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Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant tissue. PPO activity can be easily assayed in crude protein extracts from English walnut (Juglans regia) leaves and from many other plant tissue extracts. PPO activity is most commonly measured by spectrophotometric assay, in which the rate of phytomelanin production is quantified, or by oxygen electrode assay, in which the consumption of oxygen by the enzyme is quantified (Figure 1). Though simpler, the utility of the spectrophotometric assay is limited by variation in the absorption maxima of phytomelanins generated from different phenolic substrates. The oxygen electrode assay is generally considered the “gold standard” for measurement of PPO activity, but it is more time consuming and difficult to implement with monophenol substrates, since cresolase activity is typically quite low compared to catecholase activity. This protocol will describe crude protein extraction from walnut leaves, the spectrophotometric assay, and the oxygen electrode assay for determining PPO activity.

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Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue
核桃叶组织中的多酚氧化酶活性分析

植物科学 > 植物生物化学 > 蛋白质 > 活性
作者: Ross Gertzen
Ross GertzenAffiliation: Department of Biological Sciences, California State University San Marcos, San Marcos, USA
Bio-protocol author page: a1586
 and Matthew A. Escobar
Matthew A. EscobarAffiliation: Department of Biological Sciences, California State University San Marcos, San Marcos, USA
For correspondence: mescobar@csusm.edu
Bio-protocol author page: a1587
Vol 4, Iss 16, 8/20/2014, 6166 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1213

[Abstract] Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant tissue. PPO activity can be easily assayed in crude protein extracts from English walnut (Juglans regia) leaves and from many other plant tissue extracts. PPO activity is most commonly measured by spectrophotometric assay, in which the rate of phytomelanin production is quantified, or by oxygen electrode assay, in which the consumption of oxygen by the enzyme is quantified (Figure 1). Though simpler, the utility of the spectrophotometric assay is limited by variation in the absorption maxima of phytomelanins generated from different phenolic substrates. The oxygen electrode assay is generally considered the “gold standard” for measurement of PPO activity, but it is more time consuming and difficult to implement with monophenol substrates, since cresolase activity is typically quite low compared to catecholase activity. This protocol will describe crude protein extraction from walnut leaves, the spectrophotometric assay, and the oxygen electrode assay for determining PPO activity.
Keywords: Polyphenol oxidase(多酚氧化酶), Walnut(核桃), Enzyme activity(酶的活性)

[Abstract] 多酚氧化酶(PPO)是催化单酚羟基化成邻二酚(甲酚酶活性)和将β-二酚氧化成醌(儿茶酚酶活性)(图1)的酶。这些醌自发聚合形成深色的植物​​色素,最常见于受损的植物组织的褐变。 PPO活性可以容易地在来自英国胡桃(Juglans regia)叶和来自许多其它植物组织提取物的粗蛋白提取物中测定。 PPO活性最通常通过分光光度测定法测量,其中植物肉豆蔻毒素产生的速率被量化,或通过氧电极测定,其中酶对氧的消耗量化(图1)。尽管更简单,分光光度测定的效用受到从不同酚类底物产生的植物角蛋白的最大吸收的变化的限制。氧电极测定通常被认为是用于测量PPO活性的"金标准",但是与单酚底物一起实施是更费时和困难的,因为与儿茶酚酶活性相比,甲酚水解酶活性通常相当低。该方案将描述从核桃叶的粗蛋白质提取,分光光度测定法和用于测定PPO活性的氧电极测定法。


Figure 1. The activity of the polyphenol oxidase enzyme

Materials and Reagents

  1. Protein extraction
    1. Juglans regia leaf tissue
    2. Liquid nitrogen
    3. Insoluble polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: 25249-54-1 )
    4. Bovine serum albumin (BSA) (Thermo Fisher Scientific, catalog number:  9048-46-8 )
    5. Bradford reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
    6. Tris base (Thermo Fisher Scientific, catalog number:  77-86-1 )
    7. Citric acid monohydrate (Thermo Fisher Scientific, Acros Organics, catalog number: 5949-29-1 )
    8. Cysteine hydrochloride (Thermo Fisher Scientific, catalog number: 7048-04-6 )
    9. Ascorbic acid (Phyto Technology Laboratories®, catalog number: 50-81-7 )
    10. Polyethylene glycol (PEG) 8000 (Thermo Fisher Scientific, catalog number: 25322-68-3 )
    11. Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
    12. Protein extraction buffer (see Recipes)

  2. Spectrophotometric assay
    1. Catalase (Thermo Fisher Scientific, catalog number:  9001-05-2 )
    2. Kojic acid (Thermo Fisher Scientific, catalog number:  501-30-4 )
    3. Monobasic dihydrogen sodium phosphate (Thermo Fisher Scientific, catalog number: 7558-80-7 )
    4. Dibasic monohydrogen sodium phosphate (Thermo Fisher Scientific, catalog number:  7558-79-4 )
    5. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: 151-21-3 )
    6. Sodium phosphate buffer containing phenolic substrate (see Recipes)

  3. Oxygraph assay
    1. Kojic acid (Thermo Fisher Scientific, catalog number:  501-30-4)
    2. Sodium phosphate buffer containing phenolic substrate (see Recipes)
    3. Assay buffer (see Recipes)

Equipment

  1. Ceramic mortar and pestle
  2. 50 ml centrifuge tubes
  3. 30 ml Oak Ridge tubes (Thermo Fisher Scientific, Nalgene®, catalog number: 3114-0030 )
  4. 1.5 ml microfuge tubes
  5. Spectrophotometer cuvettes or clear, flat bottom 96-well microtiter plates
  6. Micropipettes
  7. Vortexer
  8. Floor centrifuge with rotor that accommodates 30 ml Oak Ridge tubes
  9. Spectrophotometer or microplate reader (e.g. Molecular Devices SpectraMax M5)
  10. Oxygen electrode with computer interface (e.g. Hansatech Instruments, Oxygraph)
  11. -80 °C freezer

Procedure

  1. Crude protein extract
    1. Collect several leaves from sample tree and gently wash off dirt with deionized (DI) water, then pat completely dry.
    2. Seal leaves into 50 ml centrifuge tube and place in liquid nitrogen to flash freeze. Frozen leaf tissue may be stored at -80 °C.
    3. Grind tissue in a liquid nitrogen cooled mortar and pestle, being sure to keep adding liquid nitrogen to ensure the tissue does not thaw. A fine powder is desired.
    4. Add 6.0 ml of protein extraction buffer and 0.5 g of insoluble PVP to a 30 ml Oak Ridge tube. Place the tube on a balance and tare.
    5. Add ~1 g of ground, frozen leaf tissue to the tube. Tissue can be added using a liquid nitrogen cooled metal spatula, with mass read directly on the balance.
    6. Vortex the tissue slurry immediately to allow the frozen tissue to thaw in the presence of the buffer. Continue vortexing for 2 min.
    7. Centrifuge sample at 20,000 x g for 20 min at 4 °C.
    8. Aliquot ~200 µl volumes of the supernatant into 1.5 ml microfuge tubes and transfer the tubes to -80 °C for storage. (It is ideal to reduce freeze/thaw cycles so only the needed volume should be thawed at one time. If a sample is thawed three times, it should be discarded.)
    9. To determine total protein concentration, perform a Bradford assay according to the reagent manufacturer’s instructions with BSA standard concentrations of 25 µg/ml, 20 µg/ml, 12.5 µg/ml, 10 µg/ml, 5 µg/ml, and 1 µg/ml.
    10. Dilute the crude protein extract to 1/400 with ultrapure water for the Bradford assay. This dilution should be within the range of the BSA standards.

  2. Spectrophotometric assay
    1. Prepare sodium phosphate buffer with phenolic substrate per recipe.
    2. Obtain protein samples from -80 °C and place on ice to thaw.
    3. Warm up the plate reader or spectrophotometer, setting at 490 nm (for tyrosine or L-DOPA substrates) and choosing the kinetic option. Read times range from 3 min (most diphenol substrates) to 3 h (many monophenol substrates). If using a plate reader, it should be set to read every two minutes, with shaking for 15 sec before each read. If using a spectrophotometer, it should be set to read every 15 sec. See notes on latency below.
    4. Determine the buffer quantity needed for the experiment (200 µl/well for plate reader or 1 ml/cuvette for spectrophotometer). 2-3 technical replicates of each sample are typically performed. In addition, parallel assays containing 1 mM Kojic acid should be performed. Kojic acid is a specific inhibitor of PPO, so these experiments provide a negative control, assuring that measured activity derives from PPO enzymes.
    5. Transfer the necessary buffer volume to a centrifuge tube and then add 280 Units of catalase per milliliter of buffer. The catalase eliminates any H2O2, preventing the activity of peroxidases, some of which also oxidize phenolic compounds.
    6. Aliquot buffer samples into a 96-well plate or a cuvette. If using the plate reader, be careful to avoid bubbles caused by the SDS since they can interfere with accurate readings. Bubbles are best avoided by placing the pipette tip against the wall of the well and steadily dispensing the buffer.
    7. Add desired volume of thawed, mixed protein extract to buffer (typically 5 µl per 200 µl of buffer).
    8. Place the plate/cuvette into the plate reader/spectrophotometer and begin the read. If using a spectrophotometer, the sample should be manually mixed between readings (with a pipet tip or stir stick) to aerate. Pre-programmed shaking provides aeration in the plate reader. Given that the reaction catalyzed by PPO enzyme consumes oxygen (Figure 1), aeration is important to ensure adequate oxygen availability and thus accurate activity data.
    9. Typical PPO activity data (plate reader assay, tyrosine substrate) is provided in Figures 2 and 3.


      Figure 2. Spectrophotometric PPO activity assay using a microplate reader. A. Assay at time 0 (just after addition of protein extract). B. Assay after two hours. All wells contain tyrosine (4 mM) as substrate. Wells 1-4 also contain 1 mM kojic acid, a PPO inhibitor.


      Figure 3. Sample data (Abs490) of well A8 from Figure 2

  3. Oxygen electrode assay
    1. Prepare sodium phosphate buffer with substrate solution as per recipe.
      Note: Omit catalase in this assay since the catalase can substantially alter dissolved oxygen levels and give inaccurate data. This makes Kojic acid controls especially critical.
    2. Obtain protein samples from -80 °C and place on ice to thaw.
    3. Set up and calibrate the oxygen electrode apparatus according to manufacturer’s instructions.
    4. Pipette 1 ml (may vary depending on equipment) of buffer into the oxygen electrode chamber.
    5. Pipette desired volume of crude protein extract (typically 25 µl) into the chamber and seal.
    6. Measure oxygen consumption. Assay time can range from 10 min-4 h. Monophenol substrates typically require long measurement times due to relatively low activity and long PPO latency times.

  4. Calculating PPO enzyme activity
    1. For spectrophotometric and plate reader assays, calculate change in Abs490/min/mg total protein. Calculate based on initial rate of takeoff because the quinone products of PPO activity will oxidize proteins (including PPO), thus reducing activity over time.
    2. For oxygen electrode assay, calculate nmoles O2 consumed per milliliter per minute per mg total protein. Again, calculate based on initial rate using software to obtain nmol/ml/min value and then dividing by the amount of protein that was added to the sample in mg.


      Figure 4. PPO activity data measured using an oxygen electrode. After a latent period of ~50 min, rapid oxygen consumption commenced. PPO activity was calculated based on this initial rapid rate of oxygen consumption (slope between the two black vertical lines). The rate slows over time due to oxidative damage of proteins (including PPO) caused by the product quinones. X axis is oxygen concentration (nmol/ml), Y axis is time elapsed.

Notes

  1. Most or all plant PPOs have a characterized property of latency (a time lag before full enzyme activity) which may affect the way tests are conducted. In walnut tissue extracts, PPO has shown latency from a few minutes to a few hours, depending on the substrate. Latency is substantially longer when using monophenol substrates than when using diphenol substrates. The inclusion of SDS in the assay buffer is designed to minimize latency.
  2. When performing the spectrophotometric assay with substrates other than L-DOPA and tyrosine, alternative absorbance wavelengths may be preferable for measuring activity. A wavelength scan can be used to determine the absorbance maximum for phytomelanins derived from other substrates.
  3. Although walnut PPO displays both cresolase and catecholase activities, many plant PPOs display catecholase activity only (i.e. they can only act upon diphenol substrates). As such, initial assays are preferably performed using a diphenol substrate such as L-DOPA to assure that PPO activity is present. Later studies can evaluate whether the examined PPO also possesses cresolase activity.

Recipes

  1. Protein extraction buffer (pH 8.3)
    1. 6.5 g/L tris base
    2. 1.5 g/L citric acid monohydrate
    3. 1.0 g/L cysteine hydrochloride
    4. 1.0 g/L ascorbic acid
    5. 10.0 g/L PEG 8000
    6. 110 ml/L glycerol
    7. Ultrapure water to volume
      Mix components well and adjust to pH to 8.3 with KOH or HCl
      May be stored at 4 °C up to one week
  2. Assay buffer (pH 7.0)
    1. 100 mM sodium phosphate buffer (pH 7.0) (monobasic dihydrogen sodium phosphate; dibasic monohydrogen sodium phosphate)
    2. 0.15% SDS
    3. 4 mM phenolic substrate
    4. Ultrapure water to volume
    5. Negative control samples only: 1 mM Kojic acid
      Amount of monobasic and dibasic sodium phosphate is calculated using the Henderson–Hasselbalch equation. A 1 M sodium phosphate buffer stock solution (pH 7.0) can be made by combining 57.7 ml of 1 M Na2HPO4 and 42.3 ml of 1 M NaH2PO4. This stock can be diluted to make the assay buffer above. See Reference 4.
      Note: The solubility of some phenolic substrates may not allow a substrate concentration of 4 mM.

Acknowledgments

Protocols for the spectrophotometric assay of PPO activity were adapted from Constabel and Ryan (1998). This work was supported in part by a University of California/California State University Collaborative Research Grant.

References

  1. Constabel, C. P. and Ryan, C. A. (1998). A survey of wound-and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry 47(4): 507-511.
  2. Escobar, M. A., Shilling, A., Higgins, P., Uratsu, S. L. and Dandekar, A. M. (2008). Characterization of polyphenol oxidase from walnut. J Am Soc Hortic Sci 133(6): 852-858.
  3. Lucia, V., Mesquita, V. and Queiroz, C. (2013). Chapter 10 - Enzymatic Browning. In: Eskin, N. A. M. and Shahidi, F. (eds). Biochemistry of Foods (Third Edition). Academic Press, 387-418.
  4. Sambrook, J., Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.) Cold Spring Harbor Laboratory Press.
  5. Stewart, R. J., Sawyer, B. J., Bucheli, C. S. and Robinson, S. P. (2001). Polyphenol oxidase is induced by chilling and wounding in pineapple. Funct Plant Biol 28(3): 181-191.

图1.多酚氧化酶的活性

材料和试剂

  1. 蛋白质提取
    1. 胡桃叶叶组织
    2. 液氮
    3. 不溶性聚乙烯聚吡咯烷酮(PVPP)(Sigma-Aldrich,目录号:25249-54-1)
    4. 牛血清白蛋白(BSA)(Thermo Fisher Scientific,目录号:9048-46-8)
    5. Bradford试剂(Bio-Rad Laboratories,目录号:500-0006)
    6. Tris碱(Thermo Fisher Scientific,目录号:77-86-1)
    7. 柠檬酸一水合物(Thermo Fisher Scientific,Acros Organics,目录号:5949-29-1)
    8. 半胱氨酸盐酸盐(Thermo Fisher Scientific,目录号:7048-04-6)
    9. 抗坏血酸( Technology Laboratories ,目录号:50-81-7)
    10. 聚乙二醇(PEG)8000(Thermo Fisher Scientific,目录号:25322-68-3)
    11. 甘油(Thermo Fisher Scientific,目录号:56-81-5)
    12. 蛋白质提取缓冲液(参见配方)

  2. 分光光度法
    1. 过氧化氢酶(Thermo Fisher Scientific,目录号:9001-05-2)
    2. 曲酸(Thermo Fisher Scientific,目录号:501-30-4)
    3. 磷酸二氢钠(Thermo Fisher Scientific,目录号:7558-80-7)
    4. 磷酸二氢钠(Thermo Fisher Scientific,目录号:7558-79-4)
    5. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientific,目录号:151-21-3)
    6. 含有酚类底物的磷酸钠缓冲液(参见配方)

  3. 氧测定法
    1. 曲酸(Thermo Fisher Scientific,目录号:501-30-4)
    2. 含有酚类底物的磷酸钠缓冲液(参见配方)
    3. 测试缓冲区(参见配方)

设备

  1. 陶瓷砂浆和杵
  2. 50ml离心管
  3. 30ml Oak Ridge管(Thermo Fisher Scientific,Nalgene ,目录号:3114-0030)
  4. 1.5 ml微量离心管
  5. 分光光度计比色皿或透明,平底96孔微量滴定板
  6. 微量移液器
  7. Vortexer
  8. 带转子的地面离心机,可容纳30毫升橡树岭管
  9. 分光光度计或酶标仪(例如 Molecular Devices SpectraMax M5)
  10. 具有计算机接口的氧电极(例如 Hansatech Instruments,Oxygraph)
  11. -80°C冰箱

程序

  1. 粗蛋白提取物
    1. 从样品树收集几个叶子,用去离子(DI)水轻轻洗去污垢,然后完全拍干
    2. 将叶子留在50ml离心管中并置于液氮中以快速冷冻。冷冻叶组织可储存于-80℃
    3. 研磨组织在液氮冷却的砂浆和杵,一定要保持加入液氮,以确保组织不解冻。需要细粉。
    4. 向30ml Oak Ridge管中加入6.0ml蛋白提取缓冲液和0.5g不溶性PVP。将管子放在天平和皮重上。
    5. 向管中加入约1g研磨的冷冻叶组织。可以使用液氮冷却的金属刮刀添加组织,直接在天平上读取质量。
    6. 立即涡旋组织浆液以允许冷冻的组织在缓冲液存在下解冻。继续涡旋2分钟。
    7. 在4℃下以20,000×g离心样品20分钟
    8. 等分〜200μl体积的上清液到1.5ml微量离心管中,并将管转移至-80°C储存。 (这是理想的减少冻/融循环,所以只有所需的体积应解冻一次。如果样品解冻三次,应该丢弃。)
    9. 为了确定总蛋白浓度,根据试剂制造商的说明书,用25μg/ml,20μg/ml,12.5μg/ml,10μg/ml,5μg/ml和1μg/ml的BSA标准浓度进行Bradford测定, ml。
    10. 用超纯水稀释粗蛋白提取物至1/400用于Bradford测定。 此稀释度应在BSA标准品的范围内。

  2. 分光光度法
    1. 按照配方制备磷酸钠缓冲液和酚类底物
    2. 从-80°C获取蛋白质样品,并置于冰上解冻
    3. 预热板读数器或分光光度计,设置在490 nm(对于酪氨酸或L-DOPA底物),并选择动力学选项。读取时间范围从3min(大多数二酚底物)至3h(许多单酚底物)。如果使用读板器,应该设置为每两分钟读取一次,每次读取前振荡15秒。如果使用分光光度计,应设置为每15秒读取一次。请参阅以下有关延迟的说明。
    4. 确定实验所需的缓冲液量(平板读数器为200μl/孔,分光光度计为1 ml/cuvette)。通常进行每个样品2-3个技术重复。此外,应进行含有1mM曲酸的平行测定。曲酸是PPO的特异性抑制剂,因此这些实验提供了阴性对照,确保测量的活性源自PPO酶。
    5. 将必要的缓冲液转移到离心管中,然后每毫升缓冲液中加入280单位的过氧化氢酶。过氧化氢酶消除任何H 2 O 2 O 2,阻止过氧化物酶的活性,其中一些还氧化酚类化合物。
    6. 将缓冲液样品等分到96孔板或比色杯中。如果使用读板器,请小心避免SDS引起的气泡,因为它们会干扰精确的读数。最好避免气泡 通过将移液管尖端放置在孔的壁上并稳定地分配缓冲液
    7. 将所需体积的解冻的混合蛋白提取物加入缓冲液(通常为每200μl缓冲液5μl)
    8. 将板/比色杯放入读板器/分光光度计,开始阅读。如果使用分光光度计,样品应在读数(用移液管吸头或搅棒)之间手动混合以充气。预编程振动在读板器中提供曝气。由于PPO酶催化的反应消耗氧(图1),充气对于确保足够的氧可用性和因此准确的活性数据是重要的。
    9. 在图2和图3中提供了典型的PPO活性数据(读板仪测定,酪氨酸底物)

      图2.使用微板读数器的分光光度测定PPO活性测定。 A.在时间0时测定(刚加入蛋白质提取物后)。 B.两小时后的测定。所有孔含有酪氨酸(4mM)作为底物。孔1-4还含有1mM曲酸,PPO抑制剂

      图3.图2中的井A8的示例数据(Abs <490> )

  3. 氧电极测定
    1. 根据配方用底物溶液制备磷酸钠缓冲液。
      注意:在该测定中省略过氧化氢酶,因为过氧化氢酶可以实质上改变溶解氧水平并给出不准确的数据。 这使Kojic酸控制尤其关键。
    2. 从-80°C获取蛋白质样品,并置于冰上解冻
    3. 根据制造商的说明设置和校准氧电极装置。
    4. 移取1 ml(可能因设备而异)缓冲液进入氧电极室
    5. 移取所需体积的粗蛋白提取物(通常为25μl)进入腔室并密封
    6. 测量氧气消耗。 测定时间可以为10分钟-4小时。 由于相对低的活性和长的PPO潜伏时间,单苯酚底物通常需要长的测量时间。

  4. 计算PPO酶活性
    1. 对于分光光度计和平板读数器测定,计算Abs <490>/min/mg总蛋白的变化。 基于初始起飞速率计算,因为PPO活性的醌产物将氧化蛋白质(包括PPO),因此随时间降低活性。
    2. 对于氧电极测定,计算每毫升每分钟每mg总蛋白消耗的O 2 O 2次。 再次,使用软件基于初始速率计算以获得nmol/ml/min值和 然后除以加入到样品中的蛋白质的量(mg)

      图4.使用氧电极测量的PPO活性数据。在〜50分钟的潜伏期后,开始快速耗氧。基于这种初始快速氧消耗速率(两条黑色垂直线之间的斜率)计算PPO活性。速率随着时间的推移而减慢,这是由于由产物醌引起的蛋白质(包括PPO)的氧化损伤。 X轴是氧浓度(nmol/ml),Y轴是经过的时间。

笔记

  1. 大多数或所有植物PPO具有潜在的特征属性(完全酶活性之前的时间滞后),其可影响进行测试的方式。在核桃组织提取物中,PPO已显示潜伏期从几分钟到几小时,这取决于底物。当使用单酚基质时,与使用二酚基质时相比,延迟显着更长。设计在测定缓冲液中包含SDS以使潜伏期最小化。
  2. 当用不同于L-DOPA和酪氨酸的底物进行分光光度测定时,可选择的吸收波长可能优选用于测量活性。波长扫描可用于确定 来源于其他底物的植物素的最大吸光度
  3. 虽然核桃PPO显示甲酚和儿茶酚酶活性,但许多植物PPO仅显示儿茶酚酶活性(即它们只能作用于二苯酚底物)。 因此,优选使用二元酚底物如L-DOPA进行初始测定以确保存在PPO活性。 后来的研究可以评估所研究的PPO是否也具有甲酚活性

食谱

  1. 蛋白提取缓冲液(pH 8.3)
    1. 6.5g/L tris碱
    2. 1.5g/L柠檬酸单水合物
    3. 1.0 g/L半胱氨酸盐酸盐
    4. 1.0g/L抗坏血酸
    5. 10.0g/L PEG 8000
    6. 110ml/L甘油
    7. 超纯水至体积
      将组分充分混合,用KOH或HCl调节pH至8.3 可在4℃下储存一周
  2. 测定缓冲液(pH7.0)
    1. 100mM磷酸钠缓冲液(pH7.0)(磷酸二氢钠;磷酸二氢钠)
    2. 0.15%SDS
    3. 4mM酚类底物
    4. 超纯水至体积
    5. 阴性对照样品:1mM曲酸
      使用Henderson-Hasselbalch方程计算一元和二元磷酸钠的量。 可以通过将57.7ml的1M Na 2 HPO 4和0.5M的Na 2 HPO 4混合来制备1M磷酸钠缓冲液储备溶液(pH 7.0) 加入42.3ml 1M NaH 2 PO 4。 可以稀释该储备液以制备上述测定缓冲液。 参见参考文献4.
      注意:一些酚类底物的溶解度可能不允许底物浓度为4mM。

致谢

用于PPO活性的分光光度测定的方案改编自Constabel和Ryan(1998)。 这项工作部分由加州大学/加利福尼亚州立大学合作研究基金资助。

参考文献

  1. Constabel,C.P。和Ryan,C.A。(1998)。 关于作物中伤口和茉莉酮酸甲酯诱导的叶多酚氧化酶的调查。 Phytochemistry 47(4):507-511。
  2. Escobar,M.A.,Shilling,A.,Higgins,P.,Uratsu,S.L.and Dandekar,A.M。(2008)。 来自核桃的多酚氧化酶的表征。 J Am Soc Hortic Sci 133(6):852-858。
  3. Lucia,V.,Mesquita,V。和Queiroz,C。(2013)。 第10章 - 酶促褐变。 In:Eskin,N.A.M.and Shahidi,F。(eds)。 食物生物化学(第三版)学术出版社,387-418
  4. Sambrook,J.,Russell,D.W。(2001)。 Molecular cloning:A laboratory manual(3rd ed。)Cold Spring Harbor Laboratory Press
  5. Stewart,R.J.,Sawyer,B.J.,Bucheli,C.S。和Robinson,S.P。(2001)。 多酚氧化酶是由菠萝中的冷冻和受伤诱导的。 Funct Plant Biol 28(3):181-191。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gertzen, R. and Escobar, M. A. (2014). Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue. Bio-protocol 4(16): e1213. DOI: 10.21769/BioProtoc.1213; Full Text
  2. Araji, S., Grammer, T. A., Gertzen, R., Anderson, S. D., Mikulic-Petkovsek, M., Veberic, R., Phu, M. L., Solar, A., Leslie, C. A., Dandekar, A. M. and Escobar, M. A. (2014). Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut. Plant Physiol 164(3): 1191-1203.




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