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Catalase Activity Assay in Candida glabrata
光滑念珠菌中过氧化氢酶活性分析   

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Abstract

Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O2. Contribution of antioxidant systems on fungal virulence is actively studied. Measurement of catalase activity can contribute to the elucidation of the factors that influence the regulation of this pivotal enzyme. Here we describe a simple spectrophotometric method in which the activity of catalase is measured in total yeast extracts. Decomposition of H2O2 by the yeast extract is followed by the decrease in absorbance at 240 nm. The difference in absorbance through time (ΔA240) is inferred as the measure of catalase activity.

Keywords: Catalase(过氧化氢酶), Candida(念珠菌), Glabrata(光滑), Yeast(酵母), CTA1(CTA1)

Materials and Reagents

  1. Yeast strains
    Note: BG14 was used as the C. glabrata parental strain. The hst1Δ and the cta1Δ null mutants were used as a positive and the negative controls, respectively.
  2. Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
  3. Bradford reagent (Sigma-Aldrich, catalog number: B6916 )
  4. Bovine serum albumin - fraction V (Sigma-Aldrich, catalog number: 85040C )
  5. Zirconia/silica beads (0.5 mm diameter) (Bio Spec Products, catalog number: 11079105z )
  6. Sterile water
  7. Ice
  8. One tablet of protease inhibitors cOmplete ULTRA Mini EASYpack is used in 10 ml of phosphate buffer (Roche Diagnostics, catalog number: 05 892 970 001 )
  9. H2O2 (Sigma-Aldrich, catalog number: 349887 )
  10. Catalase lyophilized powder (Sigma-Aldrich, catalog number: C9322)
  11. YPD broth (see Recipes)
  12. 50 mM Phosphate buffer (PB) (pH 7.0) (see Recipes)
  13. 30 mM H2O2 (see Recipes)
  14. Catalase solution (see Recipes)

Equipment

  1. Orbital incubator shaker
  2. Microfuge tubes
  3. 50 ml conical tubes
  4. Corning 96 well clear flat bottom (Corning, catalog number: 3595 )
  5. Standard 10 mm light path quartz cuvette with PTFE cover
  6. UV/Vis Spectrophotometer (Shimadzu, model: UV-1700 , catalog number: 206-55401-92)
  7. Microplate spectrophotometer system (Benchmark Plus Microplate reader) (Bio-Rad Laboratories, catalog number: 170-6931 )
  8. Centrifuge (Beckman Coulter, model: Allegra® 25R, catalog number: 369464 )
  9. Microfuge
  10. Stopwatch
  11. Parafilm

Procedure

  1. Preparation of total soluble extracts
    1. Yeast strains are grown overnight in 5 ml of Yeast extract-Peptone-Dextrose broth or selective media at 30 °C.
    2. Dilute overnight cultures in 50 ml of fresh medium in order that after seven duplications, the yeast cultures reach an OD600 = 0.5 at 30 °C.
    3. Centrifuge the cells for 5 min at 2,600 x g. Discard supernatant. Temperature of centrifuge is not relevant.
    4. Wash the cells with 25 ml of sterile water and discard supernatant.
    5. Resuspend the cells in 0.5 ml PB with protease inhibitors and transfer to a microfuge tube. Keep the samples on ice.
    6. Add 50 µl of zirconia/silica beads to each sample.
    7. Disrupt the cells by vortexing at maximum speed for 1 min and place on ice for another minute. Repeat 20 times.
    8. Centrifuge the lysate at 25,000 x g for 30 min at 4 °C to remove cell debris and zirconia/silica beads
    9. Transfer supernatant to a clean microfuge tube. At this point, lysates are ready for quantification of total protein and measurement of catalase activity. Alternatively, samples can be stored at -20 °C.

  2. Bradford assay for protein quantitation
    1. Fill the wells of a microplate with 250 µl of Bradford regent.
    2. Prepare a standard curve of absorbance versus nanograms of protein using fresh BSA standards (100, 200, 400, 600, 800 ng/µl). Use PB as solvent.
    3. Dilute the lysates 1:20 or 1:50 with sterile water.
    4. Load 5 µl of the standards and diluted lysates to the Bradford reagent. Incubate room temperature for 5 min.
    5. Measure the absorbance at OD595 in a microplate spectrophotometer.
    6. Determine the amount of protein of the samples from the standard curve. Consider the dilution factor.

  3. Catalase activity assay
    1. Set up the spectrophotometer by first turning on the instrument and then the UV light. Set up a kinetics program to record every 30 s at a wavelength of 240 nm for 2 min.
    2. Calibrate the spectrophotometer using 3 ml of PB in a 3-ml quartz cuvette as a blank.
    3. Dilute the lysate samples 1:50 with PB.
    4. In a quartz cuvette, mix 1 ml PB with 1 ml of the diluted sample. To begin the assay, add 1 ml of the H2O2 solution (H2O2 to a final concentration of 10 mM). The initial absorbance must be between 0.550 and 0.520. If necessary, add H2O2 to increase the absorbance and Phosphate Buffer to decrease the absorbance.
    5. Mix the content by inversion and Immediately place the cuvettes into the spectrophotometer. Follow the decrease in absorbance at OD240 with a stopwatch for 2 min.
    6. A catalase solution must be used as a control. Pippete 2.9 ml of PB in the cuvette, add 1 ml 30 mM H2O2 and 100 µl of the catalase solution (~10 units). Record the initial and final absorbance in a one-minute period. Use 2 ml of PB and 1 ml of 30 mM H2O2 as blank.
    7. Calculate the catalase activity using the following formula (Cuellar-Cruz et al., 2009)

      Where
      is the difference between the initial and final absorbance.
      is the total volume of the reaction (3 ml).
      is the molar extinction coefficient for H2O2 at OD240 (34.9 mol-1 cm-1).
      is the optical length path of cuvette (1 cm).
      is the volume of the sample in ml.
        is the protein concentration of the sample in mg/ml.
    8. Example of catalase activities of extracts from C. glabrata strains in exponential phase of growth:

      Note: Catalase activity of the BG14 strain is higher in stationary phase ≈ 10 U/mg. However catalase activity of a cta1Δ is always < 2 U/mg or undetectable.

Notes

  1. For catalase, the dependence of the H2O2 decomposition on temperature is small, so measurements can be carried out between 0 and 37 °C, however 20 °C is recommended.
  2. For the catalse assay, each test cuvette will need to be run one at a time, so do not prepare the next test cuvette until the run with the preceding cuvette is complete.
  3. Low concentrations of H2O2 are used to avoid bubbling.
  4. Mixing of the samples can be facilitated by the use of parafilm.

Recipes

  1. Yeast extract-Peptone-Dextrose broth (1 L)
    Dissolve 10 g yeast extract and 20 g peptone in 950 ml of distilled water
    Autoclave (121 °C, 15 lb/in2 for 15 min)
    Add 50 ml 40% (w/v) dextrose (2% final; sterilized separately by autoclaving or filtering)
  2. 50 mM Phosphate Buffer (PB) (pH 7.0) (1 L)
    Dissolve 2.724 g KH2PO4 in 400 ml of distilled water
    Dissolve 5.34 g Na2HPO4 in 600 ml of distilled water
    Mix solutions [proportion (1:1.5)]
    pH to 7.0 with 1 M KOH
    To obtain cell lysates, prepare 10 ml of PB and add one tablet of cOmplete protease inhibitors.
  3. 30 mM H2O2 (100 ml)
    Dilute 0.26 ml of 35% H2O2 with PB to 100 ml. Prepare fresh to each activity assay. The solution can be at room temperature during the experiment.
  4. Catalase solution
    Dissolve 10 mg of catalase lyophilized powder in 1 ml of cold PB
    Immediately before use, dilute 5 µl of catalase solution to 1 ml cold PB to obtain a solution with ~100 U/ml. Stored at -20 °C for 6 months.

Acknowledgments

This protocol is based on the methodology reported by Aebi (1984), and by Weydert and Cullen (2010). Our adapted method was first published in Cuellar-Cruz et al. (2009). This work was funded by a CONACYT grant no. CB-2010-153929 to A.D.L.P. Finally, we thank Guadalupe Gutierrez-Escobedo for technical assistance.

References

  1. Aebi, H. (1984). Catalase in vitro. Methods Enzymol 105: 121-126.
  2. Cuellar-Cruz, M., Castano, I., Arroyo-Helguera, O. and De Las Penas, A. (2009). Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata. Mem Inst Oswaldo Cruz 104(4): 649-654. 
  3. Weydert, C. J. and Cullen, J. J. (2010). Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 5(1): 51-66.

简介

共生和致病真菌暴露于由宿主的巨噬细胞产生的过氧化氢(H 2 O 2 O 2)。 致病真菌抵消了H 2 O 2对于过氧化氢酶(EC 1.11.1.6)的有害影响,所述过氧化氢酶分解两个分子的H 2 O 2 - O 2至两个H 2 O和O 2分子。 积极研究抗氧化系统对真菌毒力的贡献。 过氧化氢酶活性的测量可有助于阐明影响这种关键酶的调节的因素。 在这里我们描述一个简单的分光光度法,其中过氧化氢酶的活性在总酵母提取物中测量。 通过酵母提取物的H 2 O 2 O 2分解后,在240nm处的吸光度降低。 吸光度随时间的差异(ΔA240)被推断为过氧化氢酶活性的量度。

关键字:过氧化氢酶, 念珠菌, 光滑, 酵母, CTA1

材料和试剂

  1. 酵母菌株
    注意:BG14用作光滑念珠菌亲本菌株。 将hst1 Δ 和cta1 Δ em突变体分别用作阳性对照和阴性对照。/em>
  2. 来自牛肝的过氧化氢酶(Sigma-Aldrich,目录号:C9322)
  3. Bradford试剂(Sigma-Aldrich,目录号:B6916)
  4. 牛血清白蛋白 - 级分V(Sigma-Aldrich,目录号:850-40℃)
  5. 氧化锆/二氧化硅珠(直径0.5mm)(Bio Spec Products,目录号:11079105z)
  6. 无菌水
  7. 冰块
  8. 在10ml磷酸盐缓冲液(Roche Diagnostics,目录号:05 892 970 001)中使用一片蛋白酶抑制剂cOmplete ULTRA Mini EASYpack
  9. (Sigma-Aldrich,目录号:349887)。< br />
  10. 过氧化氢酶冻干粉(Sigma-Aldrich,目录号:C9322)
  11. YPD肉汤(见配方)
  12. 无菌水
  13. 冰块
  14. 在10ml磷酸盐缓冲液(Roche Diagnostics,目录号:05 892 970 001)中使用一片蛋白酶抑制剂cOmplete ULTRA Mini EASYpack
  15. (Sigma-Aldrich,目录号:349887)。< br />
  16. 过氧化氢酶冻干粉(Sigma-Aldrich,目录号:C9322)
  17. YPD肉汤(见配方)
  18. ...
  19. 50 ml conical tubes
  20. Corning 96 well clear flat bottom (Corning, catalog number: 3595)
  21. Standard 10 mm light path quartz cuvette with PTFE cover
  22. UV/Vis Spectrophotometer (Shimadzu, model: UV-1700, catalog number: 206-55401-92)
  23. Microplate spectrophotometer system (Benchmark Plus Microplate reader) (Bio-Rad Laboratories, catalog number: 170-6931)
  24. Centrifuge (Beckman Coulter, model: Allegra® 25R, catalog number: 369464)
  25. Microfuge
  26. Stopwatch
  27. Parafilm

Procedure

  1. Preparation of total soluble extracts
    1. Yeast strains are grown overnight in 5 ml of Yeast extract-Peptone-Dextrose broth or selective media at 30 °C.
    2. 在50ml新鲜培养基中稀释过夜培养物,以便在7次重复后,酵母培养物在30℃达到OD 600 = 0.5。
    3. 在2,600×g离心细胞5分钟。 弃去上清液。 离心机的温度不相关。
    4. 用25 ml无菌水清洗细胞,弃去上清液
    5. 重悬细胞在0.5毫升PB蛋白酶抑制剂和转移到微量离心管。 将样品保存在冰上。
    6. 向每个样品中加入50μl氧化锆/二氧化硅珠。
    7. 通过以最大速度涡旋1分钟并在冰上放置另一分钟来破坏细胞。 重复20次。
    8. 在4℃下以25,000×g离心裂解物30分钟以除去细胞碎片和氧化锆/二氧化硅珠子
    9. 将上清转移到干净的微量离心管中。 在这一点上,裂解物准备用于总蛋白的定量和过氧化氢酶活性的测量。 或者,样品可以储存在-20°C
  2. Bradford测定蛋白质定量
    1. 填充微孔板的250微升Bradford试剂的孔。
    2. 使用新鲜BSA标准品(100,200,400,600,800ng /μl)制备吸光度相对于纳克蛋白质的标准曲线。 使用PB作为溶剂。
    3. 用无菌水稀释裂解液1:20或1:50。
    4. 加载5μl的标准品和稀释裂解液到Bradford试剂。 孵育室温5分钟。
    5. 在微量板分光光度计中测量OD 595处的吸光度
    6. 从标准曲线确定样品的蛋白质的量。 考虑稀释因子。

  3. 过氧化氢酶活性测定
    1. 设置分光光度计首先打开仪器,然后打开紫外灯。设置动力学程序,在240nm的波长下记录每30秒2分钟。
    2. 使用3ml PB在3ml石英比色杯中作为空白校准分光光度计。
    3. 用PB稀释裂解物样品1:50。
    4. 在石英比色皿中,将1ml PB与1ml稀释的样品混合。为了开始测定,将1ml H 2 O 2 O 2溶液(H 2 O 2 O 2)加入到最终浓度为10mM)。初始吸光度必须在0.550和0.520之间。如果必要,添加H 2 O 2以增加吸光度,并且磷酸盐缓冲液降低吸光度。
    5. 通过倒置混合内容,立即将比色杯放入分光光度计。用秒表观察OD 240处的吸光度降低2分钟。
    6. 必须使用过氧化氢酶溶液作为对照。在试管中加入2.9ml PB,加入1ml 30mM H 2 O 2和100μl过氧化氢酶溶液(〜10单位)。记录1分钟内的初始和最终吸光度。使用2ml PB和1ml 30mM H 2 O 2 O 2作为空白。
    7. 使用下式计算过氧化氢酶活性(Cuellar-Cruz等人,2009)

      其中
      是初始和最终吸光度之差。
      是反应的总体积(3ml)。
      是比色皿的光程长度(1厘米)。
      是以ml为单位的样本体积。
       是样品的蛋白质浓度(mg/ml)
    8. 来自C的提取物的过氧化氢酶活性的实例。 glabrata 菌株在指数生长期:

      注意:BG14菌株的过氧化氢酶活性在稳定期≈10U/mg更高。然而,cta1 Δ 的过氧化氢酶活性总是< 2 U/mg或无法检测。

笔记

  1. 对于过氧化氢酶,H 2 O 2 O 2分解对温度的依赖性小,因此可以在0至37℃之间进行测量,然而推荐20℃ 。
  2. 对于催化剂测定,每个测试比色皿将需要一次运行一个,所以不要准备下一个测试比色皿,直到前一个比色杯的运行完成。
  3. 使用低浓度的H 2 O 2 O 2以避免起泡。
  4. 通过使用石蜡膜可以促进样品的混合。

食谱

  1. 酵母提取物 - 蛋白胨 - 右旋糖肉汤(1L)
    将10克酵母提取物和20克蛋白胨溶解在950毫升蒸馏水中
    高压灭菌(121℃,15lb/in 2 15分钟)
    加入50ml 40%(w/v)葡萄糖(最终2%;通过高压灭菌或过滤单独灭菌)
  2. 50mM磷酸盐缓冲液(PB)(pH7.0)(1L) 将2.724g KH 2 PO 4溶解在400ml蒸馏水中
    将5.34g Na 2 HPO 4溶解在600ml蒸馏水中
    混合溶液[比例(1:1.5)]
    用1M KOH pH调至7.0 为了获得细胞裂解物,制备10ml PB并加入一片cOmplete蛋白酶抑制剂。
  3. 30mM H 2 O 2(100ml) 将0.26ml 35%H 2 O 2 Sub用PB稀释至100ml。 对每个活性测定准备新鲜。 溶液在实验过程中可以在室温下进行
  4. 过氧化氢酶溶液
    将10mg过氧化氢酶冻干粉溶解在1ml冷PB/B中 使用前,稀释5微升的过氧化氢酶溶液到1毫升冷PB,得到〜100 U/ml的溶液。 -20°C储存6个月。

致谢

该协议基于Aebi(1984)和Weydert和Cullen(2010)报道的方法。 我们的适应方法首次发表在Cuellar-Cruz等人(2009)。 这项工作是由CONACYT资助的。 CB-2010-153929,A.D.L.P. 最后,我们感谢Guadalupe Gutierrez-Escobedo的技术援助。

参考文献

  1. Aebi,H。(1984)。 体外过氧化氢酶 。 方法Enzymol 105:121-126。
  2. Cuellar-Cruz,M.,Castano,I.,Arroyo-Helguera,O。和De Las Penas,A。(2009)。 在机会性真菌病原体光滑假丝酵母中对甲萘醌和氢过氧化枯烯的氧化应激反应。 em> Mem Inst Oswaldo Cruz 104(4):649-654。 
  3. Weydert,C.J。和Cullen,J.J。(2010)。 在培养的细胞和组织中测量超氧化物歧化酶,过氧化氢酶和谷胱甘肽过氧化物酶 Nat Protoc 5(1):51-66。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Orta-Zavalza, E., Briones-Martin-del-Campo, M., Castano, I. and Penas, A. D. (2014). Catalase Activity Assay in Candida glabrata. Bio-protocol 4(6): e1072. DOI: 10.21769/BioProtoc.1072.
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