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Measurement of Cellular Copper in Rhodobacter capsulatus by Atomic Absorption Spectroscopy
采用原子吸收光谱法测量荚膜红细菌细胞中的铜   

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Abstract

Copper is an essential micronutrient and functions as a cofactor in many enzymes such as heme-Cu oxygen reductases, Cu-Zn superoxide dismutases, multi-copper oxidases and tyrosinases. However, due to its chemical reactivity, free copper is highly toxic (Rae et al., 1999) and all organisms use sophisticated machineries for controlling uptake, storage and export of Cu. The strict control of the cellular Cu homeostasis prevents toxic effects but sustains synthesis of cuproproteins. Monitoring the copper levels within the cell and within different cellular compartments is an essential approach for identifying the contribution of different proteins in maintaining the cellular copper equilibrium. Therefore, whole cells and whole-cell lysates, which can be further fractionated into cytoplasm and periplasm, were digested and the protein concentration was determined by Lowry assay. Subsequently, the copper content was measured by atomic absorption spectroscopy (AAS) and the Cu content per mg of protein was calculated. This provides a simple and cost-effective method of producing quantifiable results about the cellular Cu content. To exemplify this method, we used the phototrophic α-proteobacterium Rhodobacter capsulatus, which is commonly used as a model organism for studying Cu-trafficking in bacterial cells (Ekici et al., 2012).

Keywords: Copper homeostasis(铜的平衡), Cbb3-type cytochrome oxidase(cbb3型细胞色素氧化酶), Rhodobacter capsulatus(红细胞), Copper chaperones(铜伴侣蛋白), Copper-dependent Enzymes(铜依赖酶)

Background

Due to a growing interest in cellular Cu homeostasis different methods for measuring the cellular Cu content have been developed during the past years. They include electrochemical and fluorimetric protocols, inductively coupled plasma mass spectroscopy (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES), electron microprobe analyses (EMPA), X-ray absorption spectroscopy (XAS) or synchrotron-based X-ray fluorescent microscopy (SXRF) (reviewed in Ralle et al., 2009). Although these methods allow for a reliable and accurate determination of Cu in biological and environmental samples, they usually require advanced experimental set-ups and are not generally suited for analyzing a large number of samples. Atomic absorption spectroscopy (AAS) is a well established and widely available method that allows for a quick, sensitive and cost-effective Cu determination. It is suitable for determining Cu in whole cells but also in subcellular extracts.

Materials and Reagents

  1. Petri dishes (60 x 15 mm) (SARSTEDT, catalog number: 82.1194.500 )
  2. 50 ml Falcon tubes (SARSTEDT, catalog number: 62.559.001 )
  3. 15 ml Falcon tubes (SARSTEDT, catalog number: 62.554.502 )
  4. 0.45 µm filters (Carl Roth, catalog number: P667.1 )
  5. 10 ml syringes (Carl Roth, catalog number: C542.1 )
  6. Rhodobacter capsulatus wild type (MT1131) or mutant strains
  7. BactoTM peptone (BD, catalog number: 211820 )
  8. BactoTM yeast extract (BD, catalog number: 212720 )
  9. Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239.1 )
  10. Magnesium chloride hexahydrate (MgCl2·6H2O) (Carl Roth, catalog number: 2189.2 )
  11. MPYE agar media (1.5% agar in MPYE medium; 25 ml/Petri dish)
  12. Chelex® 100 resin (Bio-Rad Laboratories, catalog number: 1422832 )
  13. Tris/HCl, pH 7.5 (Carl Roth, catalog number: 5429.2 ; P074.1 )
  14. Sucrose (MP Biomedicals, catalog number: 04821713 )
  15. Lysozyme (Sigma-Aldrich, catalog number: L2879 )
  16. EDTA (Carl Roth, catalog number: 8043.2 )
  17. Lowry protein assay reagents [Reagent A, Folin & Ciocalteu’s phenol reagent (Sigma-Aldrich, catalog number: F9252 ), 1% SDS/0.1 N NaOH]
  18. SDS (SERVA Electrophoresis, catalog number: 20765.03 )
  19. Protein assay bovine serum albumin (Carl Roth, catalog number: 8076.4 ), standards (0, 0.02, 0.04, 0.08, 0.12, 0.2 mg/ml protein)
  20. Sodium carbonate (Na2CO3) (Carl Roth, catalog number: 8563.1 )
  21. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.1 )
  22. Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Carl Roth, catalog number: P024.1 )
  23. Na-tartrate (EMD Millipore, catalog number: 106663 )
  24. 53% nitric acid in ultra-pure water (Carl Roth, catalog number: 9274.1 )
  25. 30% hydrogen peroxide (Carl Roth, catalog number: 9681.1 )
  26. Ultra-pure water (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTMGenPureTM )
  27. Palladium (II) chloride (VWR, catalog number: AA11034-09 )
  28. Magnesium chloride (MgCl2) (VWR, catalog number: AA42843-22 )
  29. Copper standards [(20, 40, 60 ppb Cu in ultra-pure water; diluted from TraceCert copper standard for AAS (1,000 mg/ml Cu in 2% nitric acid, prepared with high purity Cu metal)] (Sigma-Aldrich, catalog number: 38996 )
  30. MPYE media (see Recipes)
  31. Cu-free MPYE (see Recipes)
  32. Cu-free water (see Recipes)
  33. Spheroplast buffer (see Recipes)
  34. Lysis buffer (see Recipes)
  35. Reagent A for Lowry assay (see Recipes)
  36. AAS modifier solution (see Recipes)

Equipment

  1. 1.5 ml cuvettes (Carl Roth, catalog number: Y195.1 )
  2. Magnetic stir plate (Heidolph, model: Hei-VAP )
  3. Sterile inoculation loop (Carl Roth, catalog number: 6163.1 )
  4. Shaking incubator multitron standard for bacterial growth at 35 °C (Infors, mode: INFORS HT )
  5. 250 ml Erlenmeyer flasks (Carl Roth, catalog number: K184.1 )
  6. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Lynx 6000 )
  7. Microscope at 100x magnification with numerical aperture 1.4 (OLYMPUS, model: BX51 )
  8. 1,000 µl automatic pipet (Gilson, catalog number: F123602 )
  9. Incubator for sample incubation at 60 °C and 80 °C (Kühner, model: ISF-1X )
  10. Atomic absorption spectrophotometer (PerkinElmer, model: 4110 ZL Zeeman )
  11. VIS spectrophotometer able to read OD660 and OD685 (e.g., GE Healthcare Life Science, model: Ultrospec 3100 Pro )

Software

  1. Excel 2007 (Microsoft)
  2. Perkin-Elmer AAWinLab software

Procedure

  1. Growth of Rhodobacter capsulatus and spheroplast preparation, preparation of Cu-free MPYE and Cu-free water
    1. Grow cultures under sterile conditions of the required Rhodobacter strains on MPYE agar plates (~24 h, at 35 °C) including antibiotics if required.
    2. Prepare MPYE medium for liquid culture, Cu-free MPYE and Cu-free water (see Recipes).
    3. Pick a single colony using a sterile inoculation loop and inoculate 10 ml MPYE in a 50 ml Falcon tube. Add appropriate antibiotics if required. Grow cultures at 35 °C and 110 rpm in a rotary shaker overnight.
    4. Next day: Inoculate the main culture (100 ml MPYE in 250 ml flask) with 108 cells from the overnight culture and incubate at 35 °C, 110 rpm until the culture reaches an OD685 of 1 (approx. 24 h, assuming that an OD685 = 1 corresponds to 7.5 x 108 cells/ml). This corresponds to the late exponential growth phase of R. capsulatus.
    5. Harvest the cells using a pre-cooled Lynx 6000 centrifuge (4 °C), at 5,000 rpm (corresponds to 3,000 x g in the F12-6 x 500 LEX rotor) for 20 min.
    6. Decant the supernatant and resuspend the pellet in 40 ml pre-cooled Cu-free MPYE and centrifuge once more at 5,000 rpm (3,000 x g, F12-6 x 500 LEX rotor) for 20 min at 4 °C.
    7. Decant the supernatant and resuspend the pellet in 2 ml spheroplast buffer and take a small aliquot of 50 µl for determining the protein concentration, e.g., by the Lowry assay (see Data analysis).
    8. Split the cell suspension in two equal parts into two clean 15 ml Falcon tubes. One tube is for measuring the copper concentration in whole cells, while cells in the other tube are used for spheroplast preparation. This allows measuring the copper content in the cytoplasm (spheroplasts), periplasm (supernatant after spheroplast preparation) and whole cells.
    9. Spheroplast preparation
      1. Add lysis buffer to the spheroplast sample fraction in a dilution of 1:10 to reach a final concentration of 0.1 mg/ml of lysozyme, e.g., add 100 µl lysis buffer to 900 µl sample.
      2. Incubate the cell suspension 15 min on ice with gentle mixing i.e., turning the tubes upside down several times every few minutes. After 15 min, the majority of cells are converted into spheroplasts; longer incubations might be required for other Gram-negative bacteria.
      3. Monitor spheroplast formation by using a microscope.
      4. Collect the spheroplasts by centrifugation at 5,000 rpm (3,000 x g, F12-6 x 500 LEX rotor) for 20 min at 4 °C.
      5. Carefully collect the supernatant (periplasm), preferentially using a 1,000 µl automatic pipet into a clean 15 ml Falcon tube.
      6. Resuspend the pellet (cytoplasm) with 1 ml spheroplast buffer.
      7. Take aliquots of 50 µl from each fraction and measure the protein concentration of all samples.


        Figure 1. Monitoring the spheroplast formation. A. R.capsulatus cells observed at 100x magnification. B. Spheroplasts formation observed at 100x magnification. Spheroplasts are indicated by arrows.

  2. Determination of Cu-concentration by atomic absorption spectroscopy
    1. To approximately 1 ml of whole cells, spheroplasts and the supernatant (after the spheroplast preparation) in 15 ml Falcon tubes, add 1.5 ml of 53% nitric acid. This treatment and the following incubation at high temperatures completely denature the protein solutions and releases any bound metal. Incubate for 1 h at 80 °C and subsequently at 60 °C overnight without shaking.
    2. After overnight-incubation, stop the digestion by the addition of 300 µl H2O2 (30% in water) and adjust the samples to a final volume of 10 ml by adding Cu-free water.
    3. If any insoluble impurities are observed, the solution is filtrated through a 0.45 µm disposable syringe-filter. The prepared samples are now stable and can be stored for several days at room temperature.
    4. The copper content is measured using an atomic absorption spectrophotometer measuring 3 times for 3 sec of atomization. Load 20 µl sample and 5 µl modifier containing 0.005 mg palladium and 0.003 mg magnesium (functioning as ionization agent).
      1. Blank measurement (0.2% nitric acid in ultra-pure water), calculate mean and set to 0.
      2. Calibrate the spectrophotometer by measuring 3 standards containing 20, 40 and 60 ppb of Cu.
      3. Measure the samples. If the values are out of range, dilute the samples 1:2 or 1:3.
      4. Analysis of the copper content is achieved by the Perkin-Elmer AAWinLab software. For every value the standard deviation (SD) and relative standard deviation (RSD) is calculated by the software.
    5. Plot the data in excel using protein concentrations and copper measurements. Calculate the total copper amount of your 10 ml sample and divide it through the total protein mass, calculated by protein quantification assay and the sample volumes before adding the nitric acid using the following formula:
      µg Cu/mg protein = (X Cu ppb x 10 ml)/(X mg/ml protein x X ml sample volume)
      Note: Cu ppb refers to Cu µg/L.

Data analysis

  1. A representative experiment demonstrating the determination of the cellular copper content in Rhodobacter capsulatus can be found in Trasnea et al. (2016). Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3-type cytochrome oxidase and copper homeostasis in Rhodobacter capsulatus.
  2. An example of the excel BSA standard curve for determining protein concentration in Rhodobacter capsulatus cells/cell extracts is described below. The samples were diluted in 200 µl of 1% SDS/0.1 N NaOH, 1 ml reagent A was added, followed by 5 min of incubation at room temperature. Afterwards, 100 µl of Folin & Ciocalteu’s phenol reagent 1 N was added and after 45 min of incubation the absorbance at A660 was determined. The absorbance was correlated to a protein concentration using a standard curve (as described below).

    Table 1. BSA protein standards and their relative absorbance at A660


    Absorbance reads were determined using a VIS spectrophotometer and data were subsequently plotted as seen in Figure 2.


    Figure 2. BSA protein standard curve. The absorbance at A660 (nm) was determined for a range of BSA protein standards from 0-0.2 mg/ml. The line of the best fit was plotted using y = mx (MS Excel 2007). The protein concentration of the samples was determined by correlating their absorbance to the protein concentration on the x-axis.

  3. A representative of the Cu standards and their extinction values for atomic absorption spectroscopy using the Perkin-Elmer AAWinLab software is provided in Table 2. Figure 3 contains an exemplified standard curve using Microsoft Excel (in process automatically generated by the Perkin-Elmer AAWinLab software).

    Table 2. Cu standards and their extinctions at 324.8 nm


    The data were subsequently plotted using the Perkin-Elmer AAWinLab software or MS Excel as displayed in Figure 3.


    Figure 3. Cu concentration standard curve. The curve was generated using Microsoft Excel using the values obtained by AAS. The extinction standards at 324.8 nm were determined for a range of 0-60 ppb of Cu. Using this curve the copper concentration from cells and different cell compartments can be determined by correlating their extinction to the Cu concentration values on the x-axis.

Notes

  1. This protocol is optimized for measuring the intracellular copper concentration of Rhodobacter capsulatus. For other bacteria, use the appropriate growth media with appropriate antibiotics and growth conditions. Cu was determined in cells harvested at the late exponential growth phase of OD685 = 0.8. The method should also work with cells harvested at different growth phases, but this was not analyzed here.
  2. The lysozyme treatment for periplasm extraction is optimized for R. capsulatus and therefore for Gram-negative bacteria. As lysozyme does not reliably work on all Gram-negative bacteria, different muramidases such as mutanolysin can be tested in these cases.
  3. With the obtained copper concentrations, the molar concentration of copper in cell compartments can be calculated by dividing the copper content of the sample by the volume of the cell compartment using the following formula:
    µmol/L Cu = (X µg/L Cu x A) /(63.546 g/mol x B x C)
    A = sample volume (usually 0.01 L) [L]
    B = number of cells in the sample (determined by OD measurement or by microscopic counting of cells)
    C = estimated volume of one cell or the cell compartment [L]
    63.546 g/mol corresponds to the molecular mass of Cu
    Example:
    Assuming that the cytosol plus the inner membrane of one Rhodobacter capsulatus cell has a volume of 1.9 x 10-16 L (calculated, based on the CyberCell Database; http://ccdb.wishartlab.com/CCDB/), the molar concentration of copper in the cytoplasm is around 300 µM.

Recipes

  1. MPYE medium
    0.3% (w/v) BactoTM peptone
    0.3% (w/v) BactoTM yeast extract
    0.1% (v/v) 1 M CaCl2·2H2O
    0.1% (v/v) 1 M MgCl2·6H2O
    Distilled water, autoclave
  2. Cu-free MPYE and Cu-free water
    Add 5 g Chelex 100 resin to 100 ml solution and stir continuously for at least 1 h.
    Afterwards, filter the solution through a 0.45 µm filter and autoclave.
    After Chelex-treatment, the copper concentration is below the limit of detection.
  3. Spheroplast buffer (stored at 4 °C or freshly prepared)
    100 mM Tris-HCl, pH 7.5
    0.5 M sucrose
    Prepared with Cu free water and diluted from stock solutions (1 M Tris-HCl, pH 7.5; 2.5 M sucrose). Store at 4 °C.
  4. Lysis buffer
    10 mg/ml lysozyme dissolved in 8 mM EDTA, pH 8. Final concentration 0.1mg/ml.
  5. Reagent A for Lowry assay
    2% (w/v) Na2CO3
    0.1 N NaOH
    0.01% (w/v) CuSO4
    0.02% (w/v) Na-tartrate
    Dissolved in water
  6. AAS modifier solution
    0.005 mg palladium
    0.003 mg magnesium
    Volume up to 5 µl in ultra-pure water

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, to HGK), the German Academic Exchange Service (DAAD, to PIT), the Else-Kröner-Fresenius Stiftung (to DM and MU) and the German-French PhD College on Membrane Proteins and Biological membranes (DFDK, to HGK).

References

  1. Ekici, S., Yang, H., Koch, H. G. and Daldal, F. (2012). Novel transporter required for biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus. MBio 3(1).
  2. Rae, T. D., Schmidt, P. J., Pufahl, R. A., Culotta, V. C. and O'Halloran, T. V. (1999). Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284(5415): 805-808.
  3. Ralle, M., Lutsenko, S. (2009). Quantitative imaging of metals in tissues. Biometals 22(1): 197-205.
  4. Trasnea, P. I., Utz, M., Khalfaoui-Hassani B., Lagies, S., Daldal, F. and Koch, H. G. (2016). Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3-type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus. Mol Microbiol 100(2): 345-361.

简介

铜是必需的微量营养素并且在许多酶如辅酶Cu氧还原酶,Cu-Zn超氧化物歧化酶,多铜氧化酶和酪氨酸酶中起辅因子的作用。然而,由于其化学反应性,游离铜是高毒性的(Rae等人,1999),并且所有生物体使用复杂的机器来控制Cu的摄取,储存和输出。严格控制细胞铜稳态防止毒性作用,但维持铜蛋白的合成。监测细胞内和不同细胞区室内的铜水平是识别不同蛋白质在维持细胞铜平衡中的贡献的必要方法。因此,可以进一步分离成细胞质和周质的全细胞和全细胞裂解物被消化,并通过Lowry测定法测定蛋白质浓度。随后,通过原子吸收光谱(AAS)测量铜含量,并计算每mg蛋白质的Cu含量。这提供了产生关于细胞Cu含量的可定量结果的简单且成本有效的方法。为了举例说明这种方法,我们使用光养型α-变形杆菌荚膜红细菌(Rhodobacter capsulatus),其通常用作研究细菌细胞中Cu运输的模式生物体(Ekici等人)。 ,2012)。

[背景] 由于对细胞Cu稳态的兴趣日益增长,在过去几年中开发了用于测量细胞Cu含量的不同方法。它们包括电化学和荧光方案,电感耦合等离子体质谱(ICP-MS),电感耦合等离子体原子发射光谱(ICP-AES),电子微探针分析(EMPA),X射线吸收光谱(XAS)或同步辐射X X-射线荧光显微镜(SXRF)(综述参见Ralle等人,2009)。虽然这些方法允许在生物和环境样品中Cu的可靠和准确的测定,它们通常需要高级的实验装置,并且通常不适于分析大量样品。原子吸收光谱(AAS)是一种完善的和广泛可用的方法,其允许快速,灵敏和成本有效的Cu测定。它适用于测定全细胞中的Cu,但也适用于亚细胞提取物。

关键字:铜的平衡, cbb3型细胞色素氧化酶, 红细胞, 铜伴侣蛋白, 铜依赖酶

材料和试剂

  1. 培养皿(60×15mm)(SARSTEDT,目录号:82.1194.500)
  2. 50ml Falcon管(SARSTEDT,目录号:62.559.001)
  3. 15ml Falcon管(SARSTEDT,目录号:62.554.502)
  4. 0.45μm过滤器(Carl Roth,目录号:P667.1)
  5. 10ml注射器(Carl Roth,目录号:C542.1)
  6. 野生型(MT1131)或突变体菌株
  7. BactoTM胨蛋白胨(BD,目录号:211820)
  8. Bacto TM酵母提取物(BD,目录号:212720)
  9. 氯化钙二水合物(CaCl 2·2H 2 O)(Carl Roth,目录号:5239.1)
  10. 氯化镁六水合物(MgCl 2·6H 2 O)(Carl Roth,目录号:2189.2)
  11. MPYE琼脂培养基(1.5%琼脂在MPYE培养基中; 25ml /培养皿)
  12. Chelex 100树脂(Bio-Rad Laboratories,目录号:1422832)
  13. Tris/HCl,pH7.5(Carl Roth,目录号:5429.2; P074.1)
  14. 蔗糖(MP Biomedicals,目录号:04821713)
  15. 溶菌酶(Sigma-Aldrich,目录号:L2879)
  16. EDTA(Carl Roth,目录号:8043.2)
  17. Lowry蛋白测定试剂[Reagent A,Folin& Ciocalteu的酚试剂(Sigma-Aldrich,目录号:F9252),1%SDS/0.1N NaOH]
  18. SDS(??SERVA电泳,目录号:20765.03)
  19. 蛋白质测定牛血清白蛋白(Carl Roth,目录号:8076.4),标准品(0,0.02,0.04,0.08,0.12,0.2mg/ml蛋白质)
  20. 碳酸钠(Na 2 CO 3)(Carl Roth,目录号:8563.1)
  21. 氢氧化钠(NaOH)(Carl Roth,目录号:6771.1)
  22. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Carl Roth,目录号:P024.1)
  23. 酒石酸钠(EMD Millipore,目录号:106663)
  24. 53%硝酸的超纯水(Carl Roth,目录号:9274.1)中
  25. 30%过氧化氢(Carl Roth,目录号:9681.1)
  26. 超纯水(Thermo Fisher Scientific,Thermo Scientific TM ,型号:BarnsteadTMGenPure TM
  27. 氯化钯(II)(VWR,目录号:AA11034-09)
  28. 氯化镁(MgCl 2)(VWR,目录号:AA42843-22)
  29. 铜标准[(20,40,60ppb Cu在超纯水中;从用于AAS的TraceCert铜标准稀释(在2%硝酸中的1,000mg/ml Cu,用高纯度Cu金属制备)](Sigma-Aldrich,目录number:38996)
  30. MPYE媒体(见配方)
  31. 无铜MPYE(参见配方)
  32. 无铜水(见配方)
  33. 球状体缓冲液(参见配方)
  34. 裂解缓冲液(见配方)
  35. 用于Lowry测定的试剂A(参见配方)
  36. AAS调节剂溶液(参见配方)

设备

  1. 1.5ml比色皿(Carl Roth,目录号:Y195.1)
  2. 磁搅拌板(Heidolph,型号:Hei-VAP)
  3. 无菌接种环(Carl Roth,目录号:6163.1)
  4. 在35℃下孵育细菌生长的培养箱多细胞标准(Infors,模式:INFORS HT)
  5. 250ml锥形瓶(Carl Roth,目录号:K184.1)
  6. 离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Sorvall Lynx 6000)
  7. 显微镜在100倍放大,数值孔径1.4(奥林巴斯,型号:BX51)
  8. 1000μl自动移液管(Gilson,目录号:F123602)
  9. 在60℃和80℃下孵育样品的孵育器(Kühner,型号:ISF-1X)
  10. 原子吸收分光光度计(PerkinElmer,型号:4110ZL Zeeman)
  11. VIS分光光度计,能够读取OD 660和OD?? 685(例如,GE Healthcare Life Science,型号:Ultrospec 3100 Pro)。

软件

  1. Excel 2007(Microsoft)
  2. Perkin-Elmer AAWinLab软件

程序

  1. 毛红细菌和原生质体制备物的生长,无铜MPYE和无Cu水的制备
    1. 在MPYE琼脂板(?24小时,35℃)上,在所需的红细菌菌株的无菌条件下培养培养物,包括抗生素(如果需要)。
    2. 准备用于液体培养的MPYE培养基,无铜MPYE和无铜水(见配方)。
    3. 使用无菌接种环挑选单个菌落,接种10ml MPYE在50ml Falcon管中。如果需要,添加适当的抗生素。在35℃和110rpm下在旋转振荡器中培养过夜培养物
    4. 第二天:用来自过夜培养物的10 8个细胞接种主培养物(在250ml烧瓶中的100ml MPYE),并在35℃,110rpm温育直至培养物达到OD 685 1(约24小时,假设OD 685 = 1对应于7.5×10 8个细胞/ml)。这对应于R的晚指数生长期。 capsulatus 。
    5. 使用预冷却的Lynx 6000离心机(4℃),以5,000rpm(对应于F12-6x500LEX转子中的3,000xg),收获细胞20分钟。
    6. 倾析上清液并将沉淀物重悬于40ml预冷却的无铜MPYE中,并在4℃下以5,000rpm(3,000xg,F12-6x500LEX转子)再离心20分钟。
    7. 滗析上清液,并将沉淀物重悬在2ml原生质球缓冲液中,取一小份50μl用于通过Lowry测定法(参见数据分析)测定蛋白质浓度,例如。
    8. 将细胞悬液分成两等份,放入两个干净的15ml Falcon管中。一个管用于测量全细胞中的铜浓度,而另一个管中的细胞用于原生质球制备。这允许测量细胞质(原生质球),周质(原生质体制备后的上清液)和全细胞中的铜含量。
    9. 原生质体制备
      1. 向稀释度为1:10的原生质体样品部分中加入裂解缓冲液,以达到终浓度为0.1mg/ml的溶菌酶,例如向900μl样品中加入100μl裂解缓冲液。
      2. 将细胞悬浮液在冰上孵育15分钟,同时轻轻搅拌,即每几分钟将管颠倒几次。 15分钟后,大多数细胞被转化为原生质球;其他革兰氏阴性细菌可能需要更长时间的孵育。
      3. 通过使用显微镜监测原生质体形成。
      4. 通过在4℃下以5,000rpm(3,000xg,F12-6x500LEX转子)离心20分钟收集原生质球。
      5. 小心地收集上清液(周质),优先使用1000微升自动移液管到一个干净的15毫升Falcon管。
      6. 用1 ml原生质球缓冲液重悬沉淀(细胞质)
      7. 从每个部分取50μl的等分试样,并测量所有样品的蛋白质浓度

        图1.监测原生质体形成。以100×放大倍率观察到的 R.capsulatus 细胞。 B.在100×放大率下观察球形体形成。球状体由箭头指示。

  2. 通过原子吸收光谱法测定Cu浓度
    1. 向大约1ml全细胞,原生质球和上清液(在原生质球制备后)在15ml Falcon管中,加入1.5ml的53%硝酸。该处理和在高温下的以下温育完全使蛋白质溶液变性并释放任何结合的金属。在80℃下孵育1小时,随后在60℃下振荡过夜
    2. 过夜孵育后,通过加入300μlH 2 O 2(30%水溶液)停止消化,并通过以下方法将样品调节至10ml的最终体积:加入无Cu水。
    3. 如果观察到任何不溶性杂质,则将溶液通过0.45μm一次性注射器过滤器过滤。所制备的样品现在是稳定的,并且可以在室温下储存几天
    4. 使用原子吸收分光光度计测量3次3秒的雾化来测量铜含量。加载20μl样品和5μl含有0.005mg钯和0.003mg镁(作为电离剂)的改性剂。
      1. 空白测量(0.2%硝酸在超纯水中),计算平均值并设置为0.
      2. 通过测量包含20,40和60 ppb Cu的3个标准品校准分光光度计
      3. 测量样品。如果数值超出范围,请稀释样品1:2或1:3
      4. 铜含量的分析通过Perkin-Elmer AAWinLab软件实现。对于每个值,由软件计算标准偏差(SD)和相对标准偏差(RSD)
    5. 使用蛋白质浓度和铜测量绘制数据。计算10ml样品的总铜量,并通过蛋白质定量测定计算的总蛋白质质量和使用下式计算添加硝酸前的样品体积除以:
      μgCu/mg蛋白=(X Cu ppb×10ml)/(Xmg/ml蛋白×Xml样品体积)
      注意:Cu ppb指Cuμg/L。

数据分析

  1. 在Trasnea等人中可以找到表明在红细菌中的细胞铜含量的测定的代表性实验。 (2016年)。需要两种周质铜伴侣之间的合作以用于cbb 型细胞色素氧化酶和铜稳定红细胞中的铜稳态的完全活性。
  2. 下面描述用于确定荚膜红细菌细胞/细胞提取物中的蛋白质浓度的excel BSA标准曲线的实例。将样品在200μl的1%SDS/0.1N NaOH中稀释,加入1ml试剂A,随后在室温下温育5分钟。然后,将100μlFolin&加入Ciocalteu的酚试剂1N,孵育45分钟后,测定A 660处的吸光度。使用标准曲线(如下所述)将吸光度与蛋白质浓度相关联
    表1. BSA蛋白标准品及其在A660 的相对吸光度


    使用VIS分光光度计测定吸光度读数,随后绘制数据,如图2所示

    图2. BSA蛋白标准曲线对于0-0.2mg/ml的BSA蛋白标准品范围测定A660(nm)处的吸光度。使用y = mx绘制最佳拟合的线(MS Excel 2007)。通过将它们的吸光度与x轴上的蛋白质浓度相关联来确定样品的蛋白质浓度
  3. 使用Perkin-Elmer AAWinLab软件的原子吸收光谱法的Cu标准及其消光值的代表在表2中提供。图3包含使用Microsoft Excel(在过程中由Perkin-Elmer AAWinLab软件自动产生)的示例性标准曲线, 。

    表2. Cu标准及其在324.8 nm处的消光


    随后使用Perkin-Elmer AAWinLab软件或MS Excel绘制数据,如图3所示。


    图3.Cu浓度标准曲线。使用通过AAS获得的值,使用Microsoft Excel生成曲线。对于0-60ppb的Cu的范围确定324.8nm处的消光标准。使用该曲线,可以通过将它们的消光与x轴上的Cu浓度值相关联来确定来自细胞和不同细胞区室的铜浓度。

笔记

  1. 该方案被优化用于测量红细胞杆菌的细胞内铜浓度。对于其他细菌,使用适当的生长培养基与适当的抗生素和生长条件。在OD 685 = 0.8的晚指数生长期收获的细胞中测定Cu。该方法还应该与在不同生长阶段收获的细胞一起工作,但是这里不对其进行分析。
  2. 周质提取的溶菌酶处理针对R优化。 capsulatus ,因此对于革兰氏阴性菌。由于溶菌酶不能可靠地作用于所有革兰氏阴性细菌,因此可以在这些情况下测试不同的胞壁酰胺酶例如变溶菌素。
  3. 使用所获得的铜浓度,可以通过使用以下公式将样品的铜含量除以电池隔室的体积来计算电池隔室中的铜的摩尔浓度:
    μmol/L Cu =(Xμg/L Cu×A)/(63.546g/mol×B×C)
    A =样品体积(通常为0.01L)[L]
    B =样品中的细胞数(通过OD测量或通过细胞的显微计数确定)
    C =一个细胞或细胞室的估计体积[L]
    63.546g/mol对应于Cu的分子量 示例:
    假定细胞质液加上一个红细胞脓毒细胞的内膜具有1.9×10 16 -16μL的体积(基于Cyber??Cell数据库计算; http://ccdb.wishartlab.com/CCDB/),铜的摩尔浓度在细胞质中约为300μM

食谱

  1. MPYE媒介
    0.3%(w/v)Bacto TM sup-蛋白胨 0.3%(w/v)Bacto TM上海酵母提取物 0.1%(v/v)1 M CaCl 2 2·2H 2 O
    0.1%(v/v)1M MgCl 2 6H 2 6H 2 O 蒸馏水,高压釜
  2. 无铜MPYE和无铜水
    将5g Chelex 100树脂加入100ml溶液中,并连续搅拌至少1小时 然后,通过0.45μm过滤器过滤溶液并高压灭菌 经过Chelex处理后,铜浓度低于检测限
  3. 球状体缓冲液(储存在4℃或新鲜制备的) 100mM Tris-HCl,pH 7.5
    0.5 M蔗糖 用Cu游离水制备并从储备溶液(1M Tris-HCl,pH 7.5; 2.5M蔗糖)稀释。储存于4°C。
  4. 裂解缓冲液
    溶于8mM EDTA的10mg/ml溶菌酶,pH8。终浓度0.1mg/ml
  5. 试剂A for Lowry测定
    2%(w/v)Na 2 CO 3 3/6 0.1 N NaOH
    0.01%(w/v)CuSO 4
    0.02%(w/v)酒石酸钠
    溶于水
  6. AAS改性剂溶液
    0.005mg钯
    0.003mg镁 超纯水中体积高达5μl

致谢

这项工作得到了德国Forschungsgemeinschaft(DFG,HGK),德国学术交流服务(DAAD,到PIT),Else-Kr?ner-Fresenius基金会(DM和MU)和德国 - 法国博士学院膜蛋白和生物膜(DFDK,至HGK)。

参考文献

  1. Ekici,S.,Yang,H.,Koch,HG和Daldal,F。(2012)。  cbb3 型细胞色素c氧化酶生物发生所需的新型转运蛋白 MBio < em> 3(1)。
  2. Rae,TD,Schmidt,PJ,Pufahl,RA,Culotta,VC和O'Halloran,TV(1999)。  不可检测的细胞内游离铜:超级氧化物歧化酶的铜伴侣的要求 Science 284(5415):805-808。
  3. Ralle,M.,Lutsenko,S.(2009)。  组织中金属的定量成像。生物金属22(1):197-205。
  4. Trasnea,PI,Utz,M.,Khalfaoui-Hassani B.,Lagies,S.,Daldal,F.和Koch,HG(2016)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Trasnea, P., Marckmann, D., Utz, M. and Koch, H. (2016). Measurement of Cellular Copper in Rhodobacter capsulatus by Atomic Absorption Spectroscopy . Bio-protocol 6(19): e1948. DOI: 10.21769/BioProtoc.1948.
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