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Detection of Protein Oxidative Activity Using Reduced RNase A
使用还原性核糖核酸酶A检测蛋白质的氧化活性   

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Abstract

This assay allows to determine whether proteins possess oxidative activity-the ability to introduce disulfide bond in vitro. The substrate for potential oxidases is a ribonuclease A which, for its activity, needs 4 properly formed disulfide bonds (Raines, 1998).
RNase A activity can be detected by:

Keywords: Thiol oxidoreductases(硫醇的氧化还原酶), Disulfide bonds(二硫键), Oxidative folding(氧化折叠)

  1. Monitoring the digestion of RNA (Lambert and Freedman, 1983);
  2. Methylene Blue assay (Greiner-Stoeffele et al., 1996);
  3. Analyzing the cleavage of the cyclic CMP (Lyles and Gilbert, 1991; Lyles and Gilbert, 1991).

We here describe method for measurements of oxidative activity, based on the cleavage of cCMP. Oxidative activity will be tested by measuring spectrophotometrically RNase A cleavage of cyclic-2’, 3’-cytidinemonophosphate (cCMP) to 3’-cytidinemonophosphate (3’ CMP), which results in an increase in absorption at 296 nm.
The reaction equation: RNase A +2’ 3’-cCMP→RNase A + 3’ CMP.

Materials and Reagents

  1. Flat-bottomed clear 96-well microplates (Optimum Line, catalog number: GP700 )
  2. Ribonuclease A from bovine pancreas (RNase A) (store -20 °C) (Sigma-Aldrich, catalog number: R6513-10 mg )
  3. Desalting columns-Biorad Econo-Pac 10DG Desalting Columns, 30 units (Bio-Rad Laboratories, catalog number: 7322010 )
  4. Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad Laboratories, catalog number: 7324614 )
  5. ENrichTM SEC 70 size exclusion columns (Bio-Rad Laboratories, catalog number: 7801070 )
  6. PierceTM Protein Concentrators, 9K MWCO (7 ml) (Thermo Fisher Scientific, catalog number: 89884A ) or Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore Corporation, catalog number: UFC801008 )
  7. Proteins of interest (purified to homogeneity proteins, concentration approx 7-8 mg/ml)
  8. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-50TAB )
  9. L-Glutathione oxidized disodium salt (GSSG) (Sigma-Aldrich, catalog number: G4626-100 mg )
  10. L-Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G6529-1 g )
  11. DL-Dithiothreitol (DTT) (AppliChem GmbH, catalog number: 3483-12-3 ; Sigma-Aldrich, catalog number: 43815-1 G )
  12. Guanidine hydrochloride (Gdn-HCl) (AppliChem GmbH, catalog number: A11061000 )
  13. 1 M Tris (pH 8.0) (Eurx, catalog number: E0273-01 )
  14. 0.5 M EDTA (pH 8.0) (Eurx, catalog number: E240-01 )
  15. DTNB (Ellman’s Reagent) (5, 5-dithio-bis-(2-nitrobenzoic acid) (Thermo Fisher Scientific, catalog number: 22582 )
  16. Sodium phosphate dibasic (pH 8.0) (Sigma-Aldrich, catalog number: S3264-250 G )
  17. Cytidine 2’:3’-cyclic monophosphate monosodium salt (cCMP) (store -20 °C) (Sigma-Aldrich, catalog number: C9630-100 mg )
  18. 2x Reaction buffer (see Recipes)
  19. Reduction buffer (see Recipes)

Equipment

  1. Plate reader (Tecan, Infinite®, model: 200 PRO series )

Procedure

  1. Preparation of proteins for assay (approx 1 week) (Chim et al., 2013)
    1. Purify proteins.
      Note: We overexpressed proteins by autoinduction (Studier, 2005) and then purified by affinity chromatography using the NGC chromatography system (see Optional materials).
    2. To obtain higher purity, load your proteins onto size exclusion columns and elute with PBS.
    3. Determine the amount of protein by nanodrop.
    4. Oxidize proteins with 50 mM oxidized glutathione (GSSG) and incubate for 1 h at RT (5-7 mg in 1 ml).
    5. Exchange buffer on desalting columns according to manufacturer’s guidelines.
    6. Determine the amount of protein by nanodrop.
    7. Follow the Ellman assay to confirm proper redox state.
    8. Concentrate if necessary (using protein concentration columns). For this assay you need approx 3 mg/ml.
    9. Use fresh or in the next couple of days.

  2. Reduction and denaturation of RNase A (2 days) (Daniels et al., 2010)
    1. Resuspend 10 mg of RNase A in 1 ml of reduction buffer (in 1.5 ml tube).
      Note: 10 mg of RNase A permit to perform approx. 20 assays.
    2. Incubate overnight at RT without shaking.
    3. Desalinate on columns and elute with PBS (according to manufacturer’s guidelines).
    4. Determine the amount of protein by nanodrop.
    5. If necessary, concentrate (for Ellman assay you need 2.5 mg/ml of RNase A).
    6. Follow the Ellman assay to confirm proper redox state.
    7. Use the same day.

  3. Oxidative folding of reduced RNase A (1 day) (Daniels et al., 2010)
    1. Prepare 2x reaction buffer.
    2. Prepare mixture of protein and reduced RNase A in a volume of 100 μl (add PBS if necessary) in a 96-well plate (40 μM protein and 20 μM RNase A).
      Note: With concentration of 2.5 mg/ml you need to add 11 μl to the well to achieve 20 μM of RNase A.
    3. Add 100 μl of 2x reaction buffer to the mixture of protein and RNase A; mix gently by pipetting up and down.
    4. Insert plate into the plate reader and start program.

Representative data


Figure 1. The results from one representative experiment. EcDsbA stands for main oxidase in E. coli.

Notes

  1. Notes about microplate reader settings (Table 1).

Table 1. Universal settings for microplate reader

Recipes

  1. 2x reaction buffer
    200 mM Tris/HCl (pH 8.0)
    4 mM EDTA
    0.4 mM GSSG
    2 mM GSH
    9 mM cCMP
  2. Reduction buffer
    100 mM Tris/HCl (pH 8.0)
    6 M Gdn-HCl
    140 mM DTT

Acknowledgments

The work was supported by the National Science Centre (grant No. 2012/05/B/NZ1/00039).

References

  1. Chim, N., Harmston, C. A., Guzman, D. J. and Goulding, C. W. (2013). Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis. BMC Struct Biol 13: 23.
  2. Daniels, R., Mellroth, P., Bernsel, A., Neiers, F., Normark, S., von Heijne, G. and Henriques-Normark, B. (2010). Disulfide bond formation and cysteine exclusion in gram-positive bacteria. J Biol Chem 285(5): 3300-3309.
  3. Greiner-Stoeffele, T., Grunow, M. and Hahn, U. (1996). A general ribonuclease assay using methylene blue. Anal Biochem 240(1): 24-28.
  4. Lambert, N. and Freedman, R. B. (1983). Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction. Biochem J 213(1): 235-243.
  5. Lyles, M. M. and Gilbert, H. F. (1991). Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry 30(3): 613-619.
  6. Lyles, M. M. and Gilbert, H. F. (1991). Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase. Biochemistry 30(3): 619-625.
  7. Raines, R. T. (1998). Ribonuclease A. Chem Rev 98(3): 1045-1066.
  8. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234.

简介

该测定允许确定蛋白质是否具有氧化活性 - 在体外引入二硫键的能力。 潜在氧化酶的底物是核糖核酸酶A,其活性需要4个适当形成的二硫键(Raines,1998)。
核糖核酸酶A活性可通过以下检测:

关键字:硫醇的氧化还原酶, 二硫键, 氧化折叠

  1. 监测RNA的消化(Lambert和Freedman,1983);
  2. 亚甲基蓝测定法(Greiner-Stoeffele等人,1996);
  3. 分析环状CMP的裂解(Lyles和Gilbert,1991; Lyles和Gilbert,1991)。

我们在这里描述了基于cCMP裂解的氧化活性测量方法。氧化活性将通过测量分光光度计RNase A将环状2',3'-胞苷一磷酸(cCMP)切割成3'-胞苷一磷酸(3'CMP)来测试,其导致在296nm处的吸收增加。 反应方程式:RNase A + 2'3'-cCMP→RNase A + 3'CMP。

材料和试剂

  1. 平底透明96孔微孔板(Optimum Line,目录号:GP700)
  2. 来自牛胰腺的核糖核酸酶A(RNase A)(存储-20℃)(Sigma-Aldrich,目录号:R6513-10mg)
  3. 脱盐柱 - Biorad Econo-Pac 10DG脱盐柱,30单位(Bio-Rad Laboratories,目录号:7322010)
  4. Bio-Scale Mini Profinity IMAC Cartridges(Bio-Rad Laboratories,目录号:7324614)
  5. ENrichSup 70 SEC尺寸排阻柱(Bio-Rad Laboratories,目录号:7801070)。
  6. (Thermo Fisher Scientific,目录号:89884A)或具有Ultracel-10膜的Amicon Ultra-4离心过滤器单元(EMD Millipore Corporation,目录号:UFC801008)的9k MWCO(7ml) )
  7. 感兴趣的蛋白质(纯化至同质性蛋白质,浓度约7-8mg/ml)
  8. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417-50TAB)
  9. L-谷胱甘肽氧化二钠盐(GSSG)(Sigma-Aldrich,目录号:G4626-100mg)
  10. L-谷胱甘肽还原(GSH)(Sigma-Aldrich,目录号:G6529-1g)
  11. DL二硫苏糖醇(DTT)(AppliChem GmbH,目录号:3483-12-3; Sigma-Aldrich,目录号:43815-1G)
  12. 盐酸胍(Gdn-HCl)(AppliChem GmbH,目录号:A11061000)
  13. 1M Tris(pH8.0)(Eurx,目录号:E0273-01)
  14. 0.5M EDTA(pH8.0)(Eurx,目录号:E240-01)
  15. DTNB(Ellman's Reagent)(5,5-二硫代 - 双 - (2-硝基苯甲酸)(Thermo Fisher Scientific,目录号:22582)
  16. 磷酸氢二钠(pH8.0)(Sigma-Aldrich,目录号:S3264-250G)
  17. 胞苷2':3'-环单磷酸单钠盐(cCMP)(存储-20℃)(Sigma-Aldrich,目录号:C9630-100mg)
  18. 2x反应缓冲液(参见配方)
  19. 还原缓冲区(请参阅配方)

设备

  1. 读板器(Tecan,Infinite ,型号:200 PRO系列)

程序

  1. 用于测定的蛋白质的制备(约1周)(Chim等人,2013)
    1. 净化蛋白质。
      注意:我们通过自身诱导过表达蛋白质(Studier,2005),然后通过使用NGC层析系统的亲和层析纯化(参见任选材料)。
    2. 为了获得更高的纯度,将您的蛋白质加载到尺寸排阻柱上,并用PBS洗脱
    3. 通过nanodrop确定蛋白质的量。
    4. 用50mM氧化型谷胱甘肽(GSSG)氧化蛋白质,并在室温下孵育1小时(5-7mg在1ml中)。
    5. 根据制造商的指南,在脱盐柱上交换缓冲液。
    6. 通过nanodrop确定蛋白质的量。
    7. 按照Ellman测定法确认正确的氧化还原状态
    8. 如有必要,浓缩(使用蛋白质浓缩柱)。对于此测定,您需要约3 mg/ml
    9. 使用新鲜或未来几天。

  2. RNase A的还原和变性(2天)(Daniels et al。,2010)
    1. 重悬10毫克RNase A在1毫升还原缓冲液(在1.5毫升管中) 注意:10 mg核糖核酸酶A允许执行约。 20分析。
    2. 在室温下孵育过夜,不摇动
    3. 在柱上脱盐并用PBS洗脱(根据制造商的指导)
    4. 通过nanodrop测定蛋白质的量。
    5. 如果需要,浓缩(对于Ellman测定,需要2.5mg/ml的核糖核酸酶A)
    6. 按照Ellman测定法确认正确的氧化还原状态
    7. 使用同一天。

  3. 还原型核糖核酸酶A的氧化折叠(1天)(Daniels等人,2010)
    1. 制备2x反应缓冲液
    2. 准备蛋白质和的混合物 减少的RNA酶A体积为100μl(如有必要,加入PBS) 96孔板(40μM蛋白质和20μMRNA酶A) 注意:浓度为2.5 mg/ml时,您需要向孔中加入11μl,以达到20μM的RNA酶A.
    3. 向蛋白质和RNA酶A的混合物中加入100μl的2x反应缓冲液;通过上下吹吸轻轻混匀。
    4. 将板插入读板器并启动程序。

代表数据


图1.一个代表性实验的结果。 EcDsbA代表em中的主要氧化酶。大肠杆菌。

笔记

  1. 有关酶标仪设置的注意事项(表1)。

表1.微孔板读取器的通用设置

食谱

  1. 2x反应缓冲液
    200mM Tris/HCl(pH8.0) 4mM EDTA
    0.4 mM GSSG
    2 mM GSH
    9 mM cCMP
  2. 减少缓冲区
    100mM Tris/HCl(pH8.0)
    6 M Gdn-HCl
    140 mM DTT

致谢

这项工作得到了国家科学中心的支持(授权号2012/05/B/NZ1/00039)。

参考文献

  1. Chim,N.,Harmston,C.A.,Guzman,D.J。和Goulding,C.W。(2013)。 来自结核分枝杆菌的基本DsbA样二硫键形成蛋白的结构和生物化学特征/em>。 BMC Struct Biol 13:23.
  2. Daniels,R.,Mellroth,P.,Bernsel,A.,Neiers,F.,Normark,S.,von Heijne,G.and Henriques-Normark,B。 革兰氏阳性菌中的二硫键形成和半胱氨酸排除。 Chem 285(5):3300-3309。
  3. Greiner-Stoeffele,T.,Grunow,M。和Hahn,U.(1996)。 使用亚甲蓝的一般核糖核酸酶测定法 Anal Biochem 240(1):24-28
  4. Lambert,N。和Freedman,R.B。(1983)。 均相蛋白质二硫键异构酶在蛋白质二硫键异构化和硫醇 - 蛋白质 - 二硫键氧化还原中的动力学和特异性。 Biochem J 213(1):235-243。
  5. Lyles,M.M。和Gilbert,H.F。(1991)。 由蛋白质二硫键异构酶催化核糖核酸酶A的氧化折叠:速率对组成的依赖性氧化还原缓冲液。生物化学 30(3):613-619。
  6. Lyles,M.M。和Gilbert,H.F。(1991)。 由蛋白质二硫键异构酶催化核糖核酸酶A的氧化折叠:预稳态动力学和利用异构酶的氧化当量。生物化学 30(3):619-625。
  7. Raines,R.T。(1998)。 Ribonuclease A. Chem Rev 98(3): 1045-1066。
  8. Studier,F.W。(2005)。 在高密度摇动培养中通过自动诱导产生蛋白质。蛋白质Purif 41(1):207-234。

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引用:Grzeszczuk, M., Bocian-Ostrzycka, K., Lasica, A. and Jagusztyn-Krynicka, E. K. (2016). Detection of Protein Oxidative Activity Using Reduced RNase A. Bio-protocol 6(6): e1766. DOI: 10.21769/BioProtoc.1766.
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