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Glycolate Oxidase Activity Assay in Plants
植物中乙醇酸盐氧化酶活性测定实验   

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Abstract

Glycolate oxidase is located in the peroxisome and is involved in the photorespiratory cycle which recovers some of the carbon loss during photosynthesis. Glycolate oxidase converts glycolate to glyxoylate with the concomitant production of H2O2.In this assay, the H2O2 generated, in the presence of HRP, oxidizes O-dianisidine into a colored O-dianisidine radical cation that can be quantified spectrophotometrically. The amount of color produces is directly proportional to the glycolate oxidase activity.

Materials and Reagents

  1. Sucrose
  2. HEPES
  3. EDTA
  4. DTT
  5. L-cysteine
  6. MgCl2
  7. PVP
  8. BSA
  9. Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 04693159001 ).
  10. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
  11. Horseradish peroxidase(HRP) (Sigma-Aldrich, catalog number: P8375 )
  12. O-Dianisidine (Sigma-Aldrich, catalog number: D9143 )
  13. Sodium glycolate (Thermo Fisher Scientific, Acros Organics,catalog number: 351570250 )
  14. Potassium phosphate
  15. Triton X-100
  16. Protein extraction buffer (see Recipes)
  17. Glycolate oxidase assay buffer (see Recipes)

Equipment

  1. Microtiter plate reader (Infinite M200 Pro, Tecan)
  2. Microcentrifuge (AqquSpin Micro R) (Thermo Fisher Scientific)
  3. 96-well microtiter plate flat bottom(BD Biosciences, catalog number: 353075 )
  4. 2 ml-microcentrifuge tubes

Procedure

  1. Total Protein extraction from plant tissues
    1. Harvest tissue in liquid nitrogen. If not used immediately, keep at -80 °C until processing.
    2. Grind tissue in liquid nitrogen and weigh out 0.5 g of the ground tissues in empty falcon tube that has been pre-chilled in liquid nitrogen and used to tare the scale.
    3. Add 6 ml of ice cold protein extraction buffer to ground tissues on ice.
    4. Vortex at room temperature to mix thoroughly.
    5. Filter homogenized tissue through four layers of cheese cloth andtransfer filtrate (flow through) to 2 ml-microcentrifuge tubes, on ice.
    6. Centrifuge at 10,000 x g for 45 min at 4 °C and transfer supernatant to new tubes on ice.
    7. Use supernatant to estimate protein concentration and to measure glycolate oxidase activity.

  2. Protein estimation using Bradford microassay (160 μl)
    1. Prepare BSA standards ranging from 5 μg-25 μg/ml as follows.



    2. Use 96 well microtiter plate to estimate protein content.
    3. Prepare blank by160 μl of water to one well in triplicates.
    4. Prepare test samplesby adding 2 μl of supernatant (from section 1) to 158 μl of water.
    5. Add 40 μl of Bradford Assay reagent to BSA standards, blank and test samples.
    6. Mix and incubate at room temperature for 5 min and read absorbance at 595 nm (A595) on plate reader spectrophotometer.
      Note: If spectrophotometer does not include a software to generate standard curve to automatically estimate protein content, generate a BSA standard curve by plotting known protein concentration (X-axis) vs. Absorbance (in Y-axis). Protein concentration for a given unknown sample is estimated by plotting the A595 absorbance of the unknown (in the y-axis)and determining the intersection point with the BSA standard curve and then find the concentration associated with that particular point (in the x-axis). If using excel, after plotting concentration vsA595, obtain the trendline and use the equation for the line and the A595 of the unknown to resolve the unknown concentration.

  3. Glycolate oxidase activity assay
    1. Prepare blank by adding 10 μl of 0.1 M sodium phosphate buffer (pH 8.3), to 250 μl of glycolate oxidase assay bufferin 96 well microtiter plate.
    2. Prepare test samples by adding 10 μl of supernatant (from section 1) to 250 μl of glycolate oxidase assay buffer.
    3. Read blank and samples at 440 nm for 0 min and then at 20 min intervals for one hour or until saturation point reached, on plate reader spectrophotometer.
    4. Calculate the generation of O-dianisidine radical using the following formula:
      (ΔA440nm/min test-ΔA440nm/min blank))/ (11.60) (0.04)
      ΔA440nm/mintest =A440 (sample X) at saturation point - A440 (sample X) at 0 min
      ΔA440nm/min blank = A440nm (blank) at saturation point - A440nm (blank) at 0 min
      Where
      11.60 =extinction coefficient for O-dianisidine. (Macheroux et al, 1991)
      0.04= dilution factor (10 μl/250 μl)
      To calculate specific activity, divide the value obtained in equation by the amount of protein present in the sample (converted to mg/ml).

Recipes

  1. Protein extraction buffer
    Working solution:
    0.25 M sucrose
    50 mM HEPES
    3 mM EDTA
    1 mM DTT
    3.6 mM L-cysteine
    0.1 mM MgCl2
    0.6% PVP
    10 tablets of complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Protein extraction buffer (100 ml)
    Use following stock solutions to make working solution:
    In 80 ml of water add the following reagents:


    Add 10 tablets of complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Mix well and adjust volume to 100 ml.
  2. Glycolate oxidase assay buffer
    Working solution:
    10 μg ml-1 HRP
    0.4 mM O-dianisidine
    10 mM sodium glycolate
    in 0.1 M potassium phosphate (pH 8.3)
    Glycolate oxidase assay buffer (1 ml)
    PrepareGlycolate oxidase assay buffer using following stock solutions:


Acknowledgments

This protocol has been adapted and modified to use in Arabidopsis from Macheroux et al. (1991). This work was supported by the Samuel Roberts Noble Foundation.

References

  1. Macheroux, P., Massey, V., Thiele, D. J. and Volokita, M. (1991). Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization. Biochemistry 30(18): 4612-4619.
  2. Rojas, C. M., Senthil-Kumar, M., Wang, K., Ryu, C. M., Kaundal, A. and Mysore, K. S. (2012). Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24(1): 336-352.

简介

乙醇酸氧化酶位于过氧化物酶体中并且参与光呼吸循环,其恢复光合作用期间的一些碳损失。 乙醇酸氧化酶将乙醇酸转化为乙醛酸,同时产生H 2 O 2 Sub。在该测定中,H 2 O 2 O 2 - 在HRP的存在下,将O-联茴香胺氧化成可以用分光光度法定量的有色的O-联苯胺自由基阳离子。 产生的颜色量与乙醇酸氧化酶活性成正比。

材料和试剂

  1. 蔗糖
  2. HEPES
  3. EDTA
  4. DTT
  5. L-半胱氨酸
  6. MgCl 2
  7. PVP
  8. BSA
  9. Complete,Mini,无EDTA的蛋白酶抑制剂混合片(F.Hoffmann-La Roche,目录号:04693159001)。
  10. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:500-0006)
  11. 辣根过氧化物酶(HRP)(Sigma-Aldrich,目录号:P8375)
  12. O-二噻吩(Sigma-Aldrich,目录号:D9143)
  13. 乙醇酸钠(Thermo Fisher Scientific,Acros Organics,目录号:351570250)
  14. 磷酸钾
  15. Triton X-100
  16. 蛋白质提取缓冲液(参见配方)
  17. 乙醇氧化酶测定缓冲液(参见配方)

设备

  1. 微量滴定板读数器(Infinite M200 Pro,Tecan)
  2. 微量离心机(AqquSpin Micro R)(Thermo Fisher Scientific)
  3. 96孔微量滴定板平底(BD Biosciences,目录号:353075)
  4. 2 ml微量离心管

程序

  1. 从植物组织中提取总蛋白
    1. 收获组织在液氮。 如果不立即使用,保持在-80℃直到处理
    2. 在液氮中研磨组织,称出已在液氮中预冷却并用于去除鳞片的空的猎鹰管中的0.5g研磨组织。
    3. 加入6毫升冰冷的蛋白质提取缓冲液在冰上研磨组织
    4. 在室温下涡旋以彻底混合
    5. 通过四层奶酪布过滤匀浆的组织,并在冰上将滤液(流过)转移到2ml微量离心管中。
    6. 在4℃下以10,000×g离心45分钟,并在冰上将上清液转移到新管中。
    7. 使用上清液估计蛋白质浓度和测量乙醇酸氧化酶活性

  2. 使用Bradford微量测定法(160μl)
    1. 制备BSA标准品,范围为5μg-25μg/ml,如下所示


    2. 使用96孔微量滴定板估计蛋白质含量
    3. 用160μl水制备空白,一式三份
    4. 通过加入2μl上清液(从第1部分)到158μl水制备测试样品
    5. 向BSA标准品,空白和测试样品中加入40μlBradford测定试剂
    6. 混合并在室温下孵育5分钟,并在平板读数器分光光度计上读取595nm处的吸光度(A595)。
      注意:如果分光光度计不包括生成标准曲线以自动估计蛋白质含量的软件,则通过绘制已知蛋白质浓度(X轴)与吸光度(Y轴)绘制BSA标准曲线。通过绘制未知的A595吸光度(在y轴上)并确定与BSA标准曲线的交点,然后找到与该特定点相关的浓度(在x轴上)来估计给定未知样品的蛋白质浓度)。如果使用excel,绘制浓度vsA595后,获得趋势线,并使用线的方程和未知的A595来解决未知浓度。

  3. 乙醇氧化酶活性测定
    1. 通过加入10微升0.1 M磷酸钠缓冲液(pH 8.3)到250微升乙醇酸氧化酶测定缓冲液在96孔微量滴定板中准备空白。
    2. 通过加入10μl上清液(从第1部分)到250μl乙醇酸氧化酶测定缓冲液制备测试样品
    3. 在平板读数器分光光度计上,在440nm下读取空白和样品0分钟,然后以20分钟间隔读取1小时或直到达到饱和点。
    4. 使用下式计算邻联茴香胺自由基的生成:
      (ΔA440nm)/分钟测试空气 440nm 空白))/(11.60)(0.04) br /> 在饱和点-A A 440(样品X)处的ΔA440nm /min测试 = A 440(样品X)在0分钟
      ΔA440nm(分钟)空白= A 440nm(空白)在饱和点-A 440nm(空白)在0℃时min
      其中
      11.60 =邻联茴尼啶的消光系数。 (Macheroux et al ,1991)
      0.04 =稀释因子(10μl/250μl)
      为了计算比活性,将公式中得到的值除以样品中存在的蛋白质量(换算成mg/ml )。

食谱

  1. 蛋白提取缓冲液
    工作解决方案:
    0.25 M蔗糖 50 mM HEPES
    3 mM EDTA
    1 mM DTT
    3.6mM L-半胱氨酸 0.1mM MgCl 2·h/v 0.6%PVP
    10片完全,微型,无EDTA的蛋白酶抑制剂鸡尾酒片 蛋白提取缓冲液(100ml)
    使用以下储备溶液制作工作溶液:
    在80ml水中加入以下试剂:


    添加10片完整,迷你,无EDTA的蛋白酶抑制剂鸡尾酒片 混合均匀,调节体积至100 ml
  2. 乙醇氧化酶测定缓冲液
    工作解决方案:
    10μgml HRP
    0.4mM O-联苯胺 10mM乙醇酸钠 在0.1M磷酸钾(pH8.3)中 乙醇酸氧化酶测定缓冲液(1ml) 使用以下储备溶液制备乙醇酸氧化酶测定缓冲液:


致谢

该方案已经改编和修改以用于来自Macheroux等人(1991)的拟南芥中。 这项工作得到了Samuel Roberts Noble基金会的支持。

参考文献

  1. Macheroux,P.,Massey,V.,Thiele,D.J。和Volokita,M。(1991)。 菠菜乙醇酸氧化酶在酿酒酵母中的表达:纯化和表征。/a> Biochemistry 30(18):4612-4619
  2. Rojas,C.M.,Senthil-Kumar,M.,Wang,K.,Ryu,C.M.,Kaundal,A.and Mysore,K.S.(2012)。 乙醇酸氧化酶调节在本尼特烟草中的非宿主抗性期间的反应性氧物质介导的信号转导 em>和拟南芥。植物细胞 24(1):336-352。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kaundal, A., Rojas, C. M. and Mysore, K. S. (2012). Glycolate Oxidase Activity Assay in Plants. Bio-protocol 2(20): e277. DOI: 10.21769/BioProtoc.277.
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