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Determination of Quinone Reductase Activity
醌还原酶活性的测定   

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Abstract

We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a quinone reductase, YaiB, described here, and a hydroquinone dioxygenase, YaiA, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the yahCD-yaiAB operon. The operon is induced by copper to prevent the synergistic toxicity of quinones and copper. The quinone reductase, YaiB, reduces p-benzoquinone and a range of quinone derivatives to hydroquinone, using NADPH as a reductant, according to the reaction: p-benzoquinone + NADPH + H+ → hydroquinone + NADP+. We here describe the measurement of quinone reductase activity, based on the spectrophotometric measurement of NADPH-oxidation.

Materials and Reagents

  1. Quinone reductase purified from Escherichia coli (E. coli) by Ni-NTA chromatography as described in Mancini et al. (2015).
  2. p-Benzoquinone (Sigma-Aldrich, catalog number: B10358 )
    Note: optionally other quinone substrates like 1, 4-penzoquinone, 2-methyl-1, 4-benzoquinone, 2, 5-dimethyl-1, 4-benzoquinone, menadione, naphthoquinone, or 2, 6-dichloro-1, 4-benzoquinone, all available from Sigma-Aldrich.
  3. 50 mM NADPH in water; prepare on the day of use (Sigma-Aldrich, catalog number: N5130 )
  4. 20 mM Tris-Cl buffer (pH 7.5)
  5. Flavin mononucleotide (Sigma-Aldrich, catalog number: F1392 )
  6. p-Benzoquinone (see Recipes)
  7. NADPH (see Recipes)
  8. 20 mM Tris-Cl (see Recipes)
  9. 100 µM flavin mononucleotide (see Recipes)

Equipment

  1. Thermostated spectrophotometer (Shimadzu, model: UV2600 or similar)

Procedure

  1. Mix 970 µl of 20 mM Tris-Cl (pH 7.5), with 10 µl of 100 mM NADPH (final concentration 1 mM) and 10 µl of 100 mM p-benzoquinone (final concentration 1 mM) in a cuvette and equilibrate at 30 °C.
  2. Zero the spectrophotometer and start the reaction at 30 °C by adding purified quinone reductase and immediately start recording the decrease in absorbance at 340 nm due to the oxidation of NADPH to NADP (approximately a 0.1 OD increase at 340 nm in 10 min should be observed, requiring 1 to 10 µg of purified enzyme). In a double-beam spectrophotometer, the measurement can be conducted in the reference beam, which will result in an apparent increase in absorbance. Since YaiB is an enzyme from L. lactis which grows best at 30 °C, this temperature was chosen for the assay,
  3. A linear absorbance change should be observed within a few seconds. Let the reaction proceed for 5 to 10 min or until a steady reaction rate is observed. Calculate the activity as nmol/min, using an extinction coefficient for NADPH of 6,220 M-1 cm-1.

    Enzyme kinetics
    1. Both, NADPH and quinones are substrates of the enzyme reaction. NADPH is the reductant and quinones are the substrates being reduced. To determine the Km for a substrate (either quinones or NADPH), run enzyme reactions with one substrate at 1, 3, 10, 30, 100, 300 and 1,000 µM, while keeping the other substrate at 1 mM.
    2. Determine the initial reaction rates, v0, from the slopes of the earliest linear regions of the recordings and plot v0 versus the substrate concentrations, S.
    3. Fit the curve to obtain the affinity for the substrate, Km, and the maximal velocity, vmax. Alternatively, plot v0 versus 1/S to obtain a Lineweaver-Burk plot, from which Km and vmax can be determined.

Representative data



Figure 1. Representative data. Hanes-Woolf plot of p-benzoquinone reduction by YaiB of L. lactis. [S] is the mM substrate concentration and v the reaction rate in mmol/min. The slope of the linear regression line equals 1/vmax: vmax = 1/0.474 = 2.11 mmol/min/mg. Km is defined by the intercept of the regression line with the ordinate, which equals Km/vmax: Km = 0.1338*2.11 = 0.28 mM.

Notes

  1. Quinone reductases are flavoproteins which lose their cofactor quite readily; therefore, all the buffers used for purification or dilution of the enzyme should be supplemented with 10 µM flavin mononucleotide.
  2. NADPH tends to auto-oxidize to NADP in the presence of oxygen; therefore, it must be prepared fresh.

Recipes

  1. p-Benzoquinone (optionally other quinone substrates like 1,4-penzoquinone, 2-methyl-1,4-benzoquinone, 2,5-dimethyl-1,4-benzoquinone, menadione, naphthoquinone, or 2,6-dichloro-1,4-benzoquinone)
    Dissolve substrates at 50 mM in dimethylsulfoxide
    Stable at room temperature
  2. NADPH
    50 mM in water
    Prepare fresh on the day of use.
  3. 20 mM Tris-Cl (pH 7.5)
    Stable at room temperature.
  4. 100 µM flavin mononucleotide
    Stored frozen

Acknowledgments

This work was supported by Russian Federation Government Grant 14.Z50.31.0011 to leading scientists. The procedure has previously been described in Mancini et al. (2015).

References

  1. Mancini, S., Abicht, H. K., Gonskikh, Y. and Solioz, M. (2015). A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403. Mol Microbiol 95(4): 645-659.

简介

我们最近展示了存在于Firmicutes中的醌解毒途径。 它基于两种酶活性,即本文所述的醌还原酶,YaiB和在单独方案中描述的氢醌双加氧酶YaiA。 在乳酸乳球菌(乳酸乳杆菌)中,这些酶由yahCD-yaiAB 操纵子编码。 操纵子由铜诱导以防止醌和铜的协同毒性。 使用NADPH作为还原剂,醌还原酶,YaiB,根据反应,将对苯醌和一系列醌衍生物还原为氢醌:苯醌+ NADPH + H + +氢醌+ NADP + 。 我们在这里描述醌还原酶活性的测量,基于NADPH氧化的分光光度测量。

材料和试剂

  1. 如Mancini等人(2015)中所述通过Ni-NTA色谱从大肠杆菌(大肠杆菌)纯化的醌酮还原酶。
  2. 苯并醌(Sigma-Aldrich,目录号:B10358)
    注意:任选的其它醌类底物如1,4-苯醌,2-甲基-1,4-苯醌,2,5-二甲基-1,4-苯醌,甲萘醌,萘醌或2,6-二氯-1 ,4-苯醌,均得自Sigma-Aldrich。
  3. 50mM NADPH水溶液; 使用当天准备(Sigma-Aldrich,目录号:N5130)
  4. 20mM Tris-Cl缓冲液(pH7.5)
  5. 黄素单核苷酸(Sigma-Aldrich,目录号:F1392)
  6. p - 苯并醌(请参阅食谱)
  7. NADPH(参见配方)
  8. 20 mM Tris-Cl(参见配方)
  9. 100μM黄素单核苷酸(参见配方)

设备

  1. 恒温分光光度计(Shimadzu,型号:UV2600或类似)

程序

  1. 将970μl20mM Tris-Cl(pH7.5),10μl100mM NADPH(终浓度1mM)和10μl100mM p'-苯醌(终浓度1mM)混合并在30℃下平衡
  2. 零分光光度计并通过加入纯化的醌还原酶在30℃开始反应,并且立即开始记录在340nm的吸光度的减少,这是由于NADPH氧化为NADP(在340℃下大约增加0.1OD在10分钟内应该观察到,需要1至10μg的纯化酶)。在双光束分光光度计中,可以在参考光束中进行测量,这将导致吸光度的明显增加。由于YaiB是来自乳酸乳杆菌的酶,其在30℃下最好生长,因此选择该温度用于测定,
  3. 在几秒钟内应观察到线性吸光度变化。使反应进行5至10分钟或直到观察到稳定的反应速率。使用6,220M -1 cm -1 的NADPH的消光系数计算活性,以nmol/min表示。

    酶动力学
    1. NADPH和醌都是酶反应的底物。 NADPH是   还原剂和醌是被还原的底物。 至 确定底物(醌或NADPH),运行酶的K m 一个底物在1,3,10,30,100,300和1,000μM的反应, 同时保持另一底物为1mM
    2. 确定初始 反应速率,v 0 ,从最早的线性区域的斜率 记录和绘图 0 与底物浓度S.
    3. 拟合曲线以获得对底物的亲和力K m, 最大速度, v max 。 或者,绘制 v 0 与 1/S, Lineweaver-Burk图,从中可以确定K sub和v em。 max

代表数据



图1.代表性数据。Hanes-Woolf对 p - 苯醌的还原。 lactis 。 [S]是mM底物浓度,v是以mmol/min计的反应速率。 线性回归线的斜率等于1/v max max:1/v max = 1/0.474 = 2.11mmol/min/mg。 K m通过回归线与纵坐标的截距来定义,其等于K m/v/v max;/0.133 * 2.11 = 0.28mM。

笔记

  1. 醌还原酶是很容易失去其辅因子的黄素蛋白; 因此,用于纯化或稀释酶的所有缓冲液应补充10μM黄素单核苷酸
  2. NADPH倾向于在氧存在下自动氧化成NADP; 因此,必须准备新鲜。

食谱

  1. 苯醌(任选的其它醌类底物如1,4-苯醌,2-甲基-1,4-苯醌,2,5-二甲基-1,4-苯醌,甲萘醌,萘醌或2 ,6-二氯-1,4-苯醌) 将底物溶于50mM二甲基亚砜中 在室温下稳定
  2. NADPH
    50 mM水溶液中 在使用当天准备新鲜。
  3. 20mM Tris-Cl(pH7.5) 室温下稳定。
  4. 100μM黄素单核苷酸 保存冻结

致谢

这项工作是由俄罗斯联邦政府赠款14.Z50.31.0011支持领先的科学家。 该程序先前已在Mancini等人(2015)中描述。

参考文献

  1. Mancini,S.,Abicht,H.K.,Gonskikh,Y。和Solioz,M。(2015)。 铜诱导的醌降解途径提供针对乳酸乳球菌中铜/醌综合应激的保护 IL1403。 Mol Microbiol 95(4):645-659。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mancini, S. and Solioz, M. (2015). Determination of Quinone Reductase Activity. Bio-protocol 5(17): e1581. DOI: 10.21769/BioProtoc.1581.
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