Carl Roth, catalog number: T875.1 )
  23. Potassium phosphate monobasic (KH2PO4) (Carl Roth, catalog number: P018.1 )
  24. [14C]Retronecine
    Note: It was prepared according to Lindigkeit et al., 1997 and Hartmann et al., 2001.
  25. Ethyl acetate (Carl Roth, catalog number: 6784.1 )
  26. 2-Propanol (Carl Roth, catalog number: 9866.2 )
  27. 5% ammonia in methanol (see Recipes)
  28. 100 mM phosphate buffer, pH 7.5 (see Recipes)
  29. Mobile phase TLC (see Recipes)

Equipment

  1. Pipettes (Eppendorf, model: Research® plus, catalog number: 3123000063 )
  2. Microscale
  3. Fume hood (Thermo Fisher Scientific, catalog number: 1363 )
  4. Vacuum manifold for SPE (Agilent Technologies, catalog number: 5982-9110 )
  5. Lamp (Carl Roth, catalog number: 2986.1 )
  6. Mortar and pestle (Carl Roth, catalog number: NT80.1 )
  7. Vortex shaker (IKA, model: Vortex 2 )
  8. Micro stir bars (Carl Roth, catalog number: 0955.1 )
  9. Magnetic stirrer (IKA, model: lab disc [white] )
  10. Optima 1 MS GC column (MACHEREY-NAGEL, catalog number: 726205.15 )
  11. Minispin Microcentrifuge (Eppendorf, catalog number: 5452000018 )
  12. Radioactivity thin-layer-chromatography detector (RITA, Raytest, Straubenhardt)
  13. Tri-Carb 2910 TR LSC Low Activity Liquid Scintillation Analyzer (PerkinElmer)
  14. LiChrograph HPLC (Merck-Hitachi) connected to a fraction collector Pharmacia Frac-100 (GE Healthcare Life Science)
  15. EC 250/4 NUCLEOSIL® 120-5 C18 HPLC column (MACHEREY-NAGEL, catalog number: 720041.40 )
  16. Developing chambers for TLC (Carl Roth, catalog number: 3133.1 )
  17. Shimadzu GC-2010 gas chromatograph with SSL injector (Shimadzu, model: GC-2010 )
  18. Shimadzu AOC-20i Auto-injector (Shimadzu, model: AOC-20i )
  19. Fisons MD 800 quadrupole mass spectrometer

Software

  1. XCalibur 1.1 (Thermo Fisher Scientific, Dreieich, Germany)
  2. GinaStar TLC (Raytest, Straubenhardt)
  3. QuantaSmartTM 4.0 (PerkinElmer)

Procedure

  1. Radioactive tracer feeding experiments
    1. 169 kBq of [1,4-14C]putrescine solution is transferred into a 2 ml microcentrifuge tube and evaporated to dryness.
      Note: Make sure to follow the official guidelines for the work with radioactivity.
    2. 0.04 µmol of [12C]putrescine is weighed on a microscale and transferred into another 2 ml microcentrifuge tube.
      Note: The purpose of using also a [12C]putrescine parallel to tracer is to give you the ability of using an equally-treated sample to analyze with hyphenated techniques, like mass spectroscopy, in later stages of this experiment.
    3. 1 ml of tap water is added to each of the tubes of Step A1 and Step A2. Take 5 µl of the [14C]putrescine solution that is mixed with 3 ml of scintillation cocktail and the radioactivity is quantified using the Tri-Carb LSC (Tri-Carb Count Conditions for 14C nuclides are: ‘Nuclide: 14C, Quench Setting Low Energy: 14C; Count Time (min): 5; Count Mode: Normal; Assay Count Cycles: 1; Repeat Sample Count: 1; #Vials/Sample: 1; Calculate % Reference: Off’).
      Notes:
      1. For safety reasons, make sure to mark the tubes according to the kind of putrescine that is in solution ([12C] or [14C]) to avoid mix-up of tubes. This labeling should be continued at every consecutive step.
      2. Any commercially available liquid scintillation counter machine can be used; the mentioned program is used every time radioactivity has to be quantified.
      3. Keep in mind to always run a blank without any radioactive tracer within a quantification of radioactivity to quantify the background.
    4. Two young comfrey leaves subtending an inflorescence with unopened flower buds are cut from a comfrey plant. Immediately place one leaf in the microcentrifuge tube containing the [14C]putrescine solution, the other in the tube containing the [12C]putrescine solution (Figure 1).


      Figure 1. Work flow of tracer feeding and extraction. A. Steps A1 to A3, showing the preparation of the tracer in the microcentrifuge tubes. B. Step A4, showing the incubation of the leaves in the [12C]- and [14C]-tracer solution. C. Steps A6 to A8 refilling the tube with tap water to avoid desiccation of the leaf. D and E. Steps A9 to A10 showing pulverization of the leaves and transfer of the powder into 2 ml microcentrifuge tubes. F and G. Steps A11 to A12 showing acidic extraction of the leaves.

    5. The leaves in the tubes are placed under a 12 h/12 h light/dark regime under the lamp with an intensity of ca. 1,000 lux.
    6. As soon as the water is taken up by the leaves, another 1 ml of tap water is added. Avoid the petiole to dry out.
    7. After addition of the tap water to the [14C]putrescine-containing tube, an aliquot of 5 µl is mixed with 3 ml of scintillation cocktail and the radioactivity is quantified using the Tri-Carb LSC.
    8. Steps A6 and A7 are repeated for four days. After the final addition of 1 ml of tap water to the tube, the amount of radioactivity taken up by the leaf in the [14C]putrescine solution is calculated using this formula: uptake in percent = (169 kBq - [kilobecquerel present in last measurement x 200 + amount of radioactivity measured in the 5 µl steps])/169 kBq x 100.
    9. Both leaves are frozen separately in liquid nitrogen and pulverized with mortar and pestle.
      Note: The mortar and the pestle will retain plant debris in their pores. Especially the mortar and pestle used with the leaf sample incubated with [14C]putrescine has to be treated carefully after use to decontaminate residual radioactivity.
    10. Each of the pulverized leaves is transferred into a 2 ml microcentrifuge tube (labeled [14C]-leaf and [12C]-leaf, respectively).
    11. 1.5 ml of 0.05 M H2SO4 is added to each of the two samples in the centrifuge tubes.
    12. The closed tubes of Step A11 are vortexed for 3 min at room temperature and the cell debris is separated by centrifugation (10 min, 5,000 x g).
    13. The supernatants of Step A12 are transferred, each, into a fresh 2 ml microcentrifuge tube containing a micro stir bar.
    14. An aliquot of 5 µl is taken from each tube of Step A13, mixed with 3 ml of scintillation cocktail and the radioactivity of the sulphuric extract is quantified via Tri-Carb LSC.
    15. In each tube of Step A13 a spatula tip of zinc dust is added and stirred for three hours on a magnetic stirrer.
      Note: Zinc dust reduces the alkaloid N-oxides present in your sample to the tertiary alkaloid and may result in formation of foam rising in your tube. Avoid spilling of your sample.
    16. Each tube of Step A15 is centrifuged (10 min, 5,000 x g) and the clear supernatant is applied to a Strata SCX-SPE cartridge positioned on the vacuum manifold. The cartridges were conditioned with 6 ml of methanol and equilibrated with 6 ml of 0.05 M H2SO4 prior to use.
      Notes:
      1. Since there is most likely residual radioactivity in the Zinc make sure to dispose the Zinc as solid radioactive material.
      2. After each treatment, the Strata SCX-SPE cartridge is dried by applying vacuum for an additional minute to the cartridge.
    17. An aliquot of 5 µl of the flow-through of each of the Strata-SCX cartridges is mixed with 3 ml of scintillation cocktail to quantify the radioactivity that is incorporated into non-alkaloid metabolites using the Tri-Carb LSC (Figure 2).


      Figure 2. Solid phase extraction setup. Steps A17 to A21 are shown. On the left: SPE cartridges assembled onto the vacuum manifold loaded with sample. On the right: test tubes of the vacuum manifold with their label and the scintillation vials.

    18. The loaded Strata SCX-SPE cartridges of Step A16 are washed on the vacuum manifold first with 12 ml of deionized water and then with 12 ml of methanol.
    19. An aliquot of 5 µl of the flow-through from the washing steps of each of the Strata-SCX cartridges is mixed with 3 ml of scintillation cocktail to quantify the radioactivity using the Tri-Carb LSC.
    20. The washed cartridges of Step A18 are eluted using the vacuum manifold with a glass tube positioned under each of the two cartridges by applying three times 6 ml of methanol containing 5% (v/v) of ammonia (Recipe 1). The three elution fractions of each cartridge are combined.
    21. An aliquot of 5 µl of the combined elution fractions is mixed with 3 ml of scintillation cocktail to quantify radioactivity that was incorporated into the alkaloid compounds using the Tri-Carb LSC.
      Note: The cartridge will most likely retain some radioactive molecules (very strong bases), make sure to dispose properly.
    22. The solvent of the combined elution fractions in both tubes of Step A20 is evaporated.
    23. The dry residue of the [14C]-labeled sample (Step A22) is dissolved in 2 ml of methanol and divided into equal parts by transfer 1 ml in each of two 2 ml microcentrifuge tubes. The tubes are labeled (#23a) and (#23b).
    24. The methanol of both samples (tubes #23a and #23b) is evaporated.
    25. The dry residue of sample #23a is dissolved in 50 µl of methanol and stored at -20 °C (to be used in Step A31).
    26. The dry residue of sample #23b is dissolved in 2 ml of 2 N NaOH. The tube is closed and heated in a heating block at 60 °C for 3 h. Afterwards the tube is opened and the solvent evaporated over 2 days under a fume hood (see Notes 1 and 2).
    27. The dry residue of the [12C]-labeled sample of (Step A 22) is dissolved in 2 ml of 2 N NaOH. The tube is closed and incubated in a heating block at 60 °C for 3 h. Afterwards, the tube is opened and the solvent evaporated over 2 days under a fume hood (see Notes 1 and 2).
    28. The dry residue of hydrolyzed samples of Step A26, labeled with [14C], and Step A27, labeled with [12C], are dissolved in 50 µl of methanol, each, and stored at -20 °C (to be used in Steps A34 and B1).


      Figure 3. Example of the tracer surveillance. Data according to Kruse et al. (2017).

    29. The HPLC column is equilibrated with 85% solvent A (100 mM phosphate buffer, pH 7.5, Recipe 2) and 15% solvent B (acetonitrile) at 1 ml/min for 1.5 h.
    30. The fraction collector Frac-100 is equipped with 4 ml scintillation vials.
    31. 20 µl of the sample from Step A25 is transferred with a Hamilton syringe into the rheodyne injector-loop of the LiChrograph HPLC and the valve is changed to the inject position. The fraction collector is programmed to a fraction size of 500 µl.
    32. After the HPLC run finished (25 min), 3 ml of scintillation cocktail is added to each fraction collected in the scintillation vials. The vials are closed and radioactivity is quantified with the Tri-Carb LSC (Figure 4).
    33. The HPLC column is reequilibrated after the run for 1.5 h as described in Step A29.
    34. Steps A30 to A33 are repeated with the [14C]-labeled sample from Step A28.
    35. Steps A30 to A33 are repeated with the [14C]retronecine standard.
    36. An aliquot of 10 µl of the remaining [14C]-labeled sample from Step A28 is mixed in a 1.5 ml microcentrifuge tube with 10 µl of the [14C]retronecine standard.
    37. Steps A30 to A33 are repeated with the sample of Step A36.


      Figure 4. Example of a plotted radio- chromatograms. A. The unhydrolyzed purified extract of Step A32. B. The hydrolyzed purified extract of Step A34. C. The [14C]retronecine standard of Step A35. D. Mixture of standard and hydrolyzed extract of Step A37. Data according to Kruse et al. (2017).

  2. Product analysis
    1. The 20 µl remaining of the [14C]-labeled sample from Step A28 is applied as aliquots of 5 µl onto a TLC plate by using the microcapillary pipette. Make sure that the solvent is completely evaporated before applying the next aliquot. This lane is labeled [14C].
    2. The complete [12C]-labeled sample from Step A28 is applied to the lane parallel to that of Step B1 in the same manner as described in Step B1. The lane is labeled [12C].
    3. The TLC is developed in a presaturated TLC developing chamber with 20 ml of mobile phase (ethyl acetate:isopropyl alcohol:ammonium hydroxide [30%, v/v]: 45:35:20, Recipe 3).
    4. The TLC plate is dried under a fume hood overnight.
    5. The radioactivity of the TLC plate is detected using the RITA system with GinaStar TLC software. The area of the [12C]-lane that is parallel to the radioactive spot of the [14C]-lane is scrapped off including the area 1 cm above and 1 cm below with a spatula into a 2 ml microcentrifuge tube (Figure 5).


      Figure 5. Workflow for product analysis. A. Steps B1 to B4 41 showing the developed TLC. B. Step B5 showing the spots labeled after detection of radioactivity with the RITA system. C. Step B5 showing the spot on the [12C]-TLC lane that is parallel to the radioactivity spot and is scraped off. D. Steps B5 to B6 show the silica gel powder scraped off the TLC plate and transferred to a 2 ml microcentrifuge tube.

    6. The silica gel powder of Step B5 is suspended by adding 1 ml of the mobile phase to the microcentrifuge tube. The sample is shaken vigorously and centrifuged (1 min, 5,000 x g). The supernatant is transferred into a fresh 2 ml microcentrifuge tube and evaporated under a fume hood.
    7. Step B6 is repeated three times with transfer of the supernatant into the same 2 ml microcentrifuge tube to ensure full extraction.
    8. The dry residue resulting from Step B7 is dissolved in 25 µl of methanol and transferred into a µVial for GC-MS analysis. 1 µl is injected with the AOC-20i auto-injector and electron-impact mass spectra are recorded at 70 eV. Gas chromatography conditions are as follows: injector: 250 °C; injection mode splitless; splitless time 1 min, temperature program: 60 °C for 3 min and 60 °C to 300 °C at 16 °C min-1; carrier gas, helium at 1 ml min-1 (Figure 6).
      Note: Any commercially available GC-MS system can be used for this purpose, a direct injection of the radioactive area is not recommended, by reason of the risk for contaminating expensive equipment and of the non-availability of 14C-labeled reference spectra.


      Figure 6. Example of a GC-MS run. A. Showing the total ion chromatogram (TIC) of the sample measured at Step B8. B. Showing the extracted ion chromatogram for the molecular weight of hydrolyzed pyrrolizidine alkaloids from comfrey. C. Showing the mass spectrum at the retention index 1,478, the largest peak of the TIC and EIC, identified as retronecine by comparison with literature and NIST database. Data according to Kruse et al. (2017).

Data analysis

  1. The data resulting from measurement of radioactivity of the sample aliquots with the Tri-Carb LSC in Steps A1 to A8 allow calculation of the total uptake of [14C]putrescine as a tracer by the comfrey leaves given in decays per minute (dpm).
    The radioactivity is calculated using the following formula:
    Total Radioactivity [dpm] = (measured radioactivity) [dpm] x (total volume) [µl]/5
    The values for radioactivity resulting from aliquots taken in Steps A14 to A21 allow conclusions about the chemical properties of the metabolites that incorporated radioactivity: The amount of radioactivity in the sulfuric extract indicates the amount of radioactivity incorporated into water soluble and acidic extractable molecules. As they eluted from the SPE with ammoniacal methanol they are most likely weak bases as, for example, alkaloids (Figure 3).
  2. The HPLC runs of Steps A30 to B1 do not require an HPLC detector since the radioactivity measured for the individual fractions is plotted against the fraction time point and thereby delivering a direct radio-chromatogram. This can be done by using standard data-processing software like Microsoft Excel, Apple Numbers or Libre Calc or manually on paper. The resulting peaks deliver insights if the SPE-purified molecules that incorporated tracers carry side chains that are esterified to the [14C]-labeled core structure originating from the tracer. A comparison with standards is already the first evidence for a possible incorporation of putrescine into alkaloids.
  3. The GC-MS total ion chromatogram of the compounds extracted from the TLC spot will further be processed, in the case of pyrrolizidine alkaloids, by extracting the ion 155 m/z representing the [M]+ molecule of the retronecine core structure. In the case of a single peak, the EI mass spectrum of this peak allow comparisons with spectral libraries like NIST or data previously published of pyrrolizidine alkaloids.

Notes

  1. Make sure to close the tubes properly. For safety reasons, it is recommended to put some weight on the cap of the tubes to avoid them being opening by the heat.
  2. This process can take very long depending on the laminar air flow of the fume hood and can be speeded up by mixing the water with methanol in a 1:1 ratio, or by using a nitrogen flow or a centrifugal evaporator.

Recipes

  1. 5% ammonia in methanol
    In a volumetric flask, 16.67 ml of 30% ammonia solution is added to 83.33 ml of methanol
  2. 100 mM phosphate buffer, pH 7.5
    2.449 g KH2PO4 and 14.143 g K2HPO4 are weighed into a volumetric flask and made up to 1 L with deionized water
  3. Mobile phase TLC
    In a beaker, 45 ml of ethyl acetate is mixed with 35 ml of 2-propanol and 20 ml of 30% (v/v) ammonia solution. The mobile phase has to be prepared fresh prior to use

Acknowledgments

This work was funded by a DFG grant given to Dietrich Ober. We thank Dr. Dorothee Langel for her input and ideas contributing to this method. We thank Dr. Christoph Gelhaus and Maren Hartelt, Kiel University, for help in implementing this method. The authors declare no conflict of interest.

References

  1. Frölich, C., Ober, D. and Hartmann, T. (2007). Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species. Phytochemistry 68(7): 1026-1037.
  2. Hartmann, T., Theuring, C., Witte, L. and Pasteels, J. M. (2001). Sequestration, metabolism and partial synthesis of tertiary pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi. Insect Biochem Mol Biol 31(11): 1041-1056.
  3. Kruse, L. H., Stegemann, T., Sievert, C. and Ober, D. (2017). Identification of a second site of pyrrolizidine alkaloid biosynthesis in comfrey to boost plant defense in floral stage. Plant Physiol 174(1): 47-55.
  4. Lindigkeit, R., Biller, A., Buch, M., Schiebel, H. M., Boppre, M. and Hartmann, T. (1997). The two faces of pyrrolizidine alkaloids: the role of the tertiary amine and its N-oxide in chemical defense of insects with acquired plant alkaloids. Eur J Biochem 245(3): 626-636.
  5. Niemüller, D., Reimann, A. and Ober, D. (2012). Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the boraginales. Plant Physiol 159(3): 920-929.

简介

该协议提供了一种方法来确定紫草叶生物合成能力的吡咯烷生物碱独立于其他器官,如根或花。

该协议将放射性示踪剂实验与薄层色谱法,固相萃取法,高效液相色谱法和气相色谱 - 质谱法等标准和现代技术相结合。

【背景】据了解,紫草根能够合成吡咯里西啶生物碱(Frölichet al。,2007),生物合成的关键酶高胡亚素合成酶(HSS)定位于内皮细胞。 除了这个合成位点之外,还有一些暗示,即某个发育阶段的叶子也许能够产生吡咯里西啶生物碱(Niemüller等人,2012)。 因此,开发一种方案来确定紫草叶合成吡咯里西啶生物碱的生物合成能力,以独立于其他植物器官。

关键字:示踪剂, 放射性HPLC, 放射性TLC, SPE, GC-MS, 生物碱, 植物器官特异性

材料和试剂

  1. epT.I.P.S 枪头(Eppendorf,目录号:0030000730)
  2. Strata SCX 500 mg / 6 ml试管(Phenomenex,目录号:8B-S010-HCH)
  3. 闪烁小瓶4毫升(卡尔罗斯,目录号:HEE8.1)
  4. 1.5 ml微量离心管(SARSTEDT,目录号:72.706.700)
  5. 2 ml微量离心管(SARSTEDT,目录号:72.695.500)
  6. 手术刀(卡尔罗斯,目录号:AH88.1)
  7. 微量毛细管移液器,体积1-5微升(西格玛奥德里奇,目录号:Z611239)
  8. 硅胶G-25 TLC板20×20厘米(Merck,目录号:1003900001)
  9. Verex Vial Kit 9 mm,μViali3(Phenomenex,目录号:AR0-9974-12)
  10. 汉密尔顿注射器25微升(汉密尔顿,目录号:80400)
  11. 新鲜的紫草离开开花植物(大小约3.5厘米长)
  12. 液氮
  13. [1,4- 14 C]腐胺(3.95GBq / mmol,Amersham Int。,目录号:CFA.301,自Amersham Int。,[1,4- C]腐胺在PerkinElmer仍然可用,目录号:NEC150000MC)
  14. [12C]腐胺(Carl Roth,目录号:4141.2)
  15. 闪烁鸡尾酒,Rotiszint eco plus(Carl Roth,目录号:0016.3)
  16. 乙腈(Carl Roth,目录号:7330.1)
  17. 甲醇(Carl Roth,目录号:P717.1)
  18. 氨溶液30%(卡尔罗斯,目录号:CP17.1)
  19. 硫酸溶液体积,0.05M(VWR,目录号:319589-500ML)
  20. 锌粉(卡尔罗斯,目录号:9524.2)
  21. 氢氧化钠(NaOH)(Carl Roth,目录号:6771.1)
  22. 磷酸二氢钾(KH 2 HPO 4)(Carl Roth,目录号:T875.1)
  23. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:P018.1)
  24. [14S] Retronecine
    注:它是根据Lindigkeit等人,1997年和Hartmann等人,2001年编写的。
  25. 乙酸乙酯(Carl Roth,目录号:6784.1)
  26. 2-丙醇(Carl Roth,目录号:9866.2)
  27. 5%的氨在甲醇(见食谱)
  28. 100 mM磷酸盐缓冲液,pH 7.5(见食谱)
  29. 流动相TLC(见食谱)

设备

  1. 移液器(Eppendorf,型号:Research plus,产品目录号:3123000063)
  2. 微型
  3. 通风橱(Thermo Fisher Scientific,目录号:1363)
  4. SPE(Agilent Technologies,目录号:5982-9110)的真空管道
  5. 灯(卡尔罗斯,目录号:2986.1)
  6. 灰浆和杵(卡尔罗斯,目录号:NT80.1)
  7. 涡旋振荡器(IKA,型号:Vortex 2)
  8. 微型搅拌棒(Carl Roth,目录号:0955.1)
  9. 磁力搅拌器(IKA,型号:实验室盘[白色])
  10. Optima 1 MS GC柱(MACHEREY-NAGEL,目录号:726205.15)
  11. Minispin微量离心机(Eppendorf,目录号:5452000018)
  12. 放射性薄层色谱检测器(RITA,Raytest,Straubenhardt)
  13. Tri-Carb 2910 TR LSC低活性液体闪烁分析仪(PerkinElmer)
  14. 连接到级分收集器Pharmacia Frac-100(GE Healthcare Life Science)的LiChrograph HPLC(Merck-Hitachi)
  15. EC 250/4 NUCLEOSIL 120-5 C18 HPLC柱(MACHEREY-NAGEL,目录号:720041.40)
  16. 为TLC开发室(Carl Roth,目录号:3133.1)

  17. 带有SSL注射器的岛津GC-2010气相色谱仪(日本岛津,型号:GC-2010)
  18. 岛津AOC-20i自动注射器(岛津,型号:AOC-20i)
  19. Fisons MD 800四极质谱仪

软件

  1. XCalibur 1.1(Thermo Fisher Scientific,德国Dreieich)
  2. GinaStar TLC(Raytest,Straubenhardt)
  3. QuantaSmart TM 4.0(PerkinElmer)

程序

  1. 放射性示踪剂喂养实验
    1. 将169KBq的[1,4- 14 C]腐胺溶液转移到2ml微量离心管中并蒸发至干燥。
      注意:确保按照放射性工作的官方指导
    2. 称取0.04μmol[12C]腐胺在微量级上并转移到另一个2ml微量离心管中。
      注:使用与示踪剂平行的[ 12 C]腐胺的目的是为了让您能够平等地使用经过处理的样品在本实验的后期阶段使用联用技术(如质谱)进行分析。
    3. 在步骤A1和步骤A2的每个管中加入1ml自来水。取5μl与3ml闪烁鸡尾酒混合的[14C]腐胺溶液,用Tri-Carb LSC(Tri-Carb Count Conditions for 14℃)定量放射性, C核素为:核素:14C,淬灭设置低能量:14C计数时间(分钟):5计数模式:正常分析计数周期:1;重复样品计数:1;#样品瓶/样品:1;计算%参考:关')。
      注意:
      1. 为了安全起见,请务必根据溶液中腐胺的种类来标记管子(或称为“ 12 C> [/ 14 C])来避免混淆管子。这个标签应该在每一个连续的步骤中继续。
      2. 任何市售的液体闪烁计数器都可以使用;每次放射性必须被量化时,使用所提到的程序。
      3. 请记住在放射性量化的范围内始终运行一个没有放射性示踪剂的空白来量化背景。
    4. 从雏菊植物切下的两朵嫩雏菊叶子未开放的花蕾,立即将一片叶子放入含有[14 C]腐胺溶液的微量离心管中,另一片放入含有[12 C]腐胺溶液的管中(图1)。


      图1.示踪剂进料和萃取的工作流程A.步骤A1至A3,示出微量离心管中示踪剂的制备。 B.步骤A4,显示叶子在[12C] - 和[14C] - 示踪剂溶液中的温育。 C.步骤A6至A8用自来水重新填充管子以避免叶子干燥。 D和E.步骤A9至A10显示粉碎叶片并将粉末转移至2ml微量离心管中。 F和G.步骤A11到A12显示叶子的酸性提取。

    5. 将灯管中的叶子放置在灯下12小时/ 12小时的明/暗区域中,强度为ca。 1000勒克司。
    6. 一旦水被叶子吸收,另外加入1毫升的自来水。避免叶柄变干。
    7. 在将自来水加入到含有腐胺的管中后,将5μl的等分试样与3ml的闪烁混合物混合,并使用Tri-Carb LSC定量放射性。 br />
    8. 步骤A6和A7重复四天。最后加入1ml自来水至管中后,用该公式计算[14C]腐胺溶液中叶吸收的放射性量:吸收百分数=(169 kBq - [上次测量时出现的千烛烛x 200 +5μl步骤中测量的放射性量])/ 169kBq×100。
    9. 两个叶子分别在液氮中冷冻,并用研钵和研杵粉碎。
      注:砂浆和杵将保留毛孔中的植物碎屑。特别是与用[14E] C]腐胺培养的叶样品一起使用的研钵和研杵必须在使用后小心地处理以净化残留的放射性。
    10. 将每个粉碎的叶子转移到2ml微量离心管中(分别标记为[14C] - 叶和[12C] - 叶)。
    11. 向离心管中的两个样品中的每一个中加入1.5ml的0.05M H 2 SO 4。
    12. 步骤A11的封闭管在室温下涡旋3分钟,并通过离心(10分钟,5000×g)分离细胞碎片。
    13. 将步骤A12的上清液分别转移到含有微型搅拌棒的新鲜2ml微量离心管中。
    14. 从步骤A13的每个管中取5μl的等分试样,与3ml闪烁鸡尾酒混合,并通过Tri-Carb LSC定量硫酸提取物的放射性。
    15. 在步骤A13的每个管中,加入锌粉末刮刀尖,并在磁力搅拌器上搅拌3小时。
      注意:锌粉会将样品中存在的生物碱N-氧化物还原为三级生物碱,并可能导致管内泡沫的形成。避免溢出您的样本。
    16. 将步骤A15的每个离心管离心(10分钟,5000×g),并将澄清的上清液施加到位于真空歧管上的Strata SCX-SPE柱上。使用前用6ml甲醇调节药筒并用6ml 0.05M H 2 SO 4平衡。
      注意:
      1. 由于锌中最有可能存在残留放射性,因此务必将锌作为固体放射性物质进行处置。
      2. 每次处理后,Strata SCX-SPE药筒通过对药筒施加一分钟的真空而干燥。
    17. 将5μl的每种Strata-SCX柱的流通液的等分试样与3ml的闪烁混合物混合,以使用Tri-Carb LSC(图2)定量并入非生物碱代谢物的放射性。 br />

      图2.固相萃取设置。显示步骤A17至A21。 :左侧:将SPE小柱装配到装有样品的真空歧管上。在右侧:用标签和闪烁瓶对真空腔体进行试管。

    18. 步骤A16的加载的Strata SCX-SPE柱首先用12ml去离子水,然后用12ml甲醇在真空装置上洗涤。
    19. 将5μl来自每个Strata-SCX柱体的洗涤步骤的流出物的等分试样与3ml闪烁混合物混合,以使用Tri-Carb LSC定量放射性。
    20. 使用含有5%(v / v)氨的3ml甲醇(配方1),使用带有位于两个筒的每一个下的玻璃管的真空歧管洗脱步骤A18的洗过的柱。每个药筒的三个洗脱部分被合并。
    21. 将5μl组合洗脱级分的等分试样与3ml闪烁混合物混合,以定量使用Tri-Carb LSC掺入到生物碱化合物中的放射性。
      注意:墨盒很可能会保留一些放射性分子(非常强碱),请确保正确处置。
    22. 步骤A20的两管中的组合洗脱组分的溶剂被蒸发
    23. 将[14 C]标记的样品(步骤A22)的干燥残余物溶于2ml甲醇中,并通过在两个2ml微量离心管中分别转移1ml而分成相等的部分。 (#23a)和(#23b)。
    24. 两个样品(管#23a和#23b)的甲醇被蒸发。
    25. 将样品#23a的干燥残余物溶于50μl甲醇中并储存在-20℃(用于步骤A31)。
    26. 样品#23b的干燥残余物溶于2ml 2N NaOH中。将管关闭并在60℃的加热块中加热3小时。之后,将管打开,溶剂在通风橱中蒸发2天(见注1和注2)。
    27. (步骤A22)的[12C]标记样品的干燥残余物溶于2ml 2N NaOH中。将管关闭并在60℃的加热块中温育3小时。之后,将管打开,并在通风橱中将溶剂蒸发2天(见注1和注2)。
    28. 用[14 C]标记的步骤A26的水解样品的干燥残余物和用[12 C]标记的步骤A27溶于50μl甲醇中,每个,并储存在-20°C(在步骤A34和B1中使用)。

      “”src
      图3.示踪剂监测示例根据Kruse (2017)的数据。

    29. HPLC柱用85%溶剂A(100mM磷酸盐缓冲液,pH7.5,配方2)和15%溶剂B(乙腈)以1ml / min平衡1.5小时。
    30. 馏分收集器Frac-100装有4ml闪烁瓶。
    31. 将来自步骤A25的20μl样品用Hamilton注射器转移到LiChrograph HPLC的Rheodyne注射器环中,并将阀切换到注射位置。分数收集器被编程为500微升的分数大小。
    32. HPLC运行结束后(25分钟),将3ml闪烁鸡尾酒加入收集在闪烁小瓶中的每个馏分中。小瓶关闭,放射性用Tri-Carb LSC定量(图4)。
    33. 如步骤A29所述,HPLC柱在运行1.5小时后重新平衡。
    34. 用来自步骤A28的[14 C] - 标记的样品重复步骤A30至A33。

    35. 步骤A30至A33用[14C] retronecine标准重复
    36. 将等份的10μl来自步骤A28的剩余的[14 C] - 标记的样品在1.5ml微量离心管中与10μl[14 C] retrotine标准。
    37. 使用步骤A36的样本重复步骤A30至A33。

      “”src
      图4.绘制的放射性色谱图的实例A.步骤A32的未水解的纯化提取物。 B.步骤A34的水解纯化提取物。 C.步骤A35的[14 C] retronecine标准品。 D.步骤A37的标准和水解提取物的混合物。数据根据Kruse 等(2017)。

  2. 产品分析
    1. 将来自步骤A28的20μl剩余的[14 C] - 标记的样品以5μl的等分试样通过使用微毛细管移液管施加到TLC板上。确保溶剂完全蒸发后再施加下一个等份。这条车道标有[14C]。
    2. 以与步骤B1中所述相同的方式,将来自步骤A28的完整的[12 C] - 标记的样品应用于平行于步骤B1的泳道。该车道标有[<12> C]。
    3. TLC在20ml流动相(乙酸乙酯:异丙醇:氢氧化铵[30%,v / v]:45:35:20,配方3)的预饱和TLC显影室中显影。

    4. 薄层板在通风橱中干燥过夜。
    5. 使用带有GinaStar TLC软件的RITA系统检测TLC板的放射性。平行于[14C] - 平面的放射性斑点的[12C] - 平面的面积被刮除,包括上面1cm和1cm用刮勺将其置于2ml微量离心管中(图5)。

      “”src
      图5.产品分析的工作流程A.步骤B1到B4 41显示了开发的TLC。 B.步骤B5显示用RITA系统检测放射性后标记的斑点。 C.步骤B5显示平行于放射性点的[12C] -TLC泳道上的斑点被刮掉。 D.步骤B5到B6显示从TLC板上刮下的硅胶粉末并转移到2ml微量离心管中。

    6. 步骤B5的硅胶粉末通过向微量离心管中加入1ml的流动相而悬浮。将样品剧烈摇动并离心(1分钟,5000×g)。将上清液转移到新鲜的2ml微量离心管中并在通风橱中蒸发。
    7. 步骤B6重复三次,将上清液转移到同一个2ml微量离心管中以确保完全提取。
    8. 将步骤B7产生的干燥残余物溶解在25μl甲醇中,并转移到μVial进行GC-MS分析。用AOC-20i自动注射器注射1μl,在70eV记录电子碰撞质谱。气相色谱条件如下:注射器:250℃;喷射模式不分流;不分流时间1分钟,温度程序:60℃3分钟和60℃至300℃16℃min -1 -1;载气,氦气以1ml min -1(图6)。
      注意:任何市售的GC-MS系统都可以用于此目的,不建议直接注入放射性的区域,这是由于污染昂贵的设备的风险以及不可用的 14 C标记的参考光谱。


      图6. GC-MS运行的示例:一种。显示在步骤B8中测量的样品的总离子色谱图(TIC)。 B.显示来自紫草的水解的吡咯烷生物碱的分子量的提取离子色谱图。 C.显示保留指数为1478的质谱,TIC和EIC的最大峰,通过与文献和NIST数据库的比较确定为逆转录酶。数据根据Kruse 等(2017)。

数据分析

  1. 通过在步骤A1至A8中用Tri-Carb LSC测量样品等分试样的放射性而得到的数据允许计算作为示踪剂的[14C]腐胺的总摄取量,每分钟衰减(dpm)。
    放射性计算使用以下公式:
    总放射性[dpm] =(放射性测量值)[dpm] x(总体积)[μl] / 5
    由步骤A14至A21中得到的等分试样得到的放射性值可以得出关于掺入放射性的代谢物的化学性质的结论:硫酸提取物中放射性的量表示掺入水溶性和酸性可提取分子中的放射性的量。由于它们是用氨甲醇从SPE中洗脱下来的,因此它们很可能是弱碱,例如生物碱(图3)。
  2. 步骤A30至B1的HPLC运行不需要HPLC检测器,因为针对各个级分测量的放射性相对于分数时间点绘图并且由此递送直接放射性图谱。这可以通过使用标准的数据处理软件,如Microsoft Excel,Apple Numbers或Libre Calc或在纸上手动完成。如果掺入示踪剂的SPE纯化的分子携带侧链,所述侧链被酯化为源自示踪剂的[14 C] - 标记的核心结构,则所得到的峰提供见解。与标准物的比较已经是可能将腐胺并入生物碱的首要证据。
  3. 在吡咯里西啶生物碱的情况下,从TLC斑点提取的化合物的GC-MS总离子色谱图将通过提取代表[M] 的155 m / z离子进行处理, + retroneine核心结构的分子。在单峰的情况下,该峰的EI质谱允许与如NIST的光谱库或先前公开的吡咯里西啶生物碱的数据进行比较。

笔记

  1. 确保正确关闭管子。为了安全起见,建议在管盖上加一些重量,以免被热量打开。
  2. 这个过程可能需要很长的时间,这取决于通风橱的层流空气流动,并且可以通过将水与甲醇以1:1的比率混合,或者通过使用氮气流或离心蒸发器来加速。

食谱

  1. 5%氨的甲醇溶液
    在容量瓶中,将16.67ml 30%氨溶液加入到83.33ml甲醇中
  2. 100 mM磷酸盐缓冲液,pH 7.5
    将2.449g KH 2 PO 4和14.143g K 2 HPO 4称量到容量瓶中并补足到去离子水1升
  3. 流动相TLC
    在烧杯中,将45ml乙酸乙酯与35ml 2-丙醇和20ml 30%(v / v)氨溶液混合。流动相必须在使用前新鲜制备

致谢

这项工作由迪特里希•奥伯(Dietrich Ober)给予的DFG资助。我们感谢Dorothee Langel博士的投入和对此方法的贡献。我们感谢Kiel大学的Christoph Gelhaus博士和Maren Hartelt帮助实施这种方法。作者宣称没有利益冲突。

参考

  1. Frölich,C.,Ober,D.和Hartmann,T。(2007)。 在三个紫杉科的组织分布,lycopsamine类型的pyrrolizidine生物碱的核心生物合成和多样化。 Phytochemistry 68(7):1026-1037。
  2. Hartmann,T.,Theuring,C.,Witte,L.和Pasteels,J.M。(2001)。 新热带叶甲虫Platyphora boucardi的三级吡咯里西啶生物碱的螯合,代谢和部分合成。昆虫生化分子生物学 31(11):1041-1056。
  3. Kruse,L.H.,Stegemann,T.,Sievert,C。和Ober,D。(2017)。 确定菊花中吡咯里西啶生物碱生物合成的第二个位点以促进花期的植物防御。植物生理学“(Plant Physiol)174(1):47-55。
  4. Lindigkeit,R.,Biller,A.,Buch,M.,Schiebel,H.M.,Boppre,M。和Hartmann,T。(1997)。 吡咯里西啶生物碱的两面:叔胺及其N-氧化物在化学防御中的作用获得性植物生物碱的昆虫 Eur J Biochem 245(3):626-636。
  5. Niemüller,D.,Reimann,A。和Ober,D。(2012)。 参与吡拉西啶生物碱生物合成的高精胺酸合酶在三种双岐杆菌中的不同细胞特异性表达。 / a> Plant Physiol 159(3):920-929。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Stegemann, T., Kruse, L. H. and Ober, D. (2018). Radioactive Tracer Feeding Experiments and Product Analysis to Determine the Biosynthetic Capability of Comfrey (Symphytum officinale) Leaves for Pyrrolizidine Alkaloids. Bio-protocol 8(3): e2719. DOI: 10.21769/BioProtoc.2719.
  2. Kruse, L. H., Stegemann, T., Sievert, C. and Ober, D. (2017). Identification of a second site of pyrrolizidine alkaloid biosynthesis in comfrey to boost plant defense in floral stage. Plant Physiol 174(1): 47-55.
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