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Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration
蛋白质提取、酸性磷酸酶活性分析以及可溶性蛋白浓度的测定   

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Abstract

Acid phosphatases (APases) catalyze the hydrolysis of inorganic phosphate (Pi) from a broad range of Pi-monoesters with an acidic pH optimum. The liberated Pi is reassimilated into cellular metabolism via mitochondrial or chloroplastic ATP synthases of respiration or photosynthesis, respectively. Eukaryotic APases exist as a wide variety of tissue- and/or cellular compartment-specific isozymes that display marked differences in their physical and kinetic properties. Increases in intracellular (vacuolar) and secreted APase activities are useful biochemical markers of plant nutritional Pi deficiency. The protocols for protein extraction, APase activity determination and measurement of soluble protein concentration from plant tissues or cell suspension cultures are presented.

Materials and Reagents

  1. Plant tissues
  2. Sodium acetate (Bioshop, catalog number: SAA305 )
  3. EDTA (Bioshop, catalog number: EDT001 )
  4. Dithiothreitol (DTT) (Bioshop, catalog number: DTT 002 )
  5. Phenylmethyl sulfonyl fluoride (PMSF) (G-Biosciences, catalog number: 786-0555 )
  6. Thiourea (Sigma-Aldrich, catalog number: T-7875 )
  7. Polyvinyl (polypyrrodlidone) (PVPP) (Sigma-Aldrich, catalog number: P-6755 )
  8. β-nicotinamide adenine dinucleotide reduced form (NADH) (Bioshop, catalog number: NAD002 )
  9. Phosphoelnolpyryvate (PEP) (Biovectra, catalog number: 2552 )
  10. MgCl2 ((Biolynx, catalog number: 18641 )
  11. Rabbit muscle lactate dehydrogenase (LDH) (Sigma-Aldrich, catalog number: L-2500 )
  12. para-nitrophenyl-phosphate (pNPP, phosphatase substrate) (Sigma-Aldrich, catalog number: P-4744 )
  13. Coomassie Brilliant blue G-250 (Serva, catalog number: 35050 C.!.42655 )
  14. Bovine gamma globulin (BGG) (2.0 mg/ml) (Thermo Fisher Scientific, catalog number: 23212 )
  15. NaOH
  16. Extraction buffer (EB) (see Recipes)
  17. Bradford working solution (see Recipes)
  18. Acid phosphatase enzyme assay mix #1 (see Recipes)
  19. Acid phosphatase enzyme assay mix #2 (see Recipes)
  20. Bradford assay stock (see Recipes)

Equipment

  1. Whatman #1 filter paper
  2. 1.5 ml microfuge tubes
  3. 10 and 25 μl Hamilton syringes
  4. Pipetor
  5. Small mortar and pestle
  6. Eppendorf microfuge
  7. 96 well polystyrene microtitre plate (flat bottom)
  8. A computer supported microplate spectrophotometer (Spectromax Plus, Molecular Devices, Sunnyvale)

Procedure

  1. Extraction

    This protocol applies to extraction of intracellular (vacuolar) APases from plant tissues and suspension cell cultures (e.g., Tran et al., 2010a; Veljanovski et al., 2006). Refer to Tran et al. (2010b) and Robinson et al. (2012) for information on the isolation and analysis of plant cell wall localized and secreted APase isoforms. 

    1. Weigh tissue and freeze in liquid N2. Store at -80 °C until use.
    2. Place a small spatula of sea sand into the mortar, add small amount of liquid N2, followed by frozen tissue, and grind to a powder. Carefully add more liquid N2 if needed to keep frozen.
    3. Invert tube with extraction buffer (EB) to mix and add to sample at a ratio of 1:2, w/v (e.g. 0.5 g powdered tissue + 1.0 ml EB) although this may need to be increased to 1:3 for leaf tissue and 1:4 for roots. Grind for several minutes and place in 1.5 ml microfuge tubes.
    4. Centrifuge 5 min at 4 °C and 11,000 x g. Remove supernatant (clarified extract) to a fresh microfuge tube and keep it on ice. Measure APase activity immediately at room temperature (24 °C). Aliquots of clarified extracts can be snap frozen in liquid N2 and stored at -80 °C for future use.

  2. APase activity assay 1
    1. Conveniently measure APase activity by coupling the hydrolysis of phosphoenolpyruvate (PEP) to pyruvate to the lactate dehydrogenase (LDH) reaction at 24 °C and using a spectrophotometer to continuously monitor NADH oxidation at 340 nm. PEP seems to be an excellent APase substrate for since it occupies the highest position on the thermodynamic scale of phosphorylated intermediates (and thus its P atom is an excellent leaving group). For every PEP molecule hydrolyzed to pyruvate, one NADH molecule is oxidized to NAD+ by LDH as shown in Figure 1.


      Figure 1. APase activity can be conveniently deterdmined by coupling the hydrolysis of PEP to pyruvate to the lactate dehydrogenase (LDH) reaction and using a spectrophotometer to continuously monitor NADH oxidation to NAD+ at 340 nm

    2. Accurately pipette 1-10 μl of clarified extract into a microplate well. We prefer to use a 10 or 25 μl Hamilton syringe as opposed to automated pipetors for accurate pipetting of enzyme protein extracts into wells of the microtitre plate.
    3. Use repeat pipetor to add 200 μl APase assay mix #1 to each well and immediately place in microplate spectrophotometer. Continuously monitor NADH oxidation to NAD+ as a decline in absorbance at 340 nm (A340), taking readings every 5-10 sec for up to 5 min.
    4. Correct for background NADH oxidation by omitting PEP from the reaction mixture. Ensure that the decline in A340 (amount of NADH being oxidized; ε340 = 6,220/M/cm) is proportional to assay time and concentration of enzyme assayed. Dilution of clarified extract in extraction buffer (lacking PVPP) may be necessary for samples containing abundant APase activity.
      Note: One international unit (U) of enzyme activity is defined as the amount of enzyme resulting in the hydrolysis of 1 μmol of substrate (e.g. one μmol of NADH oxidized to NAD+) per min at 24 °C. APase activity in (U/ml clarified extract) = (ΔA340/min x clarified extract dilution factor)/6.22. Thus, if 2.0 μl of clarified extract mixed with 200 μl of APase reaction mixture yields a ΔA340/min of 0.1 at 340 nm, then the APase activity = (0.1 x 100)/6.22 U/ml = 1.6 U/ml.

  3. APase activity assay 2

    This is a ‘stopped-time’ APase assay based upon the hydrolysis of the synthetic substrate, para-nitrophenyl-phosphate (pNPP), to para-nitrophenol (pNP) and Pi (Figure 2). The pNP product forms a yellow color at alkaline pH (λmax = 410 nm; extinction coefficient = ε410 = 18.2/mM/cm; meaning a 1 mM solution of pNP should have an A410 of 18.2). The amount of yellow color formed is thus directly proportional to the amount of pNP produced and is therefore an indicator of the APase activity. This assay tends to be more popular in the APase literature. However, care needs to be taken to ensure that the amount of pNP being formed is proportional to the assay time and volume of clarified extract being assayed.


    Figure 2. APase activity is often assayed spectrophotometrically at 410 nm by determining the amount of pNP produced following the hydrolysis of Pi from pNPP. Addition of NaOH after a specified assay time (e.g., 10 min) serves to stop the APase reaction while simultaneously converting the product p-nitrophenol into the yellow colored p-nitrophenolate (λmax = 410 nm).

    1. Accurately pipette 1-10 μl of clarified enzyme extract into a well of the microtitre plate. Add 200 μl of APase assay mix #2 to each well containing enzyme extract and incubate for 10 min at room temperature (24 °C).
    2. At t = 10 min, add 50 μl of 3 M NaOH to each well containing APase reactions. This stops the reaction (denatures APase) while simultaneously converting pNP product into the yellow colored p-nitrophenolate.
    3. Determine ΔA410/min for each well to determine the amount (μmol) of pNP formed per min.

  4. Bradford Assay of Soluble Protein Concentration
    1. Prepare standard curve using the template below:
      Well #
      Vol BGG (0.4 mg/ml)
      Amt Protein (BGG)
      Vol H2O

      (μl)
      (μg)
      (μl)
      1a
      0
      0
      25 (blank)
      1b
      2
      0.8
      23
      1c
      4
      1.6
      21
      1d
      8
      3.2
      17
      1e
      12
      4.8
      13
      1f
      16
      6.4
      9
      1g
      20
      8.0
      5
      1h
      25
      10.0
      0
      2a
      0
      0
      25 (blank)
    2. Pipette 2-20 μl of the clarified extract (dilute as necessary) and adjust final volume to 25 μl in each well with H2O. Dilute sample if necessary to remain in linear range of standard curve. A 25 μl Hamilton syringe is more accurate than automated pipetors for pipetting of BGG standard and clarified extract into wells of the microtitre plate.
    3. Add 250 μl of Bradford working solution to each well using a repeat pipetor and read A595 of protein standards and unknowns. Ensure that A595 values of clarified extract samples aliquot falls within range of A595 values of the BGG standards. The absorbance maximum for an acidic solution of Coomassie Brilliant blue G-250 shifts from 465 nm to 595 when protein binding occurs. Determine amount of protein in clarified extract aliquot from the standard curve (if 2 μl aliquot of clarified extract yields an A595 value equivalent to 5 μg of protein, then the extract would have a protein concentration of 2 mg/ml).

Recipes

  1. Extraction buffer (EB, keep on ice)
    50 mM Na-acetate (pH 5.6)
    1 mM EDTA
    1 mM DTT
    1 mM PMSF (prepare 100 mM stock in absolute ethanol and store at -20 °C, add fresh to EB immediately prior to tissue extraction as PMSF is unstable in aqueous solution)
    5 mM thiourea
    1% (w/v) PVPP
  2. APase assay #1 reaction mixture (prepare freshly and keep at room temperature)
    50 mM Na-acetate (pH 5.6)
    10 mM MgCl2
    0.2 mM NADH
    5 mM PEP
    3 U/ml rabbit muscle LDH (Desalt LDH by centrifuging an aliquot 3 min at 11,000 x g. Discard supernatant and resuspend the pellet in an equal volume of EB).
  3. APase assay #2 reaction mixture
    10 mM pNPP (sodium salt), dissolved in 50 mM acetate-KOH (pH 5.8)
    3 M sodium hydroxide (NaOH) also needed to stop the reaction
  4. Bradford assay stock
    100 ml 95% EtOH
    200 ml 88% H3PO4
    350 mg Brilliant blue G-250
  5. Bradford working solution
    30 ml Bradford Stock
    425 ml H2O
    15 ml EtOH
    30 ml 88% H3PO4
    Filter through Whatman #1 filter paper and store in brown or dark glass bottle.
    Protein assay solutions are stable for months at room temperature.
  6. Bradford Protein standard
    Dilute bovine Gamma Globulin with H2O to 0.4 mg/ml
    Store 100 μl aliquots at -20 °C.

Acknowledgments

Research in our laboratory has been generously funded by research and equipment grants from The Natural Sciences and Engineering Research Council of Canada (NSERC) and Queen’s Research Chairs program to William Plaxton.

References

  1. Robinson, W. D., Park, J., Tran, H. T., Del Vecchio, H. A., Ying, S., Zins, J. L., Patel, K., McKnight, T. D. and Plaxton, W. C. (2012). The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana. J Exp Bot 63(18): 6531-6542. 
  2. Tran, H. T., Hurley, B. A. and Plaxton, W. C. (2010a). Feeding hungry plants: the role of purple acid phosphatases in phosphate nutrition. Plant Sci 179(1): 14-27.
  3. Tran, H. T., Qian, W., Hurley, B. A., She, Y. M., Wang, D. and Plaxton, W. C. (2010b). Biochemical and molecular characterization of AtPAP12 and AtPAP26: the predominant purple acid phosphatase isozymes secreted by phosphate-starved Arabidopsis thaliana. Plant Cell Environ 33(11): 1789-1803.
  4. Veljanovski, V., Vanderbeld, B., Knowles, V. L., Snedden, W. A. and Plaxton, W. C. (2006). Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings. Plant Physiol 142(3): 1282-1293.

简介

酸性磷酸酶(APase)催化来自具有酸性pH最佳值的宽范围的P 1 - 单酯的无机磷酸盐(P 1)的水解。 释放的Pi分别通过呼吸或光合作用的线粒体或叶绿体ATP合酶再吸收到细胞代谢中。 真核APase作为多种组织和/或细胞区室特异性同工酶存在,其在它们的物理和动力学性质上显示出显着的差异。 细胞内(液泡)和分泌的APase活性的增加是植物营养缺乏的有用的生物化学标记物。 提出了蛋白质提取,APase活性测定和来自植物组织或细胞悬浮培养物的可溶性蛋白质浓度的测定方案。

材料和试剂

  1. 植物组织
  2. 醋酸钠(Bioshop,目录号:SAA305)
  3. EDTA(Bioshop,目录号:EDT001)
  4. 二硫苏糖醇(DTT)(Bioshop,目录号:DTT002)
  5. 苯基甲基磺酰氟(PMSF)(G-Biosciences,目录号:786-0555)
  6. 硫脲(Sigma-Aldrich,目录号:T-7875)
  7. 聚乙烯基(聚吡咯烷酮)(PVPP)(Sigma-Aldrich,目录号:P-6755)
  8. β-烟酰胺腺嘌呤二核苷酸还原型(NADH)(Bioshop,目录号:NAD002)
  9. 磷酸芘酯(PEP)(Biovectra,目录号:2552)
  10. MgCl 2((Biolynx,目录号:18641)
  11. 兔肌肉乳酸脱氢酶(LDH)(Sigma-Aldrich,目录号:L-2500)
  12. 磷酸对硝基苯酯(pNPP,磷酸酶底物)(Sigma-Aldrich,目录号:P-4744)
  13. 考马斯亮蓝G-250(Serva,目录号:35050C,42655)
  14. 牛γ-球蛋白(BGG)(2.0mg/ml)(Thermo Fisher Scientific,目录号:23212)
  15. NaOH
  16. 提取缓冲液(EB)(参见配方)
  17. Bradford工作解决方案(参见配方)
  18. 酸性磷酸酶酶测定混合#1(参见配方)
  19. 酸性磷酸酶酶测定混合#2(参见配方)
  20. Bradford检测库存(见配方)

设备

  1. Whatman#1滤纸
  2. 1.5 ml微量离心管
  3. 10和25μlHamilton注射器
  4. 移液器
  5. 小砂浆和杵
  6. Eppendorf微量离心器
  7. 96孔聚苯乙烯微量滴定板(平底)
  8. 计算机支持的微孔板分光光度计(Spectromax Plus,Molecular Devices,Sunnyvale)

程序

  1. 萃取

    该方案适用于从植物组织和悬浮细胞培养物中提取细胞内(液泡)APase(例如,Tran等人,2010a; Veljanovski等人,/em,2006)。请参阅Tran 等。 (2010b)和Robinson等人。 (2012)关于隔离和分析植物细胞壁定位和分泌的APase同种型的信息。

    1. 称重组织并在液体N 2中冷冻。储存于-80℃直至使用。
    2. 将一小块海沙放入砂浆中,加入少量液体N 2 2,然后冷冻组织,并研磨成粉末。小心地加入更多的液体N <2>如果需要保持冷冻
    3. 用提取缓冲液(EB)倒置管以1:2,w/v(例如0.5g粉末组织+ 1.0ml EB)的比例混合并添加到样品中,尽管这可能需要对于叶组织增加至1:3 1:4为根。研磨几分钟,并置于1.5ml微量离心管中
    4. 在4℃和11,000×g离心5分钟。除去上清液(澄清的提取物)到一个新鲜的微量离心管,并保持在冰上。在室温(24℃)下立即测量APase活性。可以将澄清提取物的等分试样快速冷冻在液体N 2中并储存在-80℃以备将来使用。

  2. APase活性测定1
    1. 通过在24℃下将磷酸烯醇丙酮酸(PEP)水解为丙酮酸至乳酸脱氢酶(LDH)反应并使用分光光度计在340nm连续监测NADH氧化来方便地测量APase活性。 PEP似乎是优异的APase底物,因为它在磷酸化中间体的热力学规模上占据最高位置(因此其P原子是优异的离去基团)。对于水解为丙酮酸的每个PEP分子,一个NADH分子被LDH氧化成NAD + sup/+,如图1所示。


      APase活性可以通过将PEP的水解与丙酮酸盐偶联至乳酸脱氢酶(LDH)反应并使用分光光度计连续监测NADH氧化为NAD的方式来方便地确定。 + 在340 nm

    2. 精确吸取1-10μl澄清的提取物到微孔板孔中。我们优选使用10或25μlHamilton注射器,而不是自动移液器,用于将酶蛋白提取物精确地移液到微量滴定板的孔中。
    3. 使用重复移液器添加200微升APase测定混合#1到每个孔,并立即放置在微孔板分光光度计。持续监测NADH氧化为NAD + ,作为340nm处吸光度的下降(​​A <340>),每5-10秒读取读数,持续5分钟。
    4. 通过从反应混合物中省略PEP来校正背景NADH氧化。确保A 340(NADH被氧化的量;ε340 = 6,220/M/cm)的下降与测定时间和测定的酶浓度成比例。对于含有丰富的APase活性的样品,可能需要在提取缓冲液(缺乏PVPP)中稀释澄清的提取物。
      注意:酶活性的一个国际单位(U)定义为每分钟导致1微摩尔底物水解的酶量(例如,1微摩尔NADH被氧化成NAD + )在24℃。 (U/ml澄清提取物)中的APase活性=(ΔA 340/min×澄清提取物稀释因子)/6.22。因此,如果2.0μl澄清的提取物与200μlAPase反应混合物混合,在340nm下产生0.1的ΔΔ340min/min,则APase活性=(0.1 x 100)/6.22U/ml = 1.6U/ml。

  3. APase活性测定2

    这是基于合成底物对硝基苯基磷酸(pNPP)水解成对硝基苯酚(pNP)和Pi(图2)的"停止时间"APase测定。 pNP产物在碱性pH(λmax = 410nm;消光系数=ε410 = 18.2/mM/cm 2)下形成黄色;意味着1mM的pNP溶液应具有A 410 18.2)。因此形成的黄色的量与产生的pNP的量成正比,因此是APase活性的指示剂。该测定倾向于在APase文献中更受欢迎。然而,需要注意确保形成的pNP的量与测定的澄清提取物的测定时间和体积成比例。


    图2.通常通过测定从pNPP水解P 1后产生的pNP的量,在410nm下通过分光光度法测定APase活性。 在指定的测定时间后(例如,10分钟)加入NaOH用于停止APase反应,同时将产物对硝基苯酚转化成黄色对硝基苯酚盐(λ max = 410nm)。

    1. 准确吸取1-10微升澄清的酶提取物到微量滴定板的孔中。 向每个含有酶提取物的孔中加入200μl的APase测定混合物#2,并在室温(24℃)下孵育10分钟。
    2. 在t = 10分钟时,向含有APase反应的每个孔中加入50μl的3M NaOH。 这停止反应(使APase变性),同时将pNP产物转化为黄色对硝基苯酚盐。
    3. 确定每个孔的ΔA410/min以确定每分钟形成的pNP的量(μmol)。

  4. Bradford Assay of Soluble Protein Concentration
    1. 使用以下模板制备标准曲线:
      呃#
      Vol BGG(0.4mg/ml)
      Amt蛋白(BGG)
      Vol H <2> O

      (μl)
      (μg)
      (μl)
      1a
      0
      0
      25(空白)
      1b
      2
      0.8
      23
      1c
      4
      1.6
      21
      1d
      8
      3.2
      17
      1e
      12
      4.8
      13
      1f
      16
      6.4
      9
      1g
      20
      8.0
      5
      1h
      25
      10.0
      0
      2a
      0
      0
      25(空白)
    2. 移取2-20μl澄清的提取物(根据需要稀释),并用H 2 O在每个孔中将终体积调节至25μl。如果需要稀释样品以保持在标准曲线的线性范围内。 25μlHamilton注射器比自动移液器更精确,用于将BGG标准品和澄清提取物移液到微量滴定板的孔中。
    3. 使用重复移液器向每个孔中加入250μl的Bradford工作溶液,并读取蛋白质标准品和未知物的A 595。确保澄清的提取物样品等分试样的A 595值落入BGG标准品的A <595数值范围内。当蛋白质结合发生时,考马​​斯亮蓝G-250的酸性溶液的最大吸光度从465nm变为595。从标准曲线确定澄清的提取物等分试样中的蛋白质的量(如果2μl等分的澄清的提取物产生等于5μg蛋白质的A 595值,则提取物的蛋白质浓度为2mg/ml)。

食谱

  1. 提取缓冲液(EB,保持冰)
    50mM乙酸钠(pH5.6) 1mM EDTA
    1 mM DTT
    1mM PMSF(制备在无水乙醇中的100mM储备液并储存在-20℃,在组织提取之前立即加入EB中,因为PMSF在水溶液中不稳定)
    5mM硫脲 1%(w/v)PVPP
  2. APase测定#1反应混合物(新鲜制备并保持在室温) 50mM乙酸钠(pH5.6) 10mM MgCl 2/
    0.2 mM NADH
    5mM PEP
    3 U/ml兔肌肉LDH(通过在11,000×g离心等分试样3分钟而得到脱盐LDH)弃去上清液并将沉淀物重悬于等体积的EB中。
  3. APase测定#2反应混合物
    10mM pNPP(钠盐),溶于50mM乙酸盐-KOH(pH5.8)中 3 M氢氧化钠(NaOH)也需要停止反应
  4. Bradford检测库存
    100ml 95%EtOH
    200ml 88%H 3 PO 4 4/6 350mg亮蓝G-250
  5. Bradford工作解决方案
    30 ml Bradford股票
    425ml H 2 O 2 / 15 ml EtOH
    30ml 88%H 3 PO 4 4/6 通过Whatman#1滤纸过滤,并储存在棕色或深色玻璃瓶中。
    蛋白质测定溶液在室温下稳定数月。
  6. Bradford蛋白质标准
    用H 2 O稀释牛血清球蛋白至0.4mg/ml 将100μl等分试样储存在-20°C

致谢

我们实验室的研究由加拿大自然科学和工程研究委员会(NSERC)的研究和设备赠款以及威廉·普拉克斯顿的女王研究椅计划慷慨资助。

参考文献

  1. Robinson,W.D.,Park,J.,Tran,H.T.,Del Vecchio,H.A.,Ying,S.,Zins,J.L.,Patel,K.,McKnight,T.D.and Plaxton, 分泌的紫色酸性磷酸酶同功酶AtPAP12和AtPAP26在胞外磷酸盐清除中起着关键作用> Arabidopsis thaliana J Exp Bot 63(18):6531-6542。 
  2. Tran,H.T.,Hurley,B.A。和Plaxton,W.C。(2010a)。 喂养饥饿植物:紫色酸性磷酸酶在磷酸盐营养中的作用 Plant Sci 179(1):14-27。
  3. Tran,H.T.,Qian,W.,Hurley,B.A。,She,Y.M.,Wang,D.and Plaxton,W.C。(2010b)。 AtPAP12和AtPAP26的生物化学和分子表征:磷酸盐饥饿分泌的主要紫色酸性磷酸酶同功酶em> Arabidopsis thaliana 。 Plant Cell Environ 33(11):1789-1803。
  4. Veljanovski,V.,Vanderbeld,B.,Knowles,VL,Snedden,WA和Plaxton,WC(2006)。 AtPAP26的生物化学和分子表征,磷酸盐 - 剥夺的拟南芥悬浮细胞和幼苗中的空泡紫色酸性磷酸酶上调植物生理学 142 (3):1282-1293。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Knowles, V. and Plaxton, W. (2013). Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration. Bio-protocol 3(17): e889. DOI: 10.21769/BioProtoc.889.
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