Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins

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Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties to these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to predict. We established a method suitable to probe metal transfer between two proteins, provided the proteins are amenable to purification and in vitro handling. It consists of the loading with metals, the co-incubation and the separation of metal-exchanging proteins with subsequent determination of bound metal content. The method is exemplified by experimental data of ours probing the transfer of copper(I) between the membrane-extrinsic metal binding domain MBD2 and the transmembrane domain of CopA, a copper export ATPase from Escherichia coli (Drees et al., 2015).

Keywords: Copper-binding protein(铜结合蛋白), Copper chaperone(铜伴侣), Heavy metal associated domain(重金属相关域), Copper transporter(铜转运蛋白), P-type ATPase(型三磷酸腺脢)

Materials and Reagents

  1. Desalting NAP5 columns (e.g., GE Healthcare, HiTrap Desalt, catalog number: 17-0853-01 )
  2. Gel filtration columns
    We used prepacked GE 10/300 Tricorn columns, a Superdex 75 (GE Healthcare, catalog number: 17-5174-01 ) for small soluble proteins, or a Superose 6 for lipid-reconstituted samples (GE Healthcare, catalog number: 17-5172-01 ).
  3. Proteins
    1. Metal binding proteins of interest must be on the hand in purified native state, with aggregates having been removed prior to the experiments.
    2. Proteins should be available in milligram quantities and should preferably have different molecular weights (which is a prerequisite for gel filtration).
    3. Proteins too small to be distinguished from each other on gel filtration columns could be fused to a polypeptide tag, e.g., the maltose binding protein or glutathione-S-transferase in order to artificially increase the molecular weight difference of the metal ion transfer pair.
    4. We used an eGFP tag because it is relatively inert in redox-reactions and facilitates easy detection and concentration determination.
    5. Care has to be taken with the use of a His-tag for means of protein purification. Although it was demonstrated that His-tags do not interfere with copper binding and quantification experiments under certain conditions (González-Guerrero et al., 2008), the metal chelating property of the hexahistidine sequence may cause metal carry-overs in some experiments.
  4. 3-(N-morpholino) propanesulfonic acid (MOPS), buffer grade (Sigma-Aldrich, catalog number: M1254 )
  5. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  6. Sodium ascorbate (Sigma-Aldrich, catalog number: 11140 )
  7. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  8. Phosphatidylcholine (PC) (Sigma-Aldrich, catalog number: P3644 )
  9. Sodium dodecylsulfate (SDS) (Sigma-Aldrich, catalog number: L6026 )
  10. Bathocuproinedisulfonate (BCS) (Sigma-Aldrich, catalog number: B1125 )
  11. Bicinchoninic acid (BCA) (Sigma-Aldrich, catalog number: D8284 )
  12. β-dodecylmaltoside (β-DDM) (Sigma-Aldrich, catalog number: D5172 )
  13. Copper sulfate (CuSO4) (Sigma-Aldrich, catalog number: 451657 )
  14. Buffer A (see Recipes)
  15. BCA-copper buffer (see Recipes)
  16. SDS solution (see Recipes)
  17. Ascorbate solution (see Recipes)
  18. BCS solution (see Recipes)


  1. (Optional) A dynamic light scattering instrument [e.g., High performance particle sizer (Malvern Instruments, model: HPP 5001 or Zetasizer Nano S )]
  2. Ultrasonic homogenizer for preparation of lipid emulsions (e.g., BANDELIN electronic GmbH & Co, model: UP 400 S )
  3. Ultrasonic bath for buffer degassing (e.g., BANDELIN electronic GmbH & Co, model: Sonorex DL510H )
  4. An automated chromatography unit (e.g., GE Healthcare, Äkta Purifier or Explorer)
    Note: It is essential for the experiments. Use of a regular HPLC unit with fraction collector would be just as fine, provided that some modifications with respect to the tolerance of salt-containing elution buffers have been taken care of.
  5. A thermostated shaking incubator for 1.5 ml type reaction vials (e.g., Eppendorf, catalog number: 5384000012 )
  6. Centrifugal evaporator (e.g., Eppendorf, catalog number: 5305000304 )
  7. Calibrated micropipette (e.g., Eppendorf, catalog number: 3120000062 ), alternatively: analytical balance (e.g., Mettler-Toledo International Inc., catalog number: 11142056 )
  8. UV-VIS Spectrophotometer (e.g., Jasco, model: J-650 ) and Nanodrop spectrophotometer (Thermo Fisher Scientific), alternatively: UV-VIS spectrophotometer and small volume cuvette (e.g., Hellma, model: 105.250-QS or Eppendorf, µCuvette®, model: G1.0 )
  9. Quartz microcuvette (e.g., Hellma, catalog number: 104-10-40 104-QS )
  10. Standard SDS-PAGE equipment (e.g., Bio-Rad Laboratories, catalog number: 1658001FC )


  1. Required amount of protein
    1. The required amount of protein depends on how much metal donor and acceptor protein bind relative to their molecular weight.
    2. Using 100 nM of each donor and acceptor protein (1:1 stoichiometry) is a solid starting point.
    3. Lower amounts can be used, but accuracy will decrease.
  2. Protein oxidation state
    1. Check the putative protein metal binding sites of donor and acceptor proteins for oxidation sensitivity: Monovalent copper (amongst other heavy metal ion species) is often coordinated via cysteine thiols, which is prone to oxidation during protein preparation. In our example, the donor protein, MBD2, as well as the acceptor protein CopA feature copper binding sites involving oxidation-sensitive cysteines (Fan and Rosen, 2002; Fan et al., 2001).
    2. In case of oxidation sensitivity of either protein, add DTT from a 1 M stock solution (one may alternatively try 2-mercaptoethanol or TCEP, but we have not tested these) to a concentration of 5-20 mM and incubate for 1 h.
    3. Subsequently remove all the DTT by buffer exchange via desalting chromatography using NAP5 columns (alternatively, HiTrap desalt or PD10 columns will work as well). The handling should be performed according to manufacturer’s instructions.
    4. In addition, it is crucial to use freshly degassed buffer (e.g., use the storage buffer established for your protein of interest, or buffer A) to avoid reoxidation.
    5. After desalting, proceed immediately with your experiment, or flash-freeze the pre-treated protein in liquid nitrogen.
  3. Load the donor protein with metal (workflow illustrated in Figure 1)
    1. Copper in its divalent state (such as in CuSO4) is detrimental for proteins as it adsorbs to biosurfaces and oxidizes amide bonds. Hence, protein precipitation is often observed in the presence of Cu(II) salts.
    2. On the contrary, Cu(I) salts are highly redox-active and are almost insoluble in aqueous media. It is therefore recommended to load the donor protein by means of a colored low affinity organo-copper complex, such as [BCA2-Cu(I)]3-. The donor protein is therefore titrated with BCA-copper buffer until the mixture does not destain any longer and retains the characteristic violet color.

    Figure 1. Workflow for preparation of metal-loaded donor protein. A solution of the donor protein in apo state is titrated with a metal buffer, in this case a BCA-copper buffer. This indirect loading procedure avoids direct contact of the protein with excess copper and thereby protects it from metal-caused damage. Titrating little steps is advantageous because it allows monitoring the loading process. In case there is no destaining in the first titration steps, the donor protein may have suffered damage or is not receptive for metal loading at all. As soon as an excess of metal buffer is present in the protein solution, the mixture is loaded onto a size exclusion/desalting column (e.g., a NAP-5 column, used after manufacturer’s instructions) to yield the metal-loaded donor protein without contamination of free metal or metal chelator.

  4. Repurify donor protein
    1. To remove excess BCA, repurify the donor protein using a buffer exchange column (as in step 2c) equilibrated with buffer A.
    2. The eluted protein must not contain any blue stain (measure absorbance at 562 nm).
  5. Spectroscopic determination of protein concentration
    1. Measure the concentration of the loaded donor protein using a NanoDrop spectrophotometer and an experimental or predicted absorption coefficient (Pace et al., 1995).
    2. In case the concentration of the acceptor protein is unknown, it should be quantified likewise at this point.
  6. (Optional) Lipid emulsion preparation
    1. In case of CopA, we experienced that reconstitution into lipid phase may be required for metal receptivity. Other membrane proteins (González-Guerrero et al., 2009) or soluble proteins may not require this step.
    2. Optimum conditions for reconstitution may vary and require testing for each individual case.
    3. For the presented procedure, a uniform vesicle size is advisable. Therefore, 10 % (w/v) lipid suspensions of phosphatidylcholine were emulsified by sonication for 10 min (ultrasonic homogenizer, low intensity dipped probe). The particle size distribution may be facultatively checked by means of a dynamic light scattering instrument.
  7. (Optional) Lipid reconstitution
    Add a 10 fold (w/w) excess of the 10 % lipid suspension to a defined amount of protein and incubate at 37 °C for 20 min and gentle agitation.
  8. Equilibration of the column
    Equilibrate gel filtration column with 2 column volumes of buffer A. Meanwhile store donor and acceptor protein on ice.
  9. Ion transfer
    Mix donor and acceptor protein in molar ratio of 1:1 to 2:1 and incubate for 5 min at room temperature, then add buffer A to a total of 500 µl (for GE Healthcare Tricorn 10/300 columns).
  10. Chromatography
    Apply protein mixture to the chromatography column using the autoinjector of the unit (500 µl in 1 ml sample loop) and start the elution using a flow rate of 0.2 ml min-1 and a fraction size of 1 ml over 30 ml. An increased flow rate will work fine as well, although leading to higher dilution of the proteins.
  11. Save the chromatogram UV and (if available) conductivity traces, it should look like the diagram in Figure 2A. Collect fractions exhibiting UV absorption and store for the later analysis of copper content (see below).
  12. Electrophoretic control of separation
    1. 10-50 µl samples can be drawn from each fraction for electrophoretic analysis of separation (perform electrophoresis according to manufacturer’s instructions).
    2. If donor and acceptor proteins form a stable complex during chromatography, incomplete dissociation should be observed.
    3. In case of transient interaction during transfer, both proteins are well separated (see Figure 2B).

    Figure 2. Exemplary Data on gel filtration and electrophoresis. A. Typical UV absorption (black trace) and conductivity (gray trace) data read-out of the automated chromatography unit. The example shows a separation of MBD2 (donor) and lipid-reconstituted CopA (acceptor) using a Superose 6 column. Relevant peaks are labeled with respect to their content. B. SDS-PAGE for testing effective separation between donor and acceptor proteins is an important control experiment to rule out complex formation between donor (B-fractions) and acceptor (A-fractions).

  13. Transfer fractions to 1.5 ml polypropylene vials and add 40 µl of SDS-solution [1% SDS (w/v) final concentration] to each fraction. Incubate at 65 °C and slight agitation until protein or lipid-protein emulsions are completely homogenized and the solutions appear clear. This can take up to 1 h.
  14. Cool down the samples and centrifuge for 1 min at 1,000 x g to collect the entire liquid at the bottom of the tube.
  15. Record UV-spectra of each sample.
    1. Check for the absence of a non-desired scattering curve underneath the protein absorbance spectrum.
    2. In case of scattering, add another 40 µl of SDS solution, incubate for 1 h and re-measure.
  16. (Optional) In case the expected metal concentrations are low, samples can be concentrated using a centrifugal evaporator (“Speed Vac”) to increase sensitivity. We experienced that concentrating to 300 µl does not cause any precipitation, but the actual level to which a sample volume can be reduced really depends on the particular experiment.
  17. Volume determination
    Measure the exact volume of each sample, either volumetrically by using a calibrated micropipette or gravimetrically with an analytical balance [assumed density 1.02 (500 mM NaCl solution), error due to density mismatch and the precision of the pipette are in the same range].
  18. Detection of metal content
    1. Note that many colorimetric quantification methods for different metal ions are described, some of which are mentioned in the review by Xiao and Wedd (2010).
    2. For copper, we chose to use BCS because it has a high extinction coefficient in the visible light range. Add 1 % ascorbate (v/v) and 5 % BCS solution (v/v) to each sample and incubate 5 min at room temperature.
  19. Measure the absorbance of each sample at 482 nm.
  20. Data analysis
    Calculate protein concentrations using either the computed (Pace et al., 1995) or experimental extinction coefficients.
  21. Calculate the exact metal concentration using the extinction coefficient of the [BCS2- Cu(I)]3--complex of 13,000 M-1 cm-1 (Xiao et al., 2011). Take into account all dilution steps.
  22. Sum up total molar protein and copper concentrations in donor and acceptor fractions and calculate their ratios to determine the ion transfer efficiency.
    1. Note that in case of a reconstituted membrane protein, a 100% efficiency is very unlikely and has never been reached in our experiments. This is due to anisotropic integration of the CopA acceptor protein into the lipid phase and the resulting inaccessibility to the metal donor protein.
    2. With soluble proteins, transfer efficiencies of over 90% can be observed.

Representative data

A typical example of the separation of copper-loaded MBD2 and of lipid-reconstituted CopA is shown in Figure 2. Full exemplary experimental data, i.e., the concentrations of protein (calculated from spectrophotometric read of OD280) and of copper (determined colorimetrically as outlined above) after the transfer experiment are displayed in Figure 4 of Reference 1.


  1. Avoid loading GE Superdex analytical gel filtration columns with turbid samples. Remove precipitates by centrifugation (Eppendorf microfuge) prior to sample application.
  2. The determinations of protein and of copper are fairly reliable. However, using analytical gel filtration columns, the sample volumes after fractionation are limiting to be sufficient for single chemical determinations of both components. Data from a single transfer experiment often provide a reasonable figure of the metal transfer efficiency. For proper validation of the transfer results, experimental data from at least four chromatographic separations should be appropriate.
  3. Monitoring metal contents of both, donor and acceptor proteins before the actual exchange is a very important control experiment. We therefore recommend performing gel filtrations with either donor or acceptor protein alone. Additionally, metal carryover in the receptor protein, e.g., from purification can be detected this way. The measured values can be used as a “metal content baseline” to calculate the net amount of transferred metal resulting from co-incubation.


  1. Buffer A
    30 mM MOPS, pH 7.5
    150 mM NaCl
  2. BCA-copper buffer
    Buffer A with 2.5 mM BCA
    1 mM CuSO4
    20 mM ascorbate
  3. SDS solution
    25 % SDS (w/v) in H2O
    Prepare at 37 °C for better solubility
  4. Ascorbate solution
    100 mM sodium ascorbate, pH adjusted to 7.5
    Store in small aliquots at -20 °C
  5. BCS solution
    10 mM BCS in 30 mM MOPS, pH 7.5
    Protect from light


We are grateful to the Protein Research Department of the Ruhr-Universität Bochum for financial support.


  1. Drees, S. L., Beyer, D. F., Lenders-Lomscher, C. and Lübben, M. (2015). Distinct functions of serial metal-binding domains in the Escherichia coli P1 B -ATPase CopA. Mol Microbiol 97: 423-438.
  2. Fan, B., Grass, G., Rensing, C. and Rosen, B. P. (2001). Escherichia coli CopA N-terminal Cys(X) (2)Cys motifs are not required for copper resistance or transport. Biochemical and Biophysical Research Communications 286: 414-418.
  3. Fan, B. and Rosen, B. P. (2002). Biochemical characterization of CopA, the Escherichia coli Cu (I)-translocating P-type ATPase. Journal of Biological Chemistry 277: 46987-46992.
  4. Gonzalez-Guerrero, M., Hong, D. and Argüello, J. M. (2009). Chaperone-mediated Cu+ delivery to Cu+ transport ATPases requirement of nucleotide binding. Journal of Biological Chemistry 284: 20804-20811.
  5. Gonzalez-Guerrero, M., Eren, E., Rawat, S., Stemmler, T. L. and Argüello, J. M. (2008). Structure of the two transmembrane Cu+ transport sites of the Cu+-ATPases. Journal of Biological Chemistry 283: 29753-29759.
  6. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995). How to measure and predict the molar absorption-coefficient of a protein. Protein Science 4: 2411-2423.
  7. Xiao, Z. G., Brose, J., Schimo, S., Ackland, S. M., La Fontaine, S. and Wedd, A. G. (2011). Unification of the copper (I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins detection probes and affinity standards. Journal of Biological Chemistry 286: 11047-11055.
  8. Xiao, Z. G. and Wedd, A. G. (2010). The challenges of determining metal-protein affinities. Natural Product Reports 27: 768-789.


重金属可以以多种方式对生物分子例如蛋白质和DNA造成损害。因此,细胞努力保持细胞内(重)金属离子结合到能够处理解毒,输出或整合作为辅因子的特定蛋白质。金属结合蛋白通常提供结合某些离子的特异性配位位点,具有超高的亲和力,热力学驱动力是有机金属配合物的稳定性。然而,这些蛋白质的金属结合性质可以是高度可变的。因此,在分开的蛋白质之间或甚至在位于同一蛋白质上的不同结合位点之间的特异性离子的转移不总是遵循亲和梯度,而是取决于难以预测的特定蛋白质相互作用。我们建立了一种适合探测两种蛋白质之间的金属转移的方法,只要这些蛋白质可以进行纯化和体外处理。它由金属负载,共孵育和金属交换蛋白的分离,随后确定结合金属含量。该方法通过我们探测膜 - 外在金属结合结构域MBD2和来自大肠杆菌的铜输出ATP酶的跨膜结构域的膜 - 外部金属结合结构域MBD2之间的铜(I)转移的实验数据来举例说明(Drees < em> 。,2015)。

关键字:铜结合蛋白, 铜伴侣, 重金属相关域, 铜转运蛋白, 型三磷酸腺脢


  1. 脱盐NAP5柱(,例如GE Healthcare,HiTrap Desalt,目录号:17-0853-01)
  2. 凝胶过滤柱
    我们使用预包装的GE 10/300Tricorn柱,用于小可溶性蛋白的Superdex 75(GE Healthcare,目录号:17-5174-01)或用于脂质重建样品的Superose 6(GE Healthcare,目录号:17-5172 -01)。
  3. 蛋白质
    1. 感兴趣的金属结合蛋白必须以纯化的天然状态在手上,在实验之前聚集物已经被除去。
    2. 蛋白质应以毫克数量提供,并且应优选具有不同的分子量(这是凝胶过滤的先决条件)。
    3. 太小而不能在凝胶过滤柱上彼此区分的蛋白质可以与多肽标签例如麦芽糖结合蛋白或谷胱甘肽-S-转移酶融合,以便人为增加金属离子转移的分子量差异对。
    4. 我们使用eGFP标签,因为其在氧化还原反应中相对惰性并且便于容易的检测和浓度测定。
    5. 对于蛋白质纯化方法,必须小心使用His标签。虽然已经证明His标签在某些条件下不干扰铜结合和定量实验(González-Guerrero等人,2008),但六聚组氨酸序列的金属螯合性质可能在一些实验中引起金属携带。/em>
  4. (MOPS),缓冲级(Sigma-Aldrich,目录号:M1254)。
  5. 氯化钠(Sigma-Aldrich,目录号:S7653)
  6. 抗坏血酸钠(Sigma-Aldrich,目录号:11140)
  7. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  8. 磷脂酰胆碱(PC)(Sigma-Aldrich,目录号:P3644)
  9. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L6026)
  10. (BCS)(Sigma-Aldrich,目录号:B1125)
  11. 二辛可宁酸(BCA)(Sigma-Aldrich,目录号:D8284)
  12. β-十二烷基麦芽糖苷(β-DDM)(Sigma-Aldrich,目录号:D5172)
  13. 硫酸铜(CuSO 4)(Sigma-Aldrich,目录号:451657)
  14. 缓冲区A(参见配方)
  15. BCA铜缓冲液(见配方)
  16. SDS溶液(见配方)
  17. 抗坏血酸溶液(参见配方)
  18. BCS解决方案(参见配方)


  1. (可选)动态光散射仪器[例如,高性能粒度仪(Malvern Instruments,型号:HPP 5001或Zetasizer Nano S)]
  2. 用于制备脂质乳液的超声匀浆器(例如,BANDELIN electronic GmbH& Co,型号:UP 400S)
  3. 用于缓冲脱气的超声波浴(例如,BANDELIN electronic GmbH& Co,型号:Sonorex DL510H)
  4. 自动色谱单元(例如,GE Healthcare,?ktaPurifier或Explorer)
  5. 用于1.5ml类型反应小瓶(例如,Eppendorf,目录号:5384000012)的恒温摇动培养箱
  6. 离心蒸发器(例如,Eppendorf,目录号:5305000304)
  7. 校准的微量移液管(例如,Eppendorf,目录号:3120000062),或者:分析天平(例如,Mettler-Toledo International Inc.,目录号:11142056) />
  8. UV-VIS分光光度计(例如,Jasco,型号:J-650)和Nanodrop分光光度计(Thermo Fisher Scientific),或者:UV-VIS分光光度计和小体积比色皿(例如,Hellma,型号:105.250-QS或Eppendorf,μCuvette,型号:G1.0)
  9. 石英微池(,例如,Hellma,目录号:104-10-40 104-QS)
  10. 标准SDS-PAGE设备(例如,Bio-Rad Laboratories,目录号:1658001FC)


  1. 蛋白质需要量
    1. 蛋白质的所需量取决于相对于其分子量,多少金属供体和受体蛋白结合。
    2. 使用100nM的每种供体和受体蛋白(1:1化学计量)是固体起始点。
    3. 可以使用较低的数量,但精度会降低。
  2. 蛋白氧化态
    1. 检查供体和受体蛋白质的推定的蛋白质金属结合位点的氧化敏感性:单价铜(除其他重金属离子种类之外)经常通过半胱氨酸硫醇协调,其在蛋白质制备期间易于氧化。在我们的实施例中,供体蛋白质MBD2以及受体蛋白质CopA具有涉及氧化敏感性半胱氨酸的铜结合位点(Fan和Rosen,2002; Fan等人,2001)。
    2. 在任一蛋白质的氧化敏感性的情况下,从1M储备溶液(一个可替代地尝试2-巯基乙醇或TCEP,但我们没有测试这些)添加DTT至5-20mM的浓度并孵育1小时。
    3. 随后通过使用NAP5柱的脱盐色谱通过缓冲液交换除去所有DTT(或者,HiTrap脱盐或PD10柱也将如此)。处理应根据制造商的说明进行。
    4. 此外,使用新鲜脱气的缓冲液(例如,使用为您感兴趣的蛋白质或缓冲液A建立的储存缓冲液)以避免再氧化是至关重要的。
    5. 脱盐后,立即进行实验,或在液氮中快速冷冻预处理的蛋白质
  3. 用金属载入供体蛋白(工作流程如图1所示)
    1. 处于其二价状态(例如在CuSO 4中)的铜对于蛋白质是有害的,因为它吸附到生物表面并氧化酰胺键。因此,在Cu(II)盐的存在下通常观察到蛋白质沉淀。
    2. 相反,Cu(I)盐是高度氧化还原活性的,并且几乎不溶于水性介质。因此,推荐通过有色低亲和力有机铜络合物,例如[BCA 2 sub-Cu(I)] 3+来加载供体蛋白。因此,供体蛋白质用BCA-铜缓冲液滴定,直到混合物不再消失,并保持特征性紫色。

  4. 重新净化供体蛋白
    1. 为了除去过量的BCA,使用用缓冲液A平衡的缓冲液交换柱再次纯化供体蛋白质(如步骤2c)。
    2. 洗脱的蛋白质不能含有任何蓝色染料(测量562nm处的吸光度)
  5. 光谱测定蛋白浓度
    1. 使用NanoDrop分光光度计和实验或预测的吸收系数测量负载的供体蛋白的浓度(Pace等人,1995)。
    2. 在受体蛋白质的浓度未知的情况下,应该在这一点同样量化
  6. (可选)脂质乳剂制剂
    1. 在CopA的情况下,我们经历了重建成脂质相可能需要金属接受性。其他膜蛋白(González-Guerrero等人,2009)或可溶性蛋白质可能不需要这个步骤。
    2. 重构的最佳条件可以变化,并且需要对每种个别情况进行测试。
    3. 对于所提出的程序,均匀的囊泡大小是可取的。因此,通过超声处理10分钟(超声匀浆器,低强度浸渍探针)乳化10%(w/v)磷脂酰胆碱的脂质悬浮液。可以通过动态光散射仪来兼顾检查粒度分布
  7. (可选)脂质重建
  8. 列平衡
  9. 离子传输
    以1:1至2:1的摩尔比混合供体和受体蛋白,并在室温下孵育5分钟,然后将缓冲液A加至总共500μl(用于GE Healthcare Tricorn 10/300柱)。
  10. 色谱
    使用单位的自动注射器(500μl,在1ml样品环中)将蛋白质混合物应用于色谱柱,并使用0.2ml min -1 -1的流速和1ml级分大小开始洗脱超过30ml。增加的流速也将很好地工作,尽管导致更高的蛋白质稀释
  11. 保存色谱图UV和(如果有)电导率轨迹,它应如图2A所示。收集显示UV吸收的部分并储存以用于铜含量的后续分析(参见下文)。
  12. 分离的电泳控制
    1. 可以从每个级分中取出10-50μl样品用于电泳分离分析(根据制造商的说明进行电泳)。
    2. 如果供体和受体蛋白在层析期间形成稳定的复合物,应观察到不完全解离
    3. 在转移期间的瞬时相互作用的情况下,两种蛋白质被良好分离(参见图2B)。

    图2.凝胶过滤和电泳的示例性数据A.自动化色谱单元的典型UV吸收(黑色迹线)和电导率(灰色曲线)数据读出。该实施例显示使用Superose 6柱分离MBD2(供体)和脂质重建的CopA(受体)。相关峰针对其含量进行标记。 B.用于测试供体和受体蛋白质之间有效分离的SDS-PAGE是一个重要的对照实验,以排除供体(B级分)和受体(A级分)之间的复合物形成。
  13. 将级分转移至1.5ml聚丙烯小瓶中,并向每个级分加入40μlSDS-溶液[1%SDS(w/v)终浓度]。在65℃孵育并轻微搅拌,直到蛋白质或脂质 - 蛋白质乳液完全均匀化,并且溶液看起来澄清。这可能需要长达1小时。
  14. 冷却样品,并以1,000×g离心1分钟,收集管底部的整个液体。
  15. 记录每个样品的UV光谱。
    1. 检查在蛋白质吸收光谱下不存在非期望的散射曲线。
    2. 在分散的情况下,加入另外40μl的SDS溶液,孵育1小时并重新测量
  16. (可选)如果预期的金属浓度低,可以使用离心蒸发器("Speed Vac")浓缩样品以提高灵敏度。我们经历,集中到300微升不会导致任何降水,但实际水平,样品量可以减少真的取决于具体的实验。
  17. 音量确定
    通过使用校准的微量移液器或使用分析天平(假定密度1.02(500mM NaCl溶液),重量分析,由于密度失配引起的误差和移液管的精度在相同范围内)测量每个样品的精确体积。
  18. 金属含量的检测
    1. 注意,描述了用于不同金属离子的许多比色量化方法,其中一些在Xiao和Wedd(2010)的综述中提及。
    2. 对于铜,我们选择使用BCS,因为它在可见光范围内具有高的消光系数。向每个样品中加入1%抗坏血酸(v/v)和5%BCS溶液(v/v),并在室温下孵育5分钟。
  19. 测量每个样品在482nm的吸光度
  20. 数据分析
    使用计算的(Pace et al。,1995)或实验消光系数计算蛋白质浓度。
  21. 使用13,000M sup-1的[BCS 2-Cu(I)] 3+复合物的消光系数计算确切的金属浓度。 cm -1 (Xiao等人,2011)。考虑所有稀释步骤。
  22. 总结供体和受体部分中的总摩尔蛋白和铜浓度,并计算它们的比例以确定离子转移效率。
    1. 注意,在重组膜蛋白的情况下,100%的效率是不太可能的,并且在我们的实验中从未达到。这是由于CopA受体蛋白质向脂质相中的各向异性整合以及由此导致的对金属供体蛋白的不可接近性。
    2. 对于可溶性蛋白质,可以观察到超过90%的转移效率


载铜的MBD2和脂质重建的CopA的分离的典型实例显示在图2中。完整的示例性实验数据,即,蛋白质的浓度(从分光光度计读取的OD <和在转移实验之后的铜(如上所述以比色法测定)显示在参考文献1的图4中。


  1. 避免将GE Superdex分析凝胶过滤柱装入混浊样品。在样品施加之前通过离心(Eppendorf microfuge)除去沉淀
  2. 蛋白质和铜的测定是相当可靠的。然而,使用分析凝胶过滤柱,分级后的样品体积限制为足以用于两种组分的单一化学测定。来自单次转移实验的数据通常提供金属转移效率的合理数字。为了正确验证转移结果,来自至少四次色谱分离的实验数据应该是适当的。
  3. 在实际交换之前监测供体和受体蛋白质的金属含量是非常重要的对照实验。因此,我们建议单独使用供体或受体蛋白进行凝胶过滤。另外,可以以这种方式检测来自纯化的受体蛋白质中的金属携带,例如,。测量值可以用作"金属含量基线"以计算由共孵育产生的转移金属的净量。


  1. 缓冲区A
    30mM MOPS,pH7.5 150mM NaCl
  2. BCA铜缓冲液
    缓冲液A与2.5mM BCA
    1mM CuSO 4
  3. SDS溶液
    25%SDS(w/v)在H 2 O中 在37℃下制备以获得更好的溶解性
  4. 抗坏血酸盐溶液
    100mM抗坏血酸钠,pH调至7.5 以小等分试样储存在-20℃下
  5. BCS解决方案
    10mM BCS,在30mM MOPS,pH7.5中 避光




  1. Drees,SL,Beyer,DF,Lenders-Lomscher,C.和Lübben,M.(2015)。  大肠杆菌中的连续金属结合结构域的不同功能 P1 B -ATPase CopA Mol Microbiol 97 :423-438。
  2. Fan,B.,Grass,G.,Rensing,C.and Rosen,BP(2001)。  大肠杆菌 CopA N-末端Cys(X)(2)Cys基序不需要铜抗性或转运。 Biophysical Research Communications 286:414-418。
  3. Fan,B。和Rosen,BP(2002)。  生物化学表征的CopA,大肠杆菌Cu(I)转运P型ATP酶。生物化学杂志277:46987-46992。
  4. Gonzalez-Guerrero,M.,Hong,D. andArgüello,JM(2009)。  分子伴侣介导的Cu + 递送至Cu + 运输ATP酶核苷酸结合的要求。 em> 284:20804-20811。
  5. Gonzalez-Guerrero,M.,Eren,E.,Rawat,S.,Stemmler,TL和Argüello,JM(2008)。  如何测量和预测蛋白质的摩尔吸光系数。 蛋白质科学 4:2411-2423。
  6. Xiao,ZG,Brose,J.,Schimo,S.,Ackland,SM,La Fontaine,S.and Wedd,AG(2011)。  确定金属蛋白亲和力的挑战。天然产物报告 27:768-789。
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引用:Drees, S. L. and Lübben, M. (2016). Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins. Bio-protocol 6(15): e1888. DOI: 10.21769/BioProtoc.1888.

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