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Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor
以联二-n-甲基吡啶作为电子受体体外测定丙酮酸铁氧化还原蛋白的活性   

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Abstract

Here we describe the activity measurements of heterologous expressed pyruvate:ferredoxin oxidoreductase (Noth et al., 2013) (Noth et al.,2013) from Chlamydomonas reinhardtii. This enzyme catalyzes the reversible reaction (I) from pyruvate to acetyl CoA and CO2 generating low potential electrons which are in vivo transferred to ferredoxin.



In this assay we use methyl viologen as artificial electron acceptor which turns into dark violet (ε604 = 13.6 mM-1 cm-1) (Mayhew, 1978) in its reduced state (Figure 1).

Keywords: Fermentation(发酵), Oxidoredutase(氧化还原酶), Activity Assay(活性测定), Iron Sulfur Cluster(铁硫簇), Chlamydomonas reinhardtii(莱茵衣藻)


Figure 1. Activity assay using pyruvate, coenzyme A and methyl viologen (Ctrl+). In the absence of pyruvate, no methyl viologen reduction occurs (Ctr-).

Materials and Reagents

         Note: All Reagents are dissolved freshly in an anaerobic tent.

  1. Purified pyruvate: ferredoxin oxidoreductase (PFR1)
  2. Sodium pyruvate (≥ 99%, Stock 500 mM) (Sigma-Aldrich)
  3. Sodium coenzyme A (≥ 85%, Stock 20 mM) (Sigma-Aldrich)
  4. Thiamine pyrophosphate (≥ 95%, Stock 250 mM) (Sigma-Aldrich)
  5. Methyl viologen (98%, Stock 1 M) (Sigma-Aldrich)
  6. Dithioerythriol (≥ 99%, Stock 100 mM) (Carl Roth)
  7. 0.1 M Tris-HCl buffer (pH 8.0) (see Recipes)

Equipment

  1. Anaerobic tent (1% H2, 99% N2) (Toepffer Lab Systems)
  2. 96 well plate reader (Beckman Coulter, catalog number: Paradigm1113 )
  3. PC running Multimode analysis software (Beckman Coulter)
  4. NanoDrop (Paqlab)

Procedure

  1. Protein concentration of heterologous expressed and purified PFR1 from 2 L of cell culture is measured at A280nm using NanoDrop. (ref bio-protocol: Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins).
  2. All reduction assays are performed under anaerobic atmosphere (1% H2, 99% N2) at room temperature.
  3. The reaction mixture contains 10 mM sodium pyruvate, 2 mM sodium coenzyme A, 5 mM thiamine pyrophosphate, 10 mM methyl viologen and 16 mM dithioerythritol in 0.1 mM Tris-HCl (pH 8).
  4. To start catalysis a final concentration of 1.4 μM PFR1 is added to the reaction mixture and absorbance (A604, 96 well plate reader) can be monitored time resolved every 30 seconds until saturation is reached.
  5. To determine enzyme activity the molar extinction coefficient ε604 = 13.6 mM-1 cm-1 (Mayhew, 1978) can be used applying Lambert Beer Law (Eq.I). One unit was defined as the conversion of 1 mol of pyruvate or CoA and the reduction of 2 mol of methyl viologen, respectively, per minute.

    Eλ = ελ . c . d                  (Eq.I)
    Eλ: extinction
    ελ: extinction coefficient
    c: concentration
    d: layer thickness

Recipes

  1. 0.1 M Tris-HCl buffer (pH 8.0) (1,000 ml)
    Mix 12.114 g of Tris base with 800 ml dH2O
    Adjust pH to 8 with HCl
    Add ddH2O to 1,000 ml
    Autoclave for 20 minutes at 121 °C
    Store at 4 °C

Acknowledgments

Kinetics for enzyme dependent methyl viologen reduction is adapted from Zeikus et al. (1977). Research on the pyruvate:ferredoxin oxidoreductase from C. reinhardtii was scientifically supported by Anja Hemschemeier and Thomas Happe.

References

  1. Mayhew, S. G. (1978). The redox potential of dithionite and SO-2 from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. Eur J Biochem 85(2): 535-547.
  2. Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
  3. Noth, J. (2013). Heterologous production and anaerobic purification of His- and StrepII-tagged recombinant proteins. Bio-protocol 3(17): e881.

简介

这里我们描述了来自衣藻衣藻的异源表达丙酮酸:铁氧还蛋白氧化还原酶(Noth等,2013)(Noth等,2013)的活性测定。 该酶催化从丙酮酸向乙酰CoA的可逆反应(I)和产生低电位电子的CO 2,其将体内转移至铁氧还蛋白。

在该测定中,我们使用甲基紫精作为人造电子受体,其在其还原状态下变成深紫色(ε604= 13.6mM-1cm-1)(Mayhew,1978)(图1)。

关键字:发酵, 氧化还原酶, 活性测定, 铁硫簇, 莱茵衣藻

图1.使用丙酮酸,辅酶A和甲基紫精(Ctrl +)的活性测定。在没有丙酮酸盐的情况下,不发生甲基紫精减少(Ctr-)。

材料和试剂

         注意:所有试剂都是新鲜溶解在厌氧帐篷中

  1. 纯化丙酮酸:铁氧还蛋白氧化还原酶(PFR1)
  2. 丙酮酸钠(≥99%,储备500mM)(Sigma-Aldrich)
  3. 钠辅酶A(≥85%,库存20mM)(Sigma-Aldrich)
  4. 焦磷酸硫胺素(≥95%,Stock 250mM)(Sigma-Aldrich)
  5. 甲基紫精(98%,Stock 1M)(Sigma-Aldrich)
  6. 二硫赤藓醇(≥99%,储备100mM)(Carl Roth)
  7. 0.1 M Tris-HCl缓冲液(pH 8.0)(参见配方)

设备

  1. 厌氧性帐篷(1%H 2 O,99%N 2)(Toepffer Lab Systems)
  2. 96孔板读数器(Beckman Coulter,目录号:Paradigm1113)
  3. PC运行多模分析软件(Beckman Coulter)
  4. NanoDrop(Paqlab)

程序

  1. 使用NanoDrop在A 280nm处测量来自2L细胞培养物的异源表达和纯化的PFR1的蛋白浓度。 (ref bio-protocol: Hisologous Production and Anaerobic Purification of His-和StrepII-tagged重组蛋白) 。
  2. 所有还原测定在室温下在厌氧气氛(1%H 2 O,99%N 2)下进行。
  3. 反应混合物含有在0.1mM Tris-HCl(pH8)中的10mM丙酮酸钠,2mM钠辅酶A,5mM焦磷酸硫胺素,10mM甲基紫精和16mM二硫赤藓糖醇。
  4. 为了开始催化,将最终浓度为1.4μM的PFR1加入到反应混合物中,并且可以每30秒监测时间分辨的吸光度(A 604,96孔板读数器),直到达到饱和。 >
  5. 为了测定酶活性,可以使用摩尔消光系数ε<0.04> = 13.6mM cm -1 -1(Mayhew,1978) Law(Eq.1)。一个单位定义为每分钟分别转化1摩尔丙酮酸或CoA和还原2摩尔甲基紫精。

    E λ λ 。 c 。 d                 (Eq.1)
    E λ:消光
    ε<λ>:消光系数
    c:浓度
    d:层厚度

食谱

  1. 0.1M Tris-HCl缓冲液(pH8.0)(1000ml) 将12.114g Tris碱与800ml dH 2 O混合 用HCl
    调节pH至8 将ddH <2> O添加到1,000 ml
    在121°C高压灭菌20分钟
    存储在4°C

致谢

酶依赖性甲基紫精还原的动力学改编自Zeikus等人(1977)。 丙酮酸:来自C的铁氧还蛋白氧化还原酶的研究。 reinhardtii 得到Anja Hemschemeier和Thomas Happe的科学支持。

参考文献

  1. Mayhew,S.G。(1978)。 连二亚硫酸盐和SO-2从与黄素氧还蛋白,甲基紫精和氢加氢酶的平衡反应的氧化还原电位 。 Eur J Biochem 85(2):535-547。
  2. Noth,J.,Krawietz,D.,Hemschemeier,A.and Happe,T。(2013)。 丙酮酸:铁氧还蛋白氧化还原酶偶联于莱茵衣藻中与光无关的氢产生


    J Biol Chem 288(6):4368-4377。
  3. Noth,J。(2013)。 His和StrepII标记的重组蛋白的异源生产和厌氧纯化生物方案 3(17):e881。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Noth, J. (2013). Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor. Bio-protocol 3(17): e882. DOI: 10.21769/BioProtoc.882.
  2. Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
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