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Quantification of Uric Acid or Xanthine in Plant Samples
植物样本中尿酸或黄嘌呤的定量测定   

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Abstract

We developed this protocol to assay and quantify the content of uric acid or xanthine in various tissues of Arabidopsis thaliana mutant lines with defective urate oxidase or xanthine dehydrogenase1 and in their complementation and suppressor lines (Hauck et al., 2014).
The protocol is based on a method developed by Invitrogen Life Technologies for measuring uric acid or xanthine in human serum (see References 2 and 3). That protocol though required two adaptions for its use in plant science. Firstly by heating the plant samples, the activity of urate oxidase and xanthine dehydrogenase in the wild type samples is eliminated. Wild type extracts always serve as the proper pigmentation background when calculating the standard curves of uric acid and xanthine. Secondly, all samples are measured with and without the addition of urate oxidase or xanthine dehydrogenase to correct for any H2O2 in the samples induced by previous stress.
The assay is based on the following pair of coupled reactions:
1) Uric acid + O2 → Hydroxyisourate + H2O2 (urate oxidase reaction)
2) AR + H2O2 → Resorufin + O2 (horse radish peroxidase reaction)
Accordingly for Xanthine:
1) Xanthine + H2O + O2 → Uric acid + H2O2 (xanthine oxidase reaction)
2) AR + H2O2 → Resorufin + O2 (horse radish peroxidase reaction)

Materials and Reagents

  1. Sea sand (e.g. Merck, catalog number: 107711 ) to facilitate mechanically homogenizing the samples
  2. Liquid nitrogen
  3. Horse radish peroxidase (HRP) (e.g. Sigma-Aldrich, catalog number: P8375 )
  4. Urate oxidase (UOX) (e.g. Sigma-Aldrich, catalog number: U0880 )
  5. Xanthine oxidase (XO) (e.g. SERVA Electrophoresis GmbH, catalog number: 38418 )
  6. Amplex® ultra red (AR) (Life Technologies, InvitrogenTM, catalog number: A36006 )
  7. Uric acid (e.g. Sigma-Aldrich, catalog number: U2625 )
  8. Xanthine (e.g. Sigma-Aldrich, catalog number: X7375 )
  9. Dimethylsulfoxide (DMSO) (e.g. Sigma-Aldrich, catalog number: D4540 )
  10. Prepare 10 mM AR-stocks (see Recipes)
  11. Prepare stocks of HRP, UOX and XO (see Recipes)

Equipment

  1. Standard flat-bottom microplates (e.g. Greiner, catalog number: 655161 )
  2. Spectrophotometer equipped for reading multiwell plates (e.g. MultiSkan Go, Thermo Fisher Scientific)
  3. Heating block with shaker function
  4. Ultracentrifuges for 20,000 x g (and ideally for 40,000 x g)
  5. Rotator for grinding samples (e.g. Heidolph RZR 2020, but a simpler device may also do) with a tissue-grinder which fits neatly into a conically-tapered 1.5 ml tube (see Figure 1).


    Figure 1. Tissue grinder for microtubes (Taylor Scientific)

  6. Precision analytic balance, e.g. Mettler Toledo XS series, ideally equipped with an ErgoClip holder for 1.5 ml tubes
  7. Heated magnetic stirrer
  8. Micropipettes (1,000, 200, 100, 20 µl)
  9. Multi-channel micropipette (range 50-200 µl)
  10. 1.5 ml reaction tubes
  11. 0.5 ml reaction tubes
  12. 15 ml tubes (if dilutions of extracts become necessary)

Procedure

  1. Prepare the extraction buffer and the stocks described in Recipes (takes ~ 1 h)

  2. Reagents and equipment to prepare before measuring (~ 1 h)
    1. Prepare fresh stocks of 500 µM uric acid or 500 µM xanthine for standard curves (see Figure 2, Row A).
      Notes:
      1. Add the uric acid or xanthine to ~200 ml extraction buffer (EB) in a beaker glass. Begin heating and stirring on a heating plate (set to ~100 °C). Use a pH-meter with thermometer to monitor temperature and pH. Heating will help to dissolve the chemicals but additionally you will need to add a few drops of 1 M NaOH (do this under visual control) until all the uric acid or xanthine is dissolved. Some alkalinization (you will reach around pH 8.5-9) is necessary, because both chemicals dissolve poorly in water.
      2. Another method to prepare uric acid stocks has been described by Xinhua (2006) but was not employed by us.
    2. Label a set of three 1.5 ml tubes for each plant sample biological replicate you want to measure, you will need one tube for sample extraction (no. 1) and two to collect the supernatant of each sample extract (no. 2+3).

  3. Harvesting plant samples
    We routinely analyzed various plant parts like developing seeds (age 7 to 19 days post anthesis), mature dry seeds, seedlings (age 3 to 15 days after imbibition of the seeds on agar plates), leaves at bolting time (leaf age being defined by their position on the rosettes and root tissue. For more details see Figure 5 in Hauck et al. (2014).
    Note that you need to include wild type samples to calculate the standard curves for the uric acid or xanthine concentration.
    For each sample place the empty sample tube (no. 1) in the holder of the electronic balance and set the balance back to zero, then weigh in your sample. As a rule of thumb, use ~ 5-10 mg seeds, ~ 50-100 mg leaves, ~ 20 mg roots. Write down the weight for each sample. Close sample tube tightly and transfer it to liquid nitrogen.

  4. Extraction procedure (~ 2 h)
    1. Set up the rotator with the tissue-grinder, have EB, and a bottle of distilled water at hand.
    2. To avoid any carry-over of metabolites we recommend to first homogenize the wild type samples, which do not contain any uric acid or xanthine unless you deal with leaves of stressed plants.
    3. Take a sample out of the liquid nitrogen, carefully open the lid and add very little sea sand to facilitate the complete grinding and homogenization of the sample. To 100 mg leaves add 500 µl EB, to 5-10 mg seeds add 250 µl EB, then start grinding. Better hold on tight to those sample tubes! When the sample appears homogenous, close the tube´s lid and transfer it back to liquid nitrogen. Rinse the pistil thoroughly with distilled water and proceed to the next sample.
    4. Set up the heating block (inset must be suitable for conically-tapered 1.5 ml tubes) to 95 °C. Transfer all samples back to room temperature, carefully open the lids, wait a couple of minutes, close lids and transfer all tubes to 95 °C heat block.
    5. Open lids once more to avoid sudden popping-up which may cause loss of sample. Heat-shake for 10 min at 95 °C, subsequently centrifuge them for 15 min at room temperature at 20,000 x g.
    6. Carefully transfer the supernatants with a micropipette to the second set of tubes (no. 2), repeat as follows for each sample: add again same amount of EB as before, vortex, heat-shake and centrifuge as before, transfer supernatant to the tube (no. 2).
    7. Finally, ultracentrifuge all the tubes (no. 2), ideally at 40,000 x g for 15 min. to thoroughly sediment tissue debris and transfer all supernatants finally to the third set of fresh tubes (no. 3).
      Note: As uric acid or xanthine accumulates during seed development in A.t. uox or xdh1 mutants, use 15 ml-tubes to make dilutions (e.g. 2x, 5x) of the final extracts (tubes no. 3) when measuring dry seeds.
      When initially measuring metabolite standards we found a linear relationship between absorption at 560 nm and concentrations of uric acid or xanthine up to 250 µM.

  5. Preparing 50 µl of standards and samples (~ 30 min, per plate)
    For uric acid (or xanthine) standards use the wild type extract of the same plant part like the samples you are measuring, because this will basically eliminate possible bias caused by pigments. Wild type extracts will generally not contain uric acid or xanthine (but leaf extracts of a stressed wild type plant may contain H2O2, so better use at least two different wild type extracts). The standards will give you the slope of the regression line you need to determine the metabolite content of your samples. The plate shown in Figure 2 was prepared as follows: A1 → 50 µl wild type extract (=> 0 µM), A2 → 45 µl wild type extract + 5 µl of 500 µM uric acid (=> 50 µM) etc.
    Next 50 µl of each sample were pipetted into the respective wells on the left and right half of the plate (e.g. young leaves, plant 1 in B1, B2, B7, B8; old leaves, plant 3 in F5, F6, F11, F12 etc.).

  6. Prepare two kind of working solution (~ 15 min)
    Each sample will be measured simultaneously in two different set-ups: Firstly with UOX (or XO) and secondly without UOX (or XO) to determine the amount of H2O2 already contained in the sample. The absorption values of reaction 2 will then be subtracted from those of reaction 1. Generally the right half of your plate will stay quite colorless, except when measuring extracts of stressed leaves.

    Table 1. Composition of the working solution (WS)
    # assays/wells ( including standards)
     10
    20
    30
    35
    40
    45
    50
    55
    60
    Working solution (WS) consists of:
    Amplex
    red (AR), µl
    5
    10
    15
    17.5
    20
    22.5
    25
    27.5
    30
    HRP, µl
    2
    4
    6
    7
    8
    9
    10
    11
    12
    UOX
    (or XO), µl
    4
    (2
    8
    4
    12
    6
    14
    7
    16
    8
    18
    9
    20
    10
    22
    11
    24
    12)
    Extraction
    buffer (EB), ml
    0.5
    1.0
    1.5
    1.75
    2.0
    2.25
    2.5
    2.75
    3.0

    Table 1 will help you to prepare the working solutions.
    E.g. to measure the plate shown in Figure 2, you will need (2 x 4 + 16 x 2) x 50 µl = 40 x 50 µl = 2.0 ml (better to prepare 2.5 ml) of working solution with UOX (or XO), and 16 x 2 x 50 µl = 1.6 ml (prepare 2.0 ml) of working solution without UOX (or XO). KEEP THE WS PROTECTED FROM LIGHT (e.g. wrap into aluminum foil).

  7. Setting up a 96-well microtiter plate


    Figure 2. Setup example of a 96-well plate arranged to assay the content of uric acid in leaves of different age (Rows B-G) in four to six biological replicates, each in duplicates (column 1/2, 3/4, 5/6 etc). The above setup delivered the data shown in the EXCEL-sheet.

    1. Uric acid standard in duplicate (A1-4, A7-10): Row A contains varying concentrations of uric acid in 50 µl of a wild type extract + 50 µl working solution (WS) with UOX.
    2. Samples: Columns 1-6 contain 50 µl sample extract + 50 µl WS with UOX, columns 7-12 contain 50 µl sample extract +50 µl WS without UOX.

  8. Setting up the scanner and reading the plate (~20 min per plate)
    1. Before you start the reaction by adding the WS, make sure you can access the photometer for the next 15-20 min. Do the read-out as soon as possible after starting the reaction (by adding the WS), because O2 and light will bias your assay (see Abstract).
    2. Set the photometer to absorbance at 560 nm and read-out in 1-2 min-intervals so you can graphically follow the reaction.
    3. Ideally use a multi-channel pipette to add 50 µl WS with UOX (or XO) to the left-side wells and the standards, or 50 µl WS without UOX (or XO) to the right-side wells. Each well on the left side then contains 50 µM AR, 20 milliunits HRP and 20 milliunits UOX (or 2 milliunits XO) in 100 µl. Right-side wells will contain 50 µM AR and 20 milliunits HRP only.
    4. Aim at pipetting without introducing air-bubbles to the wells. Remove air-bubbles by pricking them with a dry pipet tip or a needle, then insert the plate into the photometer and read out the plate repeatedly until an endpoint of the reaction after approximately 15-20 min. is reached, indicated by the absorbance reaching a plateau.

  9. Calculating the uric acid/xanthine content of your sample
    Copy-paste the read-out to the provided EXCEL-Sheet, which then does steps I1-5 of the calculations for you.
    Note: Refer to Figure 2 to match the plate´s read-out with the corresponding columns of the EXCEL-Sheet.
    1. From the duplicates with/without UOX or XO calculate the arithmetic means of the absorption values, MW and MWO, for each sample.
    2. Then subtract MWO, the absorption value of reaction 2 (without UOX/XO) from MW, the absorption of reaction 1 (with UOX/XO).
      Note that this operation also gets rid of the y-axis shifts caused by background.
    3. From the uric acid or xanthine standards calculate the slope S [in 1/µM] of the regression line.
    4. For each individual assay the metabolite concentration is CA = (MW-MWO)/S [in µM] (with the assumption there was no dilution).
    5. Considering the extraction volume VE [in µl] the amount of metabolite in a given sample is NE = CA x VE [in mol]. It was contained in P mg of the sample and therefore the metabolite concentration of that sample is CP = NE / P [in µmol/g].
      We provide an EXCEL-sheet we routinely used: Rows 30-39 show a typical absorption readout of the photometer at 11-20 min. Row 39 contains the plateau value after 20 min and is the only photometric data used for the subsequent calculation. The cells with the orange filling highlight the calculation steps I1-5. For our purposes we further distinguished fresh weight, fw, from dry weight, dw (we kept reference samples for 2 days at 60 °C in a heating cabinet) of the samples and added the moisture contained in the fresh leave samples (fw-dw) to the extraction volume (see column L).

Representative data

The provided EXCEL-sheet contains the read-out of the plate set-up shown in Figure 2. Use it by pasting your own read-out data into row 39.

Notes

This protocol was developed over the course of several months and found reliable to measure uric acid and xanthine concentrations up to 250 µM in a single assay, because that is the range of a linear relationship between absorption and metabolite concentration (refer to the EXCEL-sheet). When assaying dry seeds, which accumulate the metabolites heavily in the respective enzyme mutants, it may be necessary to make 3- or 5-fold dilutions in a 15-ml tube, before loading the wells.

Recipes

  1. Prepare 10 mM AR-stocks
    Add 400 µl DMSO to a 1 mg-vial of purchased AR, mix thoroughly and store as 50 µl aliquots at -20 °C.
    One vial is sufficient for 800 wells (= 800 single reactions)
  2. Prepare stocks of HRP, UOX and XO
    Prepare ~500 ml of 0.1 M extraction buffer from an 0.5 M Tris/HCl pH 7.5 buffer stock
    Then use the extraction buffer to prepare stocks of
    100 Units/ml HRP
    50 Units/ml UOX
    10 Units/ml XO
    from the purchased enzyme preparations (Reagents 4-6), and store them in 50 µl aliquots at -20 °C. Used aliquots can be stored at 4 °C for several weeks.

Acknowledgments

This research was funded by the Deutsche Forschungsgemeinschaft (Grant DFG WI3411/1 2) and the German Academic Exchange Service from funds of the German Federal Ministry for Education and Research, program German-Chinese Research Groups.

References

  1. Hauck, O. K., Scharnberg, J., Escobar, N. M., Wanner, G., Giavalisco, P. and Witte, C. P. (2014). Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance. Plant Cell 26(7): 3090-3100.
  2. Life technologies: https://tools.lifetechnologies.com/content/sfs/manuals/mp22181.pdf.
  3. Life technologies: https://tools.lifetechnologies.com/content/sfs/manuals/mp22182.pdf.
  4. Xinhua, D. Preparation of Uric Acid Standard Stock Solution Clinical Chemistry 2006; v. 52, p.2117-2118
  5. Source Figure 1: http://www.taylorscientific.com/taylorscientific/Disposable-PELLET-PESTLES-with-Microtubes-Kimble-Chase-P13260.aspx

简介

我们开发了该方案以测定和定量具有缺陷的尿酸氧化酶或黄嘌呤脱氢酶1的拟南芥突变体系以及它们的互补和抑制系的各种组织中的尿酸或黄嘌呤的含量(Hauck等</em>,2014)。
该方案基于由Invitrogen Life Technologies开发的用于测量人血清中的尿酸或黄嘌呤的方法(参见参考文献2和3)。该协议虽然需要两个适应它在植物科学中的使用。首先通过加热植物样品,消除野生型样品中的尿酸氧化酶和黄嘌呤脱氢酶的活性。当计算尿酸和黄嘌呤的标准曲线时,野生型提取物总是作为适当的色素沉着背景。其次,在添加和不添加尿酸氧化酶或黄嘌呤脱氢酶的情况下测量所有样品,以校正由先前应激诱导的样品中的任何H 2 O 2 O 2。 >该测定基于以下一对偶联反应:</1> 1)尿酸+ O 2→羟基香草酸+ H 2 O 2 - (辣根过氧化物酶反应)的反应物(尿酸氧化酶反应)的反应。 )
因此对于黄嘌呤:
1)黄嘌呤+ H 2 O + O 2→尿酸+ H 2 - O 2(黄嘌呤氧化酶反应)2→AR + H 2→O→2→Resorufin + O 2→ (辣根过氧化物酶反应)

材料和试剂

  1. 海砂(如 Merck,目录号:107711),以便于机械均质化样品
  2. 液氮
  3. 辣根过氧化物酶(HRP)(例如Sigma-Aldrich,目录号:P8375)
  4. Urate氧化酶(UOX)(例如Sigma-Aldrich,目录号:U0880)
  5. 黄嘌呤氧化酶(XO)(例如,SERVA Electrophoresis GmbH,目录号:38418)
  6. Amplex超红(AR)(Life Technologies,Invitrogen TM ,目录号:A36006)
  7. 尿酸(例如Sigma-Aldrich,目录号:U2625)
  8. 黄嘌呤(例如Sigma-Aldrich,目录号:X7375)
  9. 二甲基亚砜(DMSO)(例如Sigma-Aldrich,目录号:D4540)
  10. 准备10 mM AR股票(见配方)
  11. 准备HRP,UOX和XO的库存(参见配方)

设备

  1. 标准平底微孔板(如 Greiner,目录号:655161)
  2. 用于读取多孔板的分光光度计(例如,ThermoScience MicroSkan Go),
  3. 带振动功能的加热块
  4. 超速离心机为20,000 x em (最好为40,000 x g )
  5. 用于研磨样品的旋转器(例如 Heidolph RZR 2020,但更简单的装置也可以),具有组织研磨机,其整齐地配合到锥形锥形1.5ml管中(参见图1) >

    图1.微管组织研磨机(Taylor Scientific)

  6. 精确分析天平,例如 Mettler Toledo XS系列,理想地配备了用于1.5 ml管的ErgoClip支架
  7. 加热磁力搅拌器
  8. 微量移液器(1,000,200,100,20μl)
  9. 多通道微量移液器(范围50-200μl)
  10. 1.5 ml反应管
  11. 0.5 ml反应管
  12. 15 ml管(如果需要稀释提取液)

程序

  1. 准备提取缓冲液和食谱中描述的种子(需要〜1小时)

  2. 测量前要准备的试剂和设备(〜1小时)
    1. 准备500μM尿酸或500μM黄嘌呤的新鲜储备液用于标准曲线(参见图2,A行)。
      注意:
      1. 将尿酸或黄嘌呤加入〜200ml提取缓冲液(EB)中   烧杯玻璃。 开始加热并在加热板上搅拌(设定为 〜100℃)。 使用带温度计的pH计监测温度和pH。   加热将有助于溶解化学品,但另外你会 需要加入几滴1M NaOH(在视觉控制下这样做),直到   所有尿酸或黄嘌呤溶解。 一些碱化(你 将达到约8.5-9)是必要的,因为两种化学品 在水中溶解差。
      2. 新华(2006)描述了另一种制备尿酸储备液的方法,但是我们没有使用它。
    2. 为每个植物样品生物学标记一组三个1.5ml管 复制你想测量,你将需要一个试管 提取(1号)和两次,收集每个样品的上清液 提取(第2 + 3)。

  3. 收获植物样品
    我们常规地分析各种植物部分,如发育种子(开花后7至19天龄),成熟干种子,幼苗(种子在琼脂平板上吸收后的3至15天龄),叶子在抽苔时间它们在莲座和根组织上的位置。更多细节参见Hauck等人(2014)中的图5。 请注意,您需要包含野生型样品,以计算尿酸或黄嘌呤浓度的标准曲线。
    对于每个样品,将空样品管(编号1)放置在电子天平的支架中,并将天平置回零,然后称重样品。根据经验,使用〜5-10mg种子,〜50-100mg叶子,〜20mg根。记下每个样品的重量。密闭样品管,将其转移到液氮中
  4. 提取程序(〜2小时)
    1. 用组织研磨机设置旋转器,有EB和一瓶蒸馏水。
    2. 为了避免任何遗留的代谢物,我们建议先 匀浆野生型样品,其不含任何尿酸或 黄嘌呤,除非你处理受胁迫植物的叶子
    3. 拿一个 样品从液氮中取出,小心打开盖子加入很多 小海砂便于完全研磨和均匀化 的样品。 对100毫克叶添加500微升EB,5-10毫克种子加250 μlEB,然后开始研磨。 更好地握住那些样品管! 当样品呈现均匀时,关闭管的盖子并将其转移   回到液氮。 用蒸馏水彻底冲洗雌蕊 水并继续下一个样品
    4. 设置加热块 (插图必须适用于锥形1.5毫升锥形管)至95℃。 将所有样品转移回室温,小心打开盖子, 等待几分钟,关闭盖子,将所有管转移到95°C 热块。
    5. 打开盖一次,以避免突然弹出 可能导致样品损失。 随后在95℃下热摇动10分钟 在室温下以20,000×g离心15分钟。
    6. 用微量移液管小心转移上清液至第二 套管(2号),每个样品重复如下:再次加入 如前所述EB的量,涡旋,热摇动和离心如前, 转移上清液到管(编号2)。
    7. 最后, 超声波离心所有管(2号),理想地在40,000×g下离心15分钟。   以彻底沉淀组织碎片并转移所有上清液 最后到第三套新鲜管(3号) 注意:作为尿酸 或黄嘌呤在A.t.中的种子发育期间积累。 uox或xdh1 突变体,使用15ml管进行稀释(例如2x,5x)的最终 测量干燥种子时提取物(管3号)。
      最初时 测量代谢物标准,我们发现之间存在线性关系 在560nm的吸收和尿酸或黄嘌呤的浓度 250μM。

  5. 准备50微升的标准品和样品(〜30分钟,每板)
    对于尿酸(或黄嘌呤)标准,使用相同植物部分的野生型提取物,如您正在测量的样品,因为这将基本消除由颜料引起的可能偏差。 野生型提取物通常不会 最后, 超声波离心所有管(2号),理想地在40,000×g下离心15分钟。   以彻底沉淀组织碎片并转移所有上清液 最后到第三套新鲜管(3号) 注意:作为尿酸 或黄嘌呤在A.t.中的种子发育期间积累。 uox或xdh1 突变体,使用15ml管进行稀释(例如2x,5x)的最终 测量干燥种子时提取物(管3号)。
    最初时 测量代谢物标准,我们发现之间存在线性关系 在560nm的吸收和尿酸或黄嘌呤的浓度 250μM。

  • 准备50微升的标准品和样品(〜30分钟,每板)
    对于尿酸(或黄嘌呤)标准,使用相同植物部分的野生型提取物,如您正在测量的样品,因为这将基本消除由颜料引起的可能偏差。 野生型提取物通常不会...  10
    20
    30
    35
    40
    45
    50
    55
    60
    Working solution (WS) consists of:
    Amplex
    red (AR), µl
    5
    10
    15
    17.5
    20
    22.5
    25
    27.5
    30
    HRP,μl
    2
    4
    6
    7
    8
    9
    10
    11
    12
    UOX
    (或XO),微孔
    4
    (2
    8
    4
    12
    6
    14
    7
    16
    8
    18
    9
    20
    10
    22
    11
    24
    12)
    提取
    缓冲区(EB),ml
    0.5
    1.0
    1.5
    1.75
    2.0
    2.25
    2.5
    2.75
    3.0

    表1将帮助您准备工作解决方案。
    例如要测量图2所示的平板,您将需要(2×4 + 16×2)×50μl= 40×50μl= 2.0ml(更好地制备2.5ml)工作溶液与UOX(或XO),和16×2×50μl= 1.6ml(制备2.0ml)不含UOX(或XO)的工作溶液。保护受光保护的WS(例如包裹铝箔)。

  • 设置96孔微量滴定板


    图2.设置为在四至六个生物重复中测定不同年龄(Rows BG)的叶中尿酸含量的96孔板的设置实施例,每个重复一次(柱1/2,3/4 ,5/6 等)。上述设置提供了EXCEL表中显示的数据。

    1. 尿酸标准品一式两份(A1-4,A7-10):A行含有不同 50μl野生型提取物中的尿酸浓度+50μl 工作解决方案(WS)与UOX
    2. 样品:柱1-6含有50 μl样品提取物+50μlWS与UOX,7-12列含有50μl样品   提取+50μlWS,无UOX。

  • 设置扫描仪和读取板(〜20分钟/板)
    1. 在通过添加WS开始反应之前,请确保您可以访问   光度计下15-20分钟。 尽快读出 可能在开始反应之后(通过添加WS),因为O 2和 光将偏向您的测定(见摘要)
    2. 将光度计设置为560nm处的吸光度,并以1-2分钟间隔读出,以便您可以以图形方式跟踪反应。
    3. 理想情况下使用多通道移液器添加50 ul WS与UOX(或XO)   到左侧孔和标准品,或50μlWS没有UOX(或 XO)到右侧井。 左侧的每个井然后包含50   μMAR,20毫单位HRP和20毫单位UOX(或2毫单位XO) 100μl。 右侧孔将含有50μMAR和20毫单位HRP 只有。
    4. 瞄准在移液而不引入气泡 井。 通过用干燥的移液管尖端刺破移除气泡 针,然后将板插入光度计并读出板   反复直到反应终点大约15-20后 min。 达到,由达到平台的吸光度指示。

  • 计算样品的尿酸/黄嘌呤含量
    将读数复制粘贴到提供的EXCEL-Sheet,然后执行计算的步骤I1-5。
    注意:参考图2,使板的读数与EXCEL-Sheet的相应列匹配。
    1. 从具有/不具有UOX或XO的重复中,对于每个样本计算吸收值的算术平均值M sub W和W sub WO。
    2. 然后减去M w,反应2(无UOX/XO)从M w的吸收值,反应1(与UOX/XO的吸收)的吸收值。 > 请注意,此操作也会消除由背景引起的y轴偏移。
    3. 从尿酸或黄嘌呤标准品计算回归线的斜率S [in 1 /μM]。
    4. 对于每个单独的测定,代谢物浓度为C sub (M -M )/S [以μM为单位](假定没有稀释)。
    5. 考虑到提取体积V sub [以μl计]代谢物的量  给定的样品是N sub = C subA x V sub [以mol计]。其含量为P mg 样品和因此该样品的代谢物浓度 C sub = P sub/P [μmol/g]。
      我们定期提供EXCEL表 使用:行30-39显示光度计的典型吸收读数 11-20分钟。行39包含20分钟后的平台值,并且是 只有用于后续计算的光度数据。细胞 用橙色填充突出显示计算步骤I1-5。对于我们 我们进一步区分鲜重,fw,从干重,dw (我们在加热箱中在60℃下保持参考样品2天) 样品并添加新鲜假样品中含有的水分 (fw-dw)到提取体积(见列L)。
  • 代表数据

    所提供的EXCEL表包含图2所示的板设置的读出。通过将自己的读出数据粘贴到行39中来使用它。

    笔记

    该方案是在几个月的过程中发展的,并且发现在单次测定中可靠地测量高达250μM的尿酸和黄嘌呤浓度,因为这是吸收和代谢物浓度之间的线性关系的范围(参见EXCEL表 )。 当测定在相应的酶突变体中大量累积代谢物的干燥种子时,可能需要在装载孔之前在15-ml管中进行3或5倍稀释。

    食谱

    1. 准备10 mM AR股票
      加入400微升DMSO至购买的AR的1毫克小瓶,充分混合,并存储为50微升等分试样在-20°C。
      一个小瓶足以容纳800个孔(= 800个单反应)
    2. 准备HRP,UOX和XO的库存
      从0.5M Tris/HCl pH 7.5缓冲液储液制备〜500ml的0.1M提取缓冲液
      然后使用提取缓冲液制备
      库存 100单位/ml HRP
      50单位/ml UOX
      10单位/ml XO
      从所购买的酶制剂(试剂4-6),并将其以50μl等分试样在-20℃下储存。 使用的等分试样可以在4℃下储存数周。

    致谢

    这项研究由德意志Forschungsgemeinschaft(格兰特DFG WI3411/1 2)和德国学术交流服务从德国联邦教育和研究部,计划德中研究组资助。

    参考文献

    1. Hauck,O.K.,Scharnberg,J.,Escobar,N.M.,Wanner,G.,Giavalisco,P.and Witte,C.P。(2014)。 拟南芥中的尿酸积累尿酸氧化酶突变体通过阻断而损害幼苗建立过氧化物酶体维持。 植物细胞 26(7):3090-3100
    2. 生命技术: https://tools.lifetechnologies.com/content/sfs/manuals/mp22181.pdf。
    3. 生命技术: https://tools.lifetechnologies.com/content/sfs/manuals/mp22182.pdf。
    4. 新华,D.尿酸标准储备液的制备临床化学2006; v。52,p.2117-2118
    5. 来源图1: http://www .taylorscientific.com/taylorscientific /一次性 - PELLET-PESTLES-with-Microtubes-Kimble-Chase-P13260.aspx
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    引用:Hauck, O. K. and Witte, C. (2015). Quantification of Uric Acid or Xanthine in Plant Samples. Bio-protocol 5(13): e1523. DOI: 10.21769/BioProtoc.1523.
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