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May 2018

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Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
黄嘌呤/黄嘌呤氧化酶法测定蛋白质中的黄素电位   

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Abstract

This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (Em = -55 mV) in the SdhA flavoprotein subunit of succinate dehydrogenase from Escherichia coli. In principle, this method can be used routinely to determine the redox potential of flavin cofactors in any simple flavoprotein from equilibrium concentrations with an appropriate reference dye of known Em without the use of sophisticated electrochemical equipment.

Keywords: Flavin (黄素), Flavoprotein (黄素蛋白), Reduction potential (还原电位), Xanthine oxidase (黄嘌呤氧化酶), Midpoint potential (中点电位)

Background

Several biophysical methods can be used to measure the reduction midpoint potential (Em) of flavins in a protein. These potentiometric methods usually rely on the electrochemical coupling between the protein of interest and an electrode. For example, in complex flavoproteins harboring additional cofactors such as iron-sulfur centers and quinones, electrochemical methods and Electron Paramagnetic Resonance (EPR) spectroscopy are often used to determine the Em of the redox centers (Kowal et al., 1995; Saenger, et al., 2005; Hudson et al., 2005; Cheng et al., 2015). However, in simple flavoproteins that contain only flavin redox centers, the co-factor can be directly studied by conventional optical spectrophotometry which does not require special electrochemistry equipment or expensive EPR instrumentation. In 1990 Vincent Massey introduced a simple method that allows determination of the flavin reduction potential from the equilibrium concentrations of the oxidized and reduced partners, i.e., flavoprotein and a reference dye (Massey, 1991). The method does not directly measure the reduction potential of the flavin but rather determines a difference between Em values of the flavin and a reference dye. The scheme in Figure 1 describes the method. The xanthine/xanthine oxidase system provides a slow continuous reduction of the indicator dye and the flavin in the presence of benzyl viologen (BV) or methyl viologen (MV) which ensures the rapid equilibration of reducing equivalents. This allows slow changes in equilibrium of the reduced and oxidized forms of the protein and dye until both are completely reduced. A series of spectra recorded over the course of the reaction is used to calculate the ratio of the oxidized and reduced forms of flavin and dye. The Nernst plot of an equilibrium concentration, the dye against the flavoprotein allows determination of the shift in the Em of the protein in comparison with the dye. Because some of the components used in this method are low potential chemicals and proteins (viologen, reference dyes, and many flavoproteins) that are readily oxidized by even trace amounts of oxygen, a requirement for this system is that strict anaerobiosis be maintained.

Here we show an example of how this method is applied to determine the reduction potential of covalently bound FAD in the SdhA flavoprotein of succinate dehydrogenase. SdhA is the flavoprotein component of the four subunit membrane-bound succinate:ubiquinone reductase (i.e., complex II) which is part of the TCA (Krebs) cycle and electron transport chain of the mitochondrion and many bacteria. Complex II couples the reaction of succinate oxidation to fumarate with ubiquinone reduction to ubiquinol. The FAD co-factor is involved in the reversible succinate-fumarate conversion. The Em for free FAD in solution is -219 mV, whereas covalent attachment of FAD to SdhA considerably raises the potential of the flavin. In this example, we use the redox dye indigo-tetrasulphonate (ITS) (Em = -46 mV) and have determined that the EmFAD for the Escherichia coli SdhA protein is -55 mV. In principle, this simple method can be used for any flavoprotein by using a redox dye with a suitable reduction potential (i.e., a potential within ±30 mV to that expected for the flavoprotein being determined). This method has also been validated for the determination of the reduction potential of many other flavoproteins (see Christgen et al., 2019) and several heme proteins that suggests that the method could be widely useful for most heme proteins (Efimov et al., 2014).


Figure 1. Spectrophotometric method for determination of the Em of a flavin using a reference dye in the presence of xanthine/xanthine oxidase. Most suitable dyes undergo 2-electron reduction (see Table 1).

Materials and Reagents

  1. 0.2 µm filter (Corning) (Sigma-Aldrich, catalog number: CLS431229 )
  2. Potassium phosphate dibasic, anhydrous (Sigma-Aldrich, catalog number: 795496 )
  3. Potassium phosphate monobasic, anhydrous (Sigma-Aldrich, catalog number: 795488 )
  4. Xanthine (Sigma-Aldrich, catalog number: X7375 )
  5. Xanthine oxidase, lyophilized powder (Sigma-Aldrich, catalog number: X4376 )
  6. E. coli SdhA
    E. coli SdhA was isolated with an N-terminally fused His-tag as described in Maklashina et al., 2018.
  7. Glucose (Sigma-Aldrich, catalog number: G8270 )
  8. Catalase (Sigma-Aldrich, catalog number: C30 )
  9. Glucose oxidase (Sigma-Aldrich, catalog number: G2133 )
  10. Potassium indigotetrasulfonate (ITS) (Sigma-Aldrich, catalog number: 340596 )
  11. Benzyl viologen (Sigma-Aldrich, catalog number: 271845 ) or Methyl Viologen (Sigma-Aldrich, catalog number: 856177 )
  12. Flavoprotein, isolated SdhA (see the expression and purification method; Maklashina et al., 2018)
  13. Ultra High Purity 5.0 Grade Argon (Airgas, catalog number: AR UHP300 ) or Ultra High Purity 5.0 Grade Nitrogen (Airgas, catalog number: NI UHP300 )
  14. Sodium Dithionite (Sigma-Aldrich, catalog number: 71699 )
  15. Apiezon High Vacuum grease type L (Fisher Scientific, catalog number: 50-365-186 )
  16. Nitrogen/argon, gas
  17. Toluylene blue
  18. Phenazine methosulfate
  19. Thionine
  20. Methylene blue
  21. Pyocyanin
  22. Indigotetrasulfonate
  23. Resorufin
  24. Indigotrisulfonate
  25. Indigodisulfonate
  26. 2-Hydroxy-1,4 naphthoquinone
  27. Cresyl violet
  28. Anthraquinone-2,6-disulphonate
  29. Anthraquinone-2-sulfonate
  30. Phenosafranine
  31. Safranine T
  32. 50 mM potassium phosphate, pH 7.0 (see Recipes)
  33. Xanthine solution, 10 mM (see Recipes)
  34. Xanthine oxidase solution (see Recipes)
  35. Glucose solution, 1 M (see Recipes)
  36. Catalase solution (see Recipes)
  37. Glucose oxidase solution (see Recipes)
  38. Reference dyes solutions (see Recipes)
  39. Benzyl viologen or methyl viologen (see Recipes)

Equipment

  1. Spectrophotometer (for example, Agilent 8453 UV-visible spectrophotometer capable of scanning between 300 and 800 nm, with a spectral resolution of 1 nm)
    Temperature control is supplied by a Peltier unit from Agilent. Any means to maintain a constant temperature is appropriate (i.e., cuvette holder where the temperature is controlled by a water bath, for example).
  2. An appropriate cuvette to conduct the reaction under strict anaerobic conditions (Figure 2)
    Considerations before the reaction:
    Choosing an anaerobic cuvette. This method relies on strict anaerobic conditions required for the duration of the reaction. Some labs may be well equipped for this type of study and have a spectrophotometer placed in an anaerobic hood. For many labs, however, this option may not be available, thus the reaction should be well isolated from an aerobic environment by using specialized optical cuvettes (Figure 2). The classical anaerobic cuvette (Figure 2A) has a side arm and air is exchanged by repeated cycles of applying a vacuum and replacing air with nitrogen/argon gas. The xanthine oxidase (XO) which is used to initiate the reaction is placed in the side arm and mixed into the reaction after the cuvette is closed and sealed using vacuum grease. Alternatively, if the specialized cuvette shown in Figure 2A is unavailable, a standard stoppered cuvette (Figure 2B) can be used. Additions are made by a gas tight syringe through an oxygen impermeable septum (Figure 2B, top). The most convenient method which was used by us and others is to use a common stoppered cuvette (Figure 2B, bottom). To achieve anaerobic conditions required for the assay, we use the glucose/glucose oxidase/catalase oxygen scavenging system that efficiently eliminates dissolved oxygen. In addition, the cuvette head space is flushed with a low flow of argon/nitrogen. To initiate the reaction, XO is added with an air tight syringe and after the injection the reaction is carefully mixed with the syringe needle. For some assays when using very low potential flavoproteins or dyes (i.e., a potential less than -300 mV) the argon or nitrogen gas should be additionally treated by an oxygen scrubbing system to remove traces of oxygen. The use of an air tight cuvette (Figure 2A) would be also beneficial. The final volume of the reaction is usually 1 ml but can be scaled up or down depending on the cuvette type and amount of protein to be studied.


    Figure 2. Optical cuvettes that can be used for anaerobic measurements. A. A sealed anaerobic cuvette with a side arm. B. A cuvette with a septum (top) and a standard optical cuvette equipped with a silicone stopper (bottom). Both cuvettes can be used with a gas line to supply argon/nitrogen (shown for the bottom cuvette).

      Choosing an appropriate dye. As a general rule, the Em of a reference dye should be within ±30 mV of the anticipated flavin potential in the protein of interest. Table 1 summarized some reference dyes that had been used for determination of flavin reduction potentials. Some tips for choosing a dye: for mutants of a flavoprotein with known potential start with a dye which has an Em closest to the wild-type. For example, a mutation may result in a change of flavin attachment to the protein. Covalently bound flavins demonstrate higher reduction potentials than non-covalent flavins (Heuts et al., 2009). Since wild-type SdhA has a flavin potential of about -50 mV, we used a dye (ITS) which has a redox potential near this value (see Table 1).

    Table 1. Reference Dyes

    *Viologens are one-electron reduction dyes, all others undergo two-electron reduction.

      Choice of the wavelength to monitor absorbance changes in the flavin and dye. As depicted in Figures 1 and 4, the oxidized and reduced forms of the flavin and dye are calculated from the spectra containing both components. Thus, wavelengths used for the analysis should represent where spectral changes are attributed to only one component and where absorbance changes contributed by the second component are negligible. For example, the maximum absorbance of FAD in SdhA is around 460 nm, however, the reduced form of ITS also absorbs at this wavelength and will interfere with correct determination of FAD concentrations. A separate spectrum of dithionite reduced ITS, however, shows that 485 nm is the isosbestic point for ITS (demonstrated in Figure 3). FAD also retains significant absorbance at 485 nm, thus this wavelength can be used for calculations of reduced/oxidized forms of FAD without any contributions from ITS. Reduction of ITS was measured at 614 nm, a peak absorbance, where there is no interference from flavin absorption. Thus, the wavelengths of 485 nm for the flavin and 614 nm for the dye can be used for the analysis.


    Figure 3. The isobestic point for ITS. The spectrum of oxidized ITS is shown as the solid line and the dithionite reduced ITS is shown as the dashed line. Both forms show the same absorbance at 485 nm, i.e., the isobestic point.

    3. An air tight syringe for making additions to the anaerobic assay

Software

  1. Microsoft Excel or any other spread sheet program for data analysis

Procedure

  1. For this assay, we utilized the conventional optical cuvette with argon shown in Figure 2B (bottom). In the cuvette add 50 mM potassium phosphate buffer (pH 7.0, 25 °C), and reagents to indicated final concentration: 10 mM glucose, 5 µg/ml catalase, 0.3 mM xanthine, 50 µg/ml glucose oxidase, and benzyl viologen (10-20 µM). A low flow of argon/nitrogen is layered on the top of the cuvette (no bubbling). Record the baseline spectra.
  2. Add sufficient flavoprotein (SdhA) to give an absorbance of 0.1-0.5 at 450-460 nm, which corresponds to 8-40 µM FAD (mix carefully). The maximum absorbance should be similar between the FAD at ~460 nm and the dye (ITS at 614 nm). The spectrum is scanned between 350 and 750 nm, and recorded. This spectrum corresponds to the oxidized states of the flavoprotein and the dye.
  3. Wait about 15 min to establish anaerobic conditions and then add xanthine oxidase in an air tight syringe (10-50 nM is the usual range). The concentration of xanthine oxidase should be adjusted so that electron transfer is sufficiently slow so that the overall system remains in equilibrium at all times. Set the spectrophotometer to record spectra with 1-3 min intervals. The concentration of reagents should be such that complete reduction of the flavin and dye occur within 1-4 h.
  4. The reaction is stopped when spectra show no further changes or the benzyl viologen begins to be reduced as indicated by an increase in absorbance in the 550-600 nm region.
  5. Finally, transfer the individual spectra (wavelength vs absorbance) from the spectrophotometer and combine them in an Excel spreadsheet. Figure 4 demonstrates the set of spectra of a single experiment for the reduction of SdhA with ITS.


    Figure 4. Optical changes observed during anaerobic reduction of SdhA and ITS in the presence of xanthine/xanthine oxidase. A. Graph combining individual spectra. B. An example of the Excel spreadsheet that combined 5 spectra taken during the measurement.

Troubleshooting

  1. Major contributors for the incomplete reduction (or lack thereof) of the flavin and the dye are an oxygen leak and/or low xanthine oxidase activity. A suggested test to check the anaerobic conditions, i.e., the reduction of benzyl viologen by XO is to omit the flavoprotein and dye from the assay. Reduction of BV (Em = -359 mV) should be observed under anaerobic conditions. The oxidized form of BV is colorless and a broad peak forms at 550-600 nm when BV begins to undergo reduction.
  2. If the almost complete reduction of either the flavin or dye precedes the reduction of the second partner, then the difference in reduction potentials between the flavin and the dye has exceeded the requirements for this method and different dye should be chosen. The component with the higher potential will be reduced first and this will help to determine the reduction potential of the next dye to try.

Data analysis

The difference in Em potentials of the protein and dye, is calculated from a plot where the ratio of oxidized/reduced forms for the protein and the dye corresponding for each spectrum are plotted against each other.
  For this calculation, the data is fitted to the Nernst plot. The step by step derivation is taken from Efimov et al. (2014).
  The reduction potentials of the flavin containing protein (P) and dye (D) are determined by using the Nernst equation:




  Slow rates of electron input by XO ensure the equilibrium of the oxidized and reduced forms of the protein and dye at any given time point. Thus, their electrochemical potentials also equal:



  Defining x as a Nernst concentration term for the protein, and y as a Nernst concentration term for the dye.



  When the protein is in redox equilibrium [Pox] = [Pred], x = 0; y is defined as ΔE, the difference in mid-point potentials of the protein and the dye.



  To determine ΔE, the Nernst concentration term for the dye (y) and the Nernst concentration term for the protein(x) are calculated from each spectrum and plotted against each other. The ratio of [oxidized]/[reduced] concentrations for the protein and the dye is determined using the wavelengths designated for each component. In our experiment, we used 485 nm for FAD and 614 nm for ITS.



where Amax is the absorbance of the oxidized forms, Amin is the absorbance of the reduced forms, and A is the absorbance taken from each spectrum. Now using the values of the thermodynamic constants RT/nF is equal to 12.5; (R = the gas constant; T = the temperature in Kelvin, F = Faraday’s constant; n = number of electrons for FAD and ITS reduction, n = 2 for both FAD and ITS) and obtained the final equations for x and y.



Below, we use the data from the set of spectra (Figure 5) to calculate EmFAD in SdhA.

Calculations

  1. We determine Amax and Amin for the protein FAD (column B) and the dye (column D) (Figure 4) from the oxidized spectrum and from the spectrum at the end of the reaction.
  2. Next, using absorbance at a chosen wavelength (columns B and D) in each spectrum the Nernst concentrations terms for the protein FAD, (column C) and dye (column E) are calculated (Figure 5A). The graphic representation of y vs. x gives a straight line with a slope of one and an intercept equal to y, or ΔE, a shift in the midpoint potentials between the protein FAD and dye (Figure 5B). Thus, the EmFAD in SdhA is -55.5 mV. This value is similar to the EmFAD determined for the four-subunit succinate dehydrogenase complex determined by potentiometric titration after correction for the different pH used for analysis (Cheng et al., 2015). Since the FAD is attached covalently in SdhA, the midpoint potential is significantly higher than the EmFAD of free flavin (-219 mV).


    Figure 5. Final Calculations. A. An example of the Excel spreadsheet with calculations of the Nernst concentration terms for SdhA and ITS. B. The corresponding Nernst plot.

    Notes:
    1. It is often observed that the points at the far ends of the plot are out of alignment. This corresponds to the states of the reaction where the protein and dye are predominately in their oxidized and reduced forms, and equilibrium takes longer than the rate of the XO reaction allows. Therefore, these points are often omitted from the graph until the experimental points form a straight line with a slope equal to one.
    2. This experiment demonstrates the Em determination of the 2e- reduction of FAD/FADH2 in SdhA. In some flavoproteins 1e- reduction of FAD may produce a stable semiquinone radical FAD that can be observed as an intermediate form before reduction to hydroquinone.

Recipes

  1. 50 mM sodium phosphate (pH 7.0)
    Dissolve 4.67 g of K2HPO4 (dibasic potassium phosphate) and 3.15 g of KH2PO4 (monobasic potassium phosphate) and bring the final volume to 1-liter with distilled water
  2. Xanthine solution, 10 mM
    Dissolve 15.2 mg of xanthine in 9.9 ml of distilled water and 0.1 ml of 1 M sodium hydroxide (stored at -20 °C)
  3. Xanthine oxidase solution
    0.2 mM in 50 mM potassium phosphate pH 7.0 (stored at -20 °C)
  4. Glucose solution, 1 M
    1 M made in distilled water
    For longer storage, filter sterilize using a 0.2 µm filter (Corning) (stored at -20 °C)
  5. Catalase solution
    Catalase (5 mg/ml) in 50 mM potassium phosphate pH 7.0 (stored at -20 °C)
  6. Glucose oxidase solution
    Glucose oxidase (20 mg/ml) in 50 mM potassium phosphate pH 7.5 (stored at -20 °C)
  7. Reference dyes solutions
    10 to 20 mM solutions in water (stored at 4 °C)
  8. Benzyl viologen or methyl viologen
    Benzyl viologen or methyl viologen to a final concentration of 10 mM in water

Acknowledgments

Research in our laboratory was supported by the National Institutes of Health grant RO1 GM61606. GC is also supported by the Department of Veterans Affairs Research Senior Career Scientist award #1K6BX004215. This protocol was adapted from the method of Vincent Massey (Massey, 1991) as described in Maklashina et al. (2018) and the redox calculations were adapted from Efimov et al. (2014).

Competing interests

The authors declare no competing interests.

References

  1. Cheng, V. W., Piragasam, R. S., Rothery, R. A., Maklashina, E., Cecchini, G. and Weiner, J. H. (2015). Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase. Biochemistry 54(4): 1043-1052.
  2. Christgen, S. L., Becker, S. M. and Becker, D. F. (2019). Methods for determining the reduction potentials of flavin enzymes. Methods Enzymol 620: 1-25.
  3. Efimov, I., Parkin, G., Millett, E. S., Glenday, J., Chan, C. K., Weedon, H., Randhawa, H., Basran, J. and Raven, E. L. (2014). A simple method for the determination of reduction potentials in heme proteins. FEBS Lett 588(5): 701-704.
  4. Heuts, D. P., Scrutton, N. S., McIntire, W. S. and Fraaije, M. W. (2009). What's in a covalent bond? On the role and formation of covalently bound flavin cofactors. FEBS J 276(13): 3405-3427.
  5. Hudson, J. M., Heffron, K., Kotlyar, V., Sher, Y., Maklashina, E., Cecchini, G. and Armstrong, F. A. (2005). Electron transfer and catalytic control by the iron-sulfur clusters in a respiratory enzyme, E. coli fumarate reductase. J Am Chem Soc 127(19): 6977-6989.
  6. Kowal, A. T., Werth, M. T., Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P. and Johnson, M. K. (1995). Effect of cysteine to serine mutations on the properties of the [4Fe-4S] center in Escherichia coli fumarate reductase. Biochemistry 34(38): 12284-12293.
  7. Maklashina, E., Rajagukguk, S., Iverson, T. M. and Cecchini, G. (2018). The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS. J Biol Chem 293(20): 7754-7765.
  8. Massey, V. (1991). A simple method for the determination of redox potentials. In: Flavins and Flavoproteins 1990. Curti, B., Ronchi, S. and Zanetti, G. (Eds.) Walter de Gruyter & Co., Berlin, pp. 59-66.
  9. Saenger, A. K., Nguyen, T. V., Vockley, J. and Stankovich, M. T. (2005). Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding. Biochemistry 44(49): 16043-16053.

简介

[摘要] 该协议描述了一种简单的黄嘌呤/黄嘌呤氧化酶酶平衡方法,用于测定黄素的氧化还原电位。作为使用此方法的一个例子,我们确定了大肠杆菌中琥珀酸脱氢酶的SdhA 黄素亚基中共价结合的FAD辅因子的还原电位(E m = -55 mV)。原则上,该方法可常规用于通过平衡浓度和已知E m 的适当参考染料确定任何简单黄素蛋白中黄素辅因子的氧化还原电位。 无需使用复杂的电化学设备。


[背景] 几种生物物理方法可用于测量蛋白质中黄素的还原中点电位(E m )。这些电位测量方法通常依赖于目标蛋白质和电极之间的电化学偶联。例如,在带有其他辅助因子(例如铁硫中心和醌)的复杂黄素蛋白中,常常使用电化学方法和电子顺磁共振(EPR)光谱来确定氧化还原中心的E m (Kowal 等,1995;Saenger等,等人,2005; Hudson 等人,2005; Cheng 等人,2015)。然而,在只包含黄素氧化还原中心简单黄素蛋白,辅助因子,可以直接通过它不需要特殊的设备电化学或昂贵的EPR仪表传统光学分光光度法研究。1990年,文森特·梅西(Vincent Massey)提出了一种简单的方法,该方法可以从氧化和还原伴侣(即黄素蛋白和参考染料)的平衡浓度中确定黄素的还原电位(Massey,1991)。该方法不直接测量黄素的还原电势,而是确定黄素和参考染料的E m 值之间的差异。图1中的方案描述了该方法。黄嘌呤/黄嘌呤氧化酶系统提供了指示剂染料的缓慢连续降低和苄基紫精(BV)或甲基紫精(MV)的存在保证了黄素小号还原当量的快速平衡。这允许缓慢还原蛋白质和染料的还原形式和氧化形式的平衡,直到两者完全还原为止。一系列记录在所述反应的过程中光谱被用于计算的黄素和染料的氧化和还原形式的比率。相对于黄酮蛋白的等库浓度的能斯特图,可以确定与染料相比,蛋白质的E m 的变化。因为一些在该方法中使用的组分是低电位的化学品和蛋白易于被氧气甚至痕量氧化(紫精,参考染料,和许多黄素蛋白),用于该系统中的要求是严格厌氧被维持。

在这里,我们展示了如何使用此方法确定琥珀酸脱氢酶SdhA 黄素蛋白中共价结合的FAD还原电位的示例。SdhA 是四个亚基膜结合的琥珀酸盐:泛醌还原酶(即复合物II)的黄素蛋白成分,它是线粒体和许多细菌的TCA(Krebs)循环和电子传输链的一部分。配合物Ⅱ将琥珀酸氧化为富马酸酯的反应与泛醌还原为泛醇的反应结合在一起。FAD辅助因子参与可逆的琥珀酸酯-富马酸酯转化。溶液中游离FAD 的E m 为-219 mV,而FAD与SdhA的共价结合大大提高了黄素的潜力。在这个例子中,我们使用的氧化还原染料靛蓝四磺酸(ITS)(Ë 米= -46毫伏),并已确定该Ë 米FAD 为所述大肠杆菌SDHA 蛋白为-55毫伏。原则上,该简单方法可通过使用具有合适还原电位(即,与确定的黄素蛋白预期值相差± 30 mV 以内)的氧化还原染料用于任何黄素蛋白。这种方法也被验证了的d 许多其他的还原电位的etermination 黄素(见Christgen 等,2019)和几个血红素蛋白质表明,该方法可广泛实用于大多数血红素蛋白(叶菲莫夫等, 2014)。



D:\ Reformatting \ 2020-2-7 \ 1902880 Gary Cecchini 636980 \ Figs jpg \图1.jpg

图1 分光光度法测定的Ë 米一个的黄素在黄嘌呤/黄嘌呤氧化酶存在下,使用参比染料。最合适的染料会进行2-电子还原(请参见表1)。

关键字:黄素, 黄素蛋白, 还原电位, 黄嘌呤氧化酶, 中点电位

材料和试剂


 


0.2 µm 过滤器(Corning)(Sigma-Aldrich ,目录号:CLS431229)
无水磷酸氢二钾(Sigma-Aldrich ,目录号:795496)
磷酸钾一元,无水(Sigma-Aldrich公司,目录号:795488)
黄嘌呤(Sig ma-Aldrich ,目录号:X7375)
黄嘌呤氧化酶,冻干POW d ER(Sigma-Aldrich公司,目录号:X4376)
大肠杆菌SdhA
如Maklashina 等人(2018年)所述,用N末端融合的His-tag分离大肠杆菌SdhA 。


葡萄糖(Sigma-Aldrich ,目录号:G8270)
过氧化氢酶(Sigma-Aldrich ,目录号:C30)
葡萄糖氧化酶(Sigma-Aldrich ,目录号:G2133)
靛蓝四磺酸钾(ITS)(Sigma-Aldrich ,目录号:340596)
苄基紫精(Sigma-Aldrich ,目录号:271845)或甲基紫精(Sigma-Aldrich ,目录号:856177)
黄素蛋白,分离的SdhA (参见表达和纯化方法; Maklashina 等人,2018)
超高纯5.0级氩气(Airgas ,货号:AR UHP300)或超高纯5.0级氮气(Airgas ,货号:NI UHP300)
连二亚硫酸钠(Sigma-Aldrich ,目录号:71699)
Apiezon L型高真空润滑脂(Fisher Scientific ,目录号:50-365-186)
氮气/氩气
甲苯蓝
甲基硫酸吩嗪
硫氨酸
亚甲蓝
花青素
靛蓝四磺酸盐
试卤灵
靛果磺酸
靛二磺酸盐
2-羟基-1,4萘醌
甲酚紫
2,6-二磺酸蒽醌
蒽醌-2-磺酸盐
酚红素
番红花T
50 mM磷酸钾,pH 7.0(请参阅食谱)
黄嘌呤溶液,10 mM(请参阅食谱)
黄嘌呤氧化酶溶液(请参阅食谱)
葡萄糖溶液,1 M(请参阅食谱)
过氧化氢酶溶液(请参见食谱)
葡萄糖氧化酶溶液(请参阅食谱)
参考染料溶液(请参阅配方)
苄基紫精或甲基紫精(请参阅食谱)
 


设备


 


分光光度计(例如,Agilent 8453紫外可见分光光度计,能够在300 至800 nm 之间扫描,光谱分辨率为1 nm)
温度控制由安捷伦的Peltier装置提供。任何手段以保持恒定的温度是合适的(即,ç uvette保持器,其中所述温度由一控制水浴,例如)。


适当的比色杯在严格的厌氧条件下进行反应(图2)
反应之前的注意事项:


选择一个厌氧比色皿。该方法依赖于反应持续时间所需的严格厌氧条件。一些实验室可以很好地进行此类研究,并在厌氧罩中放置一个分光光度计。但是,对于许多实验室而言,此选项可能不可用,因此应使用专用的光学比色杯将反应与有氧环境良好隔离(图2)。经典的厌氧比色皿(图2A)具有一个侧臂,并且通过反复施加真空并用氮气/氩气代替空气的循环来交换空气。将用于引发反应的黄嘌呤氧化酶(XO)放在侧臂中,并在将比色皿关闭后用真空油脂密封后混入反应中。替代地,如果在图2A所示的专门反应杯不可用,一个标准的塞子塞住试管(图2B)可以被使用。通过气密性注射器穿过不透氧气的隔膜进行添加(图2B,顶部)。我们和其他人使用的最方便的方法是使用普通的带塞比色皿(图2B,底部)。为了达到测定所需的厌氧条件,我们使用了可以有效消除溶解氧的葡萄糖/葡萄糖氧化酶/过氧化氢酶除氧系统。另外,用低流量的氩气/氮气冲洗比色皿的顶部空间。为了引发反应,用气密注射器添加XO,注射后将反应物小心地与注射器针头混合。对于某些测定,当使用极低电位的黄素蛋白或染料(即,电位低于-300 mV)时,应通过氧气洗涤系统另外处理氩气或氮气,以除去痕量的氧气。使用气密比色皿(图2A)也将是有益的。反应的最终体积通常为1 ml,但可以根据比色皿类型和要研究的蛋白质量按比例放大或缩小。


 


D:\ Reformatting \ 2020-2-7 \ 1902880 Gary Cecchini 636980 \ Figs jpg \图2.jpg


图2.可用于厌氧测量的光学比色皿。一。带有侧臂的密封厌氧比色皿。B.带有隔垫的试管(顶部)和配有硅胶塞的标准光学试管(底部)。两个比色皿均可与气体管线一起使用,以供应氩气/氮气(显示在底部比色皿中)。


 


选择合适的染料。通常,参考染料的E m 应在目标蛋白的预期黄素电位上±30 mV以内。表1总结了一些用于测定黄素还原电位的参考染料。选择染料的一些技巧:对于具有已知潜力的黄素蛋白突变体,应从E m 最接近野生型的染料开始。例如,突变可导致黄素对蛋白质的附着改变。共价结合的黄素比非共价的黄素具有更高的还原潜力(Heuts等。,2009)。由于野生型SdhA 的黄素电位约为-50 mV ,因此我们使用的染料(ITS)的氧化还原电位接近此值(请参见表1)。






表1.参考染料


染料


E m (mV )


甲苯蓝


+115


甲基硫酸吩嗪


+80


硫氨酸


+64


亚甲蓝


+11


花青素


-34


靛蓝四磺酸盐


-46


试卤灵


-79


靛果磺酸


-81


靛二磺酸盐


-125


2-羟基-1,4萘醌


-1 42


甲酚紫


-166


2,6-二磺酸蒽醌


-184


蒽醌-2-磺酸盐


-225


酚红素


-252


番红花T


-289


苄基紫精*


-359


甲基紫精*


-446


*紫罗兰是一种单电子还原染料,所有其他均经过两电子还原。


 


选择波长以监测黄素和染料的吸光度变化。如该图所示URES 1 一个ND 4,黄素和染料的氧化和还原形式从含有两个成分的频谱进行计算。因此,用于分析的波长应表示光谱变化仅归因于一种成分,而第二种成分引起的吸光度变化可忽略不计。例如,SdhA 中FAD的最大吸光度约为460 nm,但是,ITS的还原形式也在该波长下吸收,并且会干扰FAD浓度的正确确定。然而,连二亚硫酸盐还原的ITS的单独光谱显示485 nm是ITS的等压点(如图3所示)。FAD在485 nm处也保留了显着的吸光度,因此该波长可用于计算FAD的还原/氧化形式,而ITS没有任何贡献。[R 的排出ITS在614纳米,峰值吸收,那里是F无干扰测量ROM 黄素吸收。因此,黄素的485 nm和染料的614 nm的波长可用于分析。


 


D:\ Reformatting \ 2020-2-7 \ 1902880 Gary Cecchini 636980 \ Figs jpg \图3.jpg


图3. ITS 的等量点。氧化的ITS的光谱显示为实线,连二亚硫酸盐还原的ITS 显示为虚线。这两种形式显示以485nm,同样的吸光度即,在等吸收点。


 


气密注射器,用于厌氧分析
 


软件


 


Microsoft Excel或任何其他电子表格程序以进行数据分析
 


程序


 


对于该测定,我们使用了图2B(底部)所示的常规光学比色杯和氩气。在比色杯中,添加50 mM磷酸钾缓冲液(pH 7.0,25°C)和指示最终浓度的试剂:10 mM葡萄糖,5 µg / ml过氧化氢酶,0.3 mM黄嘌呤,50 µg / ml葡萄糖氧化酶和苄基紫精( 10-20 µM)。低流量的氩气/氮气层积在比色皿的顶部(不鼓泡)。记录基线光谱。
加入足够的黄素蛋白(SDHA )在450-460纳米,这对应于100,得到0.1-0.5的吸光度ONDS 到8-40μMFAD(仔细混合)。在〜460 nm的FAD和染料(614 nm的ITS)之间,最大吸光度应相似。频谱350之间扫描和750纳米,和记录。该光谱对应于黄素蛋白和染料的氧化态。
等待约15分钟以建立厌氧条件,然后在气密注射器中添加黄嘌呤氧化酶(通常范围为10-50 nM )。应调节黄嘌呤氧化酶的浓度,以使电子转移足够缓慢,以使整个系统始终保持平衡。设置分光光度计以1-3分钟的间隔记录光谱。试剂的浓度应使黄素和染料在1-4小时内完全还原。
如光谱显示没有进一步变化或苄基紫精开始减少,反应就停止了,如550-600 nm区域吸光度的增加所示。
最后,从分光光度计传输单个光谱(波长与吸光度),并将其合并到Excel电子表格中。图4展示了使用ITS 还原SdhA 的单个实验的光谱组。
 


D:\ Reformatting \ 2020-2-7 \ 1902880 Gary Cecchini 636980 \ Figs jpg \图4.jpg


图4. 在黄嘌呤/黄嘌呤氧化物存在下,SdhA 和ITS 厌氧还原过程中观察到的光学变化。A. ģ 拍摄和组合个别光谱。B. Excel电子表格的一个示例,该示例结合了在测量过程中获得的5 倍采样率。


 


故障排除


黄素和染料不完全还原(或缺乏还原)的主要原因是氧气泄漏和/或黄嘌呤氧化酶活性低。建议的检查厌氧条件的测试,即XO还原苄基紫精的方法是从测定中省略黄素蛋白和染料。在厌氧条件下应观察到BV的降低(E m = -359 mV)。BV的氧化形式是无色的,当BV开始还原时,在550-600 nm处形成一个宽峰。
如果黄素或染料的几乎完全还原先于第二配偶体的还原,则黄素和染料之间还原电位的差异已超过该方法的要求,应选择不同的染料。具有较高电势的组分将首先被还原,这将有助于确定下一个要尝试的染料的还原电势。
 


数据分析


 


蛋白质和染料的E m 电位差是从图中计算得出的,其中将蛋白质和每种光谱对应的染料的氧化/还原形式之比相互绘制在一起。


对于此计算,将数据拟合到能斯特图。逐步推导来自(Efimov 等,2014)。


含有黄素的蛋白质(P)和染料(D)的还原电位通过使用能斯特方程确定:


 






 






 


XO缓慢的电子输入速率可确保在任何给定时间点蛋白质和染料的氧化形式和还原形式的平衡。因此,它们的电化学势也相等:


 






 






 


将x 定义为蛋白质的能斯特浓度项,将y 定义为染料的能斯特浓度项。


 






 






 






 


当蛋白质处于氧化还原平衡时[ P ox ] = [ P red ],x = 0;y定义为ΔE,即蛋白质和染料的中点电势之差。


 






 


为了确定ΔE ,从每个光谱计算染料(y)的能斯特浓度项和蛋白质(x )的能斯特浓度项并相互作图。蛋白质和染料的[氧化] / [还原]浓度之比是使用为每种组分指定的波长确定的。在我们的实验中,我们将d 4 85 nm用于FAD,将614 nm用于ITS。


 






w 在此,A max 是氧化形式的吸光度,A min 是还原形式的吸光度,A是从每个光谱获得的吸光度。现在使用热力学常数RT / nF 等于12.5。(R =气体常数;T = 开氏温度,F = 法拉第常数;n = FAD和ITS还原的电子数,FAD和ITS 均为n = 2)并获得了x 和y 的最终方程。


 






 






 


下面,我们使用来自组光谱(数据图5 )来计算È 米FAD 在SDHA 。


 


计算方式


我们从氧化光谱和反应结束时的光谱中确定蛋白质FAD(列B)和染料(列D)(图4)的A max 和A min 。
接下来,使用每个光谱中选定波长(B和D列)的吸光度,计算蛋白质FAD(C列)和染料(E列)的能斯特浓度项(图5A)。y vs的图形表示。x给出一条斜率为1的直线,其截距等于y或ΔE,即蛋白质FAD和染料之间的中点电势偏移(图5B)。因此,ê 米FAD 在SDHA 为-55 0.5 毫伏。该值类似于通过校正用于分析的不同pH值后通过电位滴定确定的四亚基琥珀酸脱氢酶复合物的E m FAD (Cheng 等人,2015)。由于FAD在SdhA中共价连接,因此中点电位明显高于游离黄素的E m FAD (-219 mV)。
 


 






图5. 最终计算。A. Excel电子表格的示例,其中包含SdhA和ITS的能斯特浓度项的计算。B.对应的能斯特图。


 


笔记:


经常观察到,曲线远端的点未对齐。这对应于反应的状态,在该状态下,蛋白质和染料主要以其氧化和还原形式存在,而平衡所需的时间比XO反应的速度要长。因此,在实验点形成斜率等于1 的直线之前,通常会从图中省略这些点。
该实验证明了Ë 米的2e的判定- 还原FAD的/ FADH 2 中SDHA 。在一些黄素蛋白1E - 还原FAD可以产生稳定的半醌自由基FAD 可以观察到的作为还原成氢醌之前的中间形式。
 


菜谱


 


50 mM磷酸钠(pH 7.0 )
溶解4.67 g K 2 HPO 4 (磷酸氢二钾)和3.15 g KH 2 PO 4 (磷酸氢二钾),并用蒸馏水将最终体积调至1升


黄嘌呤溶液,10 mM
将15.2 mg的黄嘌呤溶于9.9 ml的蒸馏水和0.1 ml的1 M氢氧化钠中(储存在-20°C)


黄嘌呤氧化酶溶液
在50 mM磷酸钾pH 7.0中为0.2 mM (储存在-20 °C )


葡萄糖溶液,1 M
1 M蒸馏水制成


对于较长的存储,过滤器使用0.2μ除菌米过滤器(Corning)中(储存于-20 ℃下)


过氧化氢酶溶液
50 mM磷酸钾pH 7.0中的过氧化氢酶(5 mg / ml)(储存在-20 °C )


葡萄糖氧化酶溶液
50 mM磷酸钾pH 7.5中的葡萄糖氧化酶(20 mg / ml)(储存在-20 °C )


参考染料溶液
水中的10至20 mM 溶液(在4 °C下储存)


苄基紫精或甲基紫精
水中的苄基紫精或甲基紫精的终浓度为1 0 mM


 


Acknowledg 发言:


 


我们实验室的研究得到了美国国立卫生研究院RO1 GM61606的资助。退伍军人事务研究部高级职业科学家奖#1K6BX004215也支持GC。如Maklashina 等人所述,该方案改编自Vincent Massey(Massey,1991 )的方法。(2018 )和氧化还原计算改编自Efimov 等。(2014年)。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


Cheng,VW,Piragasam ,RS,Rothery ,RA,Maklashina ,E.,Cecchini,G. and Weiner,JH(2015)。黄素腺嘌呤二核苷酸的氧化还原状态驱动大肠杆菌琥珀酸脱氢酶中的底物结合和产物释放。生物化学54(4):1043-1052。
Christgen ,SL,Becker,SM和Becker,DF(2019)。确定黄素酶还原电位的方法。方法酶620:1-25。
埃菲莫夫(Efimov ,I.),帕金(G.Parkin),米勒(Millett),ES,格兰德(J.Glenday ),陈(Chan),CK ,韦顿(Weedon),H。,兰德哈瓦(Randhawa),H 。一种确定血红素蛋白还原电位的简单方法。FEBS Lett 588(5):701-704。
Heuts ,DP,Scrutton ,NS,McIntire,WS和Fraaije ,MW(2009)。共价键中有什么?关于共价结合的黄素辅因子的作用和形成。FEBS J 276(13):3405-3427。
Hudson,JM,Heffron,K.,Kotlyar ,V.,Sher,Y.,Maklashina ,E.,Cecchini,G。和Armstrong,FA(2005)。呼吸酶大肠杆菌富马酸还原酶中的铁硫簇进行电子转移和催化控制。J Am Chem Soc 127(19):6977-6989。
科瓦尔,AT,韦斯,MT,Manodori ,A.,切基尼,G.,薛定谔ö 德,一,Gunsalus ,RP和约翰逊,MK(1995年)。半胱氨酸到丝氨酸突变对大肠杆菌富马酸还原酶中[4Fe-4S]中心特性的影响。生物化学34(38):12284-12293。
Maklashina ,E.,Rajagukguk ,S.,Iverson,TM和Cecchini,G.(2018)。人类和细菌复合物II的未组装的黄素蛋白亚基损害了催化活性,仅产生少量的ROS。生物化学杂志293(20):7754-7765。
Massey,V。(1991)。一种确定氧化还原电势的简单方法。在:黄素和黄素蛋白1990 柯蒂斯,B.,龙基,S。和萨内蒂,G. (ê DS)德格鲁伊特公司,柏林,第59-66。
Saenger ,AK,Nguyen,TV,Vockley ,J。和Stankovich ,MT(2005)。通过底物和产物结合对人短链酰基辅酶A脱氢酶的热力学调节。生物化学44(49):16043-16053。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Maklashina, E. and Cecchini, G. (2020). Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method. Bio-protocol 10(7): e3571. DOI: 10.21769/BioProtoc.3571.
  2. Maklashina, E., Rajagukguk, S., Iverson, T. M. and Cecchini, G. (2018). The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS. J Biol Chem 293(20): 7754-7765.
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