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Extraction and Reglucosylation of Barbarea vulgaris Sapogenins
欧洲山芥皂苷配基的提取和葡糖基化修饰   

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Abstract

Plants produce a vast array of natural compounds. Many of them are not commercially available, and are thus lacking to be tested as substrates for enzymes. This protocol describes the extraction and acidic hydrolysis of metabolites from Barbarea vulgaris with special focus on saponins and their agylcones (sapogenins). It was developed to determine if some B. vulgaris UDP-glucosyltransferases (UGTs) that were shown to glucosylate commercially available sapogenins, would also accept additional sapogenins from this plant as substrate, which are yet chemically uncharacterized and/or commercially unavailable (Figure 1).



Figure 1. Glucosylation reaction catalyzed by UGT73C10-UGT73C13 from Barbarea vulgaris (Augustin et al., 2012). All four enzymes utilize uridine diphosphate glucose (UDP-glc) as glucosyl-moiety donor and different sapogenins such as the oleanane sapogenins oleanolic acid and hederagenin as glucosyl-moiety acceptor. Oleanolic acid and hederagenin both naturally occur in G-type B. vulgaris, where they are predominantly found in their 3-O-cellobiosylated form. Additional saponins from G-type B. vulgaris have been identified by Nielsen et al., 2010. However, the majority of saponins and sapogenins that occur in B. vulgaris remain unidentified.

Materials and Reagents

  1. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  2. Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: 77627 )
  3. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  4. Tris(hydroxymethyl)aminomethane (Tris base) (Sigma-Aldrich, catalog number: T1503 )
  5. Ethyl acetate (Sigmal-Aldrich, catalog number: 34972 )
  6. N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) (Sigma-Aldrich, catalog number: T5130 )
  7. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  8. Uridine-5’-diphosphoglucose (UDP-Glc) (Sigma-Aldrich, catalog number: S451649 )
  9. Silica gel 60 F254 TLC plates (EMD Millipore, catalog number: 1055540001 )
  10. Polyvinylidene difluoride (PVDF) filter plate (0.45 μm pore diameter) (EMD Millipore, catalog number: MAHVN4510 )
  11. FRETWorks S-tag assay kit (EMD Millipore, catalog number: 70724 )

Equipment

  1. Water bath
  2. Centrifuge for 50 ml and 15 ml conical centrifugation tubes (VWR international, catalog number: 89004-368 )
  3. Thermomixer (VWR international, catalog number: 21516-168 )
  4. pH indicator paper (Whatman, catalog number: 2600-100A )
  5. Vacuum centrifuge (Labogene, catalog number: 7.008.100.777 )
  6. Thin layer chromatography (TLC) developing chamber (VWR international, catalog number: 21432-739 )
  7. Aldrich flask-type sprayer (Sigma-Aldrich, catalog number: Z190373 )
  8. Heat block (VWR international, catalog number: 12621-120 )
  9. LC-MS analysis was carried out on an Agilent 1100 Series LC (Agilent Technologies), equipped with a Gemini NX column (Phenomenex), and coupled to a Bruker HCT-Ultra ion trap mass spectrometer (Bruker Daltonics)

Software

  1. DataAnalysis 4.0 (Bruker Daltonics)

Procedure


I.   Preparation of the crude metabolite extract

  1. Freshly harvested Barbarea vulgaris leaves were weighed and transferred to 15 ml centrifugation tubes.
  2. Following addition of 5 ml 55% ethanol per g fresh leaf material the leaves were boiled in a water bath for 10 min.
  3. To increase the extraction efficiency, the tubes were occasionally shaken while boiling.
  4. After heating the extracts were chilled on ice before they were centrifuged for 5 min (3,000 x g, room temperature) to precipitate insoluble leaf debris.
  5. The supernatant was transferred to fresh centrifugation tubes and stored at -20 °C until further usage. A minimum incubation time of 4 h at -20 °C is recommended to cause further unwanted compounds to precipitate from the solution.
  6. Newly emerged precipitates were removed by centrifugation (3,000 x g, 5 min, 4 °C).
    Notes:
    1. Usage of the protocol has been limited so far to rosette leaves of 1-3 month old Barbarea vulgaris plants with a typical weight of 1.5-2 g fresh weight.
    2. Saponins can be extracted with this protocol from both fresh and ground plant material.
    3. 55% ethanol has been determined in pre-experiments to be hydrophobic enough to still extract B. vulgaris saponins, while being hydrophilic enough to lower the amount of some hydrophobic compounds that were previously seen to interfere with TLC analysis. However, it should be noted that these extracts still contains many more compounds than just saponins.


II.  Acidic hydrolysis and purification:

  1. 2 x 1.25 ml of the crude saponin extract were transferred into 2 ml microcentrifuge tubes and mixed with 250 μl 6 M HCl to adjust the final HCl concentration to 1 M.
  2. The acidified extracts were incubated for 24 h in a thermomixer adjusted to a temperature of 99 °C and shaking at 1,400 rpm.
  3. After heating the extracts were chilled for approximately 1 h at -20 °C before they were combined in 50 ml centrifugation tubes.
  4. Remaining precipitates in both microcentrifuge tubes were recovered by washing each tube three times with 250 μl 96% ethanol. The resulting ethanol solutions of these three wash steps were added to the hydrolysate in the 50 ml centrifugation tubes.
  5. 1 M Tris base solution was added to the hydrolysate until the pH shifted from acidic to basic conditions (here: 4.5 ml).
  6. Subsequently, 13.55 ml water was added to lower the ethanol concentration to 14%. 1.125 g PVPP and 225 mg BSA were added to the solution to adjust their final concentrations to 5% (w/v) and 10 mg ml-1, respectively.
  7. The mixture was six times extracted ethyl acetate using 5 ml ethyl acetate per extraction step.
  8. Phase separation was achieved by centrifugation for 20 min at 5,200 x g. The ethyl acetate fraction will be the upper phase.
  9. The combined ethyl acetate fractions were evaporated to dryness in a vacuum centrifuge.
  10. Dried extracts were dissolved in 500 μl 96% ethanol and transferred to 15 ml centrifugation tubes. For a second round of purification 3,720 μl water, 480 μl 500 mM TAPS pH 9.1, 240 mg PVPP and 48 mg BSA were added in the given order and 5-fold ethyl acetate extraction performed with 2 ml ethyl acetate per extraction step.
  11. After evaporation of the solvent of the combined ethyl acetate fractions in a vacuum centrifuge, the dried extracts were dissolved in 1 ml 96% ethanol.
    Notes:
    1. Brief spinning in a tabletop microcentrifuge was found sufficient during the washing steps to recover precipitates from the hydrolysate.
    2. Due to a lack of investigations if sapogenins will remain solubilized in the chosen hydrolysation conditions or are among the observed precipitates both fractions combined were subjected to subsequent purification steps.
    3. The pH of the hydrolysate was shifted to basic conditions by addition of Tris base prior extraction, since ethyl acetate extraction carries over low amounts of water/ions, which caused the initial hydrolysate extracts to be of slightly acidic pH. The UGTs investigated by us had a slightly basic pH optimum and a weakly basically buffered sapogenin extract was considered to have a lower effect on the pH of the final enzyme assay.
    4. pH changes were monitored by spotting 1 μl of the hydrolysate to pH indicator paper.
    5. The ethanol concentration of the hydrolysate had to be lowered prior ethyl acetate, extraction to enable formation of an organic phase upon addition of ethyl acetate.
    6. Early ethyl acetate extracts of hydrolysated crude Barbarea vulgaris leaf extracts generated without the PVPP/BSA purification step were seen to completely inhibit the activity of the investigated UGTs. PVPP was used to adsorb phenolic compounds. BSA was added in the purification step, since in enzyme assays using the early hydrolysation extracts proteins were seen to become brownish by binding to compounds from the extract. The addition of BSA was intended to remove such protein binding compounds.
    7. While drying down the ethyl acetate fractions in the vacuum centrifuge, new aqueous phases emerged, which were removed in the process.


III. Re-glucosylation assay:

  1. In preparation of the re-glucosylation assays 500 μl of the hydrolysated and purified B. vulgaris leaf metabolite extracts were dried out in a vacuum centrifuge and subsequently dissolved in 78.13 μl dimethyl sulfoxide (DMSO).
  2. Additionally, the recombinant expressed UGTs were directly quantified within E. coli lysates applying the FRETWorks S-tag assay kit.
  3. Following quantification, UGT concentrations were adjusted to 50 ng μl-1 by diluting the E. coli lysates with 10 mg ml-1 BSA in 10 mM TAPS pH 8.0.
  4. Enzymatic activity assays were performed in 1.5 ml microcentrifugation tubes in a final volume of 50 μl.
  5. Reaction conditions were adjusted to 25 mM TAPS pH 8.6 (UGT73C9-C11), pH 7.9 (UGT73C12/C13) or pH 8.2 (combination of UGT73C9, UGT73C10 or UGT73C11 with UGT73C12 or UGT73C13), 1 mM DTT and 1 mM UDP-Glc. The final UGT amount per reaction was 750 ng.
  6. Reactions were preincubated for 3 min at 30 °C and started by addition of 3.13 μl hydrolysated and purified B. vulgaris leaf metabolite extract in DMSO.
  7. The assays were incubated for 30 (LC-MS only) or 120 (TLC and LC-MS) min at 30 °C, and stopped by addition of 325 μl ice cold methanol (LC-MS) or 50 μl ice cold ethyl acetate (TLC).
    Notes:
    1. The solvent of the hydrolysated extracts was exchanged from ethanol to DMSO prior to the re-glucosylation assays, as ethanol was found to act as substrate for the applied UGTs itself.
    2. Quantification with the FRETWorks S-tag assay kit is based on regeneration of RNase S activity due the interaction of the S protein (included in the kit) and the S-tag N-terminally fused to the recombinant expressed UGTs.
    3. The E. coli lysates were diluted with a BSA solution instead of pure buffer, since the recombinant UGTs were seen to lose specific activity upon reduction of  the total protein concentration.
    4. Whenever combinations of different UGTs were tested, the individual enzymes were applied in equimolar amounts.


IV. Analysis by thin layer chromatography (TLC)

  1. Stopped enzymatic reactions were three times extracted with ethyl acetate (50 μl, 185 μl and 50 μl):
    1. Ethyl acetate was added to the enzymatic reaction and the sample thoroughly mixed for approximately 10-20 sec with a vortex shaker. (The ethyl acetate added to stop the reaction is at the same time also used for the first extraction step.)
    2. The samples were centrifuged for 5 min (16,100 x g, room temperature) to achieve phase separation. The ethyl acetate fraction will be the upper phase.
  2. The combined ethyl acetate fractions were evaporated to dryness in a vacuum centrifuge and the dried extracts dissolved in 20 μl 96% ethanol.
  3. The re-dissolved extracts were stepwise, completely (3.5 μl per step) loaded to a silica gel TLC plate.
  4. TLC plates were pre-run in 100% methanol until the solvent front was approximately 1 cm above the loading line.
  5. The methanol was left to evaporate in a fume hood, and the TLC plates were subsequently developed using dichloromethane: methanol: water (80: 19: 1) as mobile phase.
  6. Sapogenins and sapogenin-glucosides were visualized by spraying TLC plates with 10% sulfuric acid in methanol using a flask-type sprayer (or similar) and subsequent heating to 100 °C on a heat block (Figure 2).
    Notes: The amount of developing solution needed depends on the size of the used TLC plate. The plate should be consistently and homogeneously wetted. However, spraying of too much developing solution may cause the bands to diffuse.


    Figure 2. TLC plate with the (1) G-type B. vulgaris crude metabolite extract, the (2) corresponding acidic hydrolyzed metabolite extract and the (3)-(7) hydrolyzed metabolite extract treaded with different B. vulgaris UGTs.
    The TLC plate was evaluated under (A) visible (colored) as well as under (B) long wave UV (366 nm, black/white). The applied UGTs for the reglucosylation assays were (3) UGT73C9, (4) UGT73C10, (5) UGT73C11, (6) UGT73C12, (7) UGT73C13. For comparison purpose were authentic (oa) oleanolic acid, (he) hederagenin, (oa-glc) 3-O-glc oleanolic acid, (he-glc) 3-O-glc-hederagenin loaded to the (ref) reference lane (2 nmol each). Additionally are (oa-cell) oleanolic acid cellobioside and (he-cell) hederagenin cellobioside, the naturally in G-type B. vulgaris occurring di-glucosidic forms of these two sapogenins, marked in the crude metabolite extract. The accordingly estimated migration rate of (agly) aglycones, (m-glc) mono-glucosides and (di-glc) di-glucosides are shown on the right of Figure 2B.

V.  Analysis by liquid chromatography-mass spectrometry (LC-MS)

  1. Stopped enzymatic reactions were centrifuged for 5 min (16,100 x g, room temperature) to precipitate proteins.
  2. Supernatants were transferred to fresh 1.5 ml microcentrifugation tubes and evaporated to dryness in a vacuum centrifuge.
  3. Dried extracts were dissolved in 30 μl methanol and the solvent subsequently diluted to a final concentration of 50% methanol by addition of 30 μl water.
  4. The methanol extracts were filtered (PVDF, 0.45 μm pore diameter) and transferred to 1.5 ml glass sample vials for LC-MS analysis.
  5. LC-MS analysis was carried out on an Agilent 1100 Series LC, equipped with a Gemini NX column (35 °C) (2.0 x 150 mm, 3.5 μm), and coupled to a Bruker HCT-Ultra ion trap mass spectrometer.
  6. Mobile phases in the LC were water with 0.1% (v/v) formic acid (eluent A) and acetonitrile with 0.1% (v/v) formic acid (eluent B). The gradient program was as follows: 0 to 1 min, isocratic 12% B; 1 to 33 min, linear gradient 12 to 80% B; 33 to 35 min, linear gradient 80 to 99% B; 35 to 38 min isocratic 99% B; 38 to 45 min isocratic 12% B at a constant flow rate of 0.2 ml min-1.
  7. The MS detector was operated in negative electrospray mode, and MS2 ( = MS/MS) and MS3 (=MS/MS of MS2 fragments) fragmentations were performed to obtain additional structural information of the detected ions.
  8. Run files were analyzed with DataAnalysis 4.0, a software to display the LC chromatograms and the corresponding MS spectrums. Please refer to Augustin et al., 2012 (and Online Supplemental Data) to see the LC chromatograms of crude metabolite extracts from G- and P-type B. vulgaris, the acidic hydrolyzed metabolite extracts from both plants as well as chromatograms of the corresponding reglucosylation assays with different B. vulgaris UGTs.

References

  1. Augustin, J. M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J. K., Khakimov, B., Olsen, C. E., Hansen, E. H., Kuzina, V., Ekstrom, C. T., Hauser, T. and Bak, S. (2012). UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol 160(4): 1881-1895.
  2. Nielsen, N. J., Nielsen, J. and Staerk, D. (2010). New resistance-correlated saponins from the insect-resistant crucifer Barbarea vulgaris. J Agric Food Chem 58(9): 5509-5514.

简介

植物产生大量的天然化合物。 其中许多不是可商购的,因此缺乏作为酶的底物的测试。 该协议描述了从寻常型巴布巴属植物中提取和酸性水解代谢物,特别关注皂苷及其苷元(皂苷元)。 它被开发以确定是否有一些。 显示用于使市售皂苷元葡萄糖基化的普通UDP葡萄糖基转移酶(UGT)也将接受来自该植物的另外的皂苷元作为底物,其仍然是化学上未表征的和/或商业上不可获得的(图1)。



图1.来自巴巴多巴的UGT73C10-UGT73C13催化的葡萄糖基化反应(Augustin等人,2012)。 所有四种酶利用尿苷二磷酸葡萄糖(UDP-glc)作为葡萄糖基部分供体和不同的皂苷元,例如齐墩果烷醇皂甙元齐墩果酸和常春藤苷元作为葡糖基部分接受体。齐墩果酸和常春藤苷酸都天然存在于G型B中。 vulgaris,其中它们主要以其3-碳/β-半乳糖基化形式存在。来自G型的附加皂苷B. vulgaris 已经由Nielsen等人2010鉴定。然而,发生在B中的大多数皂苷和皂苷元。 vulgaris 仍然不明。

材料和试剂

  1. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  2. 聚乙烯聚吡咯烷酮(PVPP)(Sigma-Aldrich,目录号:77627)
  3. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  4. 三(羟甲基)氨基甲烷(Tris碱)(Sigma-Aldrich,目录号:T1503)
  5. 乙酸乙酯(Sigmal-Aldrich,目录号:34972)
  6. N-三(羟甲基)甲基-3-氨基丙磺酸(TAPS)(Sigma-Aldrich,目录号:T5130)
  7. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  8. 尿苷-5'-二磷酸葡萄糖(UDP-Glc)(Sigma-Aldrich,目录号:S451649)
  9. 硅胶60F 254 TLC板(EMD Millipore,目录号:1055540001)
  10. 聚偏二氟乙烯(PVDF)滤板(孔径0.45μm)(EMD Millipore,目录号:MAHVN4510)
  11. FRETWorks S标签测定试剂盒(EMD Millipore,目录号:70724)

设备

  1. 水浴
  2. 离心机用于50ml和15ml锥形离心管(VWR international,目录号:89004-368)
  3. Thermomixer(VWR international,目录号:21516-168)
  4. pH指示剂纸(Whatman,目录号:2600-100A)
  5. 真空离心机(Labogene,目录号:7.008.100.777)
  6. 薄层色谱(TLC)显影室(VWR international,目录号:21432-739)
  7. Aldrich烧瓶型喷雾器(Sigma-Aldrich,目录号:Z190373)
  8. 热块(VWR international,目录号:12621-120)
  9. 在配备有Gemini NX柱(Phenomenex)的Agilent 1100系列LC(Agilent Technologies)上进行LC-MS分析,并与Bruker HCT-Ultra离子阱质谱仪(Bruker Daltonics)偶联,

软件

  1. DataAnalysis 4.0(Bruker Daltonics)

程序


I.   制备粗代谢物提取物

  1. 称重新鲜收获的野螟叶,并转移到15ml离心管中。
  2. 在每克新鲜叶材料加入5ml 55%乙醇后,将叶子在水浴中煮沸10分钟。
  3. 为了提高提取效率,偶尔在沸腾时摇动管。
  4. 加热后,将提取物在冰上冷却,然后将其离心5分钟(3,000xg,室温),以沉淀不溶性叶碎片。
  5. 将上清液转移至新鲜离心管中并在-20℃下储存直至进一步使用。 建议在-20°C下最少孵育时间为4小时,以使更多的不需要的化合物从溶液中沉淀。
  6. 通过离心(3,000×g,5分钟,4℃)除去新出现的沉淀物。
    注意:


    1. 在预实验中已经确定55%的乙醇是足够疏水以仍然提取普通皂甙,同时亲水性足以降低之前见到的干扰TLC分析的一些疏水化合物的量。 然而,应该注意,这些提取物仍然含有比仅仅皂苷更多的化合物。


II。  酸性水解和纯化:

  1. 将2×1.25ml粗皂甙提取物转移到2ml微量离心管中,并与250μl6M HCl混合,将最终HCl浓度调节至1M。
  2. 将酸化的提取物在调节至99℃的温度并以1,400rpm振摇的热混合器中温育24小时。
  3. 加热后,将提取物在-20℃下冷冻约1小时,然后将其在50ml离心管中合并。
  4. 通过用250μl96%乙醇洗涤每个管3次回收在两个微量离心管中的剩余沉淀。 将这三个洗涤步骤的所得乙醇溶液加入50ml离心管中的水解产物中。
  5. 将1M Tris碱溶液加入到水解产物中直至pH从酸性变为碱性条件(这里为4.5ml)。
  6. 随后,加入13.55ml水以将乙醇浓度降低至14%。将1.125g PVPP和225mg BSA加入到溶液中,以将它们的最终浓度分别调节至5%(w/v)和10mg m -1 s -1。
  7. 将混合物用乙酸乙酯萃取6次,每次萃取使用5ml乙酸乙酯。
  8. 通过在5,200×g离心20分钟实现相分离。乙酸乙酯部分为上层相
  9. 将合并的乙酸乙酯级分在真空离心机中蒸发至干。
  10. 将干燥的提取物溶于500μl96%乙醇中,并转移至15ml离心管中。对于第二轮纯化,以给定的顺序加入3,720μl水,480μl500mM TAPS pH 9.1,240mg PVPP和48mg BSA,并且每次提取步骤用2ml乙酸乙酯进行5倍乙酸乙酯萃取。
  11. 在真空离心机中蒸发合并的乙酸乙酯级分的溶剂后,将干燥的提取物溶于1ml 96%乙醇中。 注意:
    1. 在洗涤步骤期间,发现在台式微量离心机中短暂旋转足以从水解产物中回收沉淀。
    2. 由于缺乏调查,如果皂草苷元在所选择的水解条件下保持溶解或者在观察到的沉淀物中,则将两种组分进行后续的纯化步骤。
    3. 通过在萃取前加入Tris碱将水解产物的pH转变为碱性条件,因为乙酸乙酯萃取带有低量的水/离子,这导致初始水解产物萃取物具有微酸性pH。我们研究的UGT具有略微碱性的pH最佳值,并且弱碱性缓冲的皂苷元提取物被认为对最终酶测定的pH具有较低的影响。
    4. 通过在pH指示剂纸上点样1μl水解产物来监测pH变化。
    5. 必须在乙酸乙酯之前降低水解产物的乙醇浓度,萃取以在加入乙酸乙酯时形成有机相。
    6. 在没有PVPP/BSA纯化步骤的情况下产生的水解的粗制巴巴巴树叶提取物的早期乙酸乙酯提取物被认为完全抑制所研究的UGT的活性。 PVPP用于吸附酚类化合物。在纯化步骤中加入BSA,因为在使用早期水解提取物的酶测定中,观察到蛋白质通过与来自提取物的化合物结合而变为褐色。加入BSA是为了除去这种蛋白质结合化合物。
    7. 在真空离心机中干燥乙酸乙酯级分的同时,出现新的水相,在该过程中将其除去。


III。重糖基化测定:

  1. 在再葡糖基化测定的制备中,将500μl水解和纯化的B。寻常的叶代谢物提取物在真空离心机中干燥,随后溶于78.13μl二甲基亚砜(DMSO)中。
  2. 此外,重组表达的UGT在E内直接定量。应用FRETWorks S-tag测定试剂盒的大肠杆菌裂解物。
  3. 定量后,通过稀释E将UGT浓度调节至50ng /μl。大肠杆菌裂解物与在10mM TAPS pH 8.0中的10mg/ml BSA反应。
  4. 酶活性测定在1.5ml微量离心管中进行,终体积为50μl。
  5. 将反应条件调节至25mM TAPS pH 8.6(U​​GT73C9-C11),pH7.9(UGT73C12/C13)或pH8.2(UGT73C9,UGT73C10或UGT73C11与UGT73C12或UGT73C13的组合),1mM DTT和1mM UDP-Glc。每次反应的最终UGT量为750ng。
  6. 反应在30℃下预温育3分钟,并通过加入3.13μl水解和纯化的B开始。寻常的叶代谢物提取物在DMSO中。
  7. 将测定在30℃下孵育30分钟(仅LC-MS)或120分钟(TLC和LC-MS),并通过加入325μl冰冷甲醇(LC-MS)或50μl冰冷的乙酸乙酯TLC) 注意:
    1. 在再葡糖基化测定之前,将水解的提取物的溶剂从乙醇交换为DMSO,因为发现乙醇作为所应用的UGT本身的底物。
    2. 使用FRETWorks S标签测定试剂盒的定量是基于RNA酶S活性的再生,因为S蛋白(包含在试剂盒中)的相互作用和N末端融合的S标签重组表达的UGTs。
    3. 用BSA溶液代替纯缓冲液稀释大肠杆菌裂解物,因为看到重组UGT在减少后丧失比活性。 总计 蛋白质浓度。
    4. 每当测试不同UGT的组合时,以等摩尔量施用各个酶。


IV。 通过薄层色谱法(TLC)

  1. 停止的酶反应用乙酸乙酯(50μl,185μl和50μl)萃取三次:
    1. 向酶反应和样品中加入乙酸乙酯 用涡旋振荡器充分混合约10-20秒。 (The 加入乙酸乙酯停止反应的同时也使用 对于第一提取步骤。)
    2. 将样品离心   5分钟(16,100×g,室温),以实现相分离。 乙酸乙酯级分将是上相。
  2. 将合并的乙酸乙酯级分在真空离心机中蒸发至干,将干燥的提取物溶于20μl96%乙醇中
  3. 将再溶解的提取物逐步完全(每步3.5μl)加载到硅胶TLC板上。
  4. TLC板在100%甲醇中预运行,直到溶剂前沿在装载线上方约1cm。
  5. 将甲醇在通风橱中蒸发,随后使用二氯甲烷:甲醇:水(80:19:1)作为流动相显影TLC板。
  6. 通过使用烧瓶型喷雾器(或类似物)在TLC板上喷洒10%硫酸的甲醇溶液,随后在加热块(图2)上加热至100℃,使皂苷配基和皂苷元 - 葡糖苷显色。 注意: 所需的显影液量取决于所使用的TLC板的尺寸。板应该一致且均匀地润湿。 但是,喷洒过多的显影液可能会导致色带扩散。

    src ="/attached/image/20130713/20130713055933_2643.png" alt =""/>
    图2.具有(1)G型B的TLC板。 (2)相应的酸性水解代谢物提取物和(3) - (7)水解的代谢物提取物与不同的B接触。 vulgaris UGTs。 在(A)可见(有色)以及(B)长波UV(366nm,黑/白)下评价TLC板。应用的用于葡萄糖基化测定的UGT是(3)UGT73C9,(4)UGT73C10,(5)UGT73C11,(6)UGT73C12,(7)UGT73C13。为了比较的目的,使用了真正的(oa)齐墩果酸,(he)常春配基,(oa-glc)3-氨基-glc齐墩果酸,(he-glc)3-em (参考)参考泳道(每个2nmo​​l)上的β-glc-常春配基。另外是(oa细胞)齐墩果酸纤维二糖苷和(he-细胞)常春配基纤维二糖苷,天然存在于G型胚中。寻找这两种皂苷元的二糖苷形式,在粗代谢物提取物中标记。因此,(agly)糖苷配基,(m-glc)单葡萄糖苷和(二葡萄糖)二糖苷的迁移率显示在图2B的右侧。

V.  通过液相色谱 - 质谱(LC-MS)

  1. 将停止的酶反应离心5分钟(16,100×g,室温)以沉淀蛋白质。
  2. 将上清液转移到新鲜的1.5ml微量离心管中,并在真空离心机中蒸发至干。
  3. 将干燥的提取物溶于30μl甲醇中,随后通过加入30μl水将溶剂稀释至50%甲醇的最终浓度。
  4. 过滤甲醇提取物(PVDF,0.45μm孔径),并转移到1.5ml玻璃样品瓶中用于LC-MS分析。
  5. 在配备有Gemini NX柱(35℃)(2.0×150mm,3.5μm)的Agilent 1100系列LC上进行LC-MS分析,并与Bruker HCT-Ultra离子阱质谱联用。
  6. LC中的流动相是含有0.1%(v/v)甲酸(洗脱液A)和含有0.1%(v/v)甲酸的乙腈的水(洗脱液B)。梯度程序如下:0至1分钟,等度12%B; 1〜33分钟,线性梯度12〜80%B; 33至35分钟,线性梯度80至99%B; 35至38分钟等度99%B; 38至45分钟等度12%B,以0.2ml min <-1> 的恒定流速。
  7. MS检测器以负电喷雾模式操作,并且MS 2(= MS/MS)和MS (= MS/MS的MS/MS) >碎片)碎裂以获得检测离子的另外的结构信息。
  8. 运行文件用DataAnalysis 4.0分析,该软件显示LC色谱图和相应的MS谱图。请参见Augustin等人(2012)(和在线补充数据)以查看来自G-和P型B的粗代谢物提取物的LC色谱图。来自两种植物的酸性水解代谢物提取物以及用不同B的相应的葡萄糖基化测定的色谱图。 vulgaris UGT。

参考文献

  1. Augustin,JM,Drok,S.,Shinoda,T.,Sanmiya,K.,Nielsen,JK,Khakimov,B.,Olsen,CE,Hansen,EH,Kuzina,V.,Ekstrom,CT,Hauser, Bak,S。(2012)。 来自寻常型巴布达UGT73C亚家族的UDP-糖基转移酶催化皂苷元3- O-葡糖基化在皂苷介导的昆虫抗性中。植物生理学 160(4):1881-1895。
  2. Nielsen,N.J.,Nielsen,J。和Staerk,D。(2010)。 来自抗虫十字花科植物的抗性相关皂苷 Barbarea vulgaris 。 J Agric Food Chem 58(9):5509-5514。
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Copyright: © 2013 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. Augustin, J. M., Olsen, C. E. and Bak, S. (2013). Extraction and Reglucosylation of Barbarea vulgaris Sapogenins. Bio-protocol 3(14): e826. DOI: 10.21769/BioProtoc.826.
  2. Augustin, J. M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J. K., Khakimov, B., Olsen, C. E., Hansen, E. H., Kuzina, V., Ekstrom, C. T., Hauser, T. and Bak, S. (2012). UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol 160(4): 1881-1895.
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