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
发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1766 浏览次数: 6982
评审: Valentine V TrotterKristin ShinglerAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2015
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:
- Monitoring the digestion of RNA (Lambert and Freedman, 1983);
- Methylene Blue assay (Greiner-Stoeffele et al., 1996);
- Analyzing the cleavage of the cyclic CMP (Lyles and Gilbert, 1991; Lyles and Gilbert, 1991).
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.
Keywords: Thiol oxidoreductases (硫醇的氧化还原酶)
Disulfide bonds (二硫键)
Oxidative folding (氧化折叠)
Materials and Reagents
- Flat-bottomed clear 96-well microplates (Optimum Line, catalog number: GP700 )
- Ribonuclease A from bovine pancreas (RNase A) (store -20 °C) (Sigma-Aldrich, catalog number: R6513-10 mg )
- Desalting columns-Biorad Econo-Pac 10DG Desalting Columns, 30 units (Bio-Rad Laboratories, catalog number: 7322010 )
- Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad Laboratories, catalog number: 7324614 )
- ENrichTM SEC 70 size exclusion columns (Bio-Rad Laboratories, catalog number: 7801070 )
- 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 )
- Proteins of interest (purified to homogeneity proteins, concentration approx 7-8 mg/ml)
- Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-50TAB )
- L-Glutathione oxidized disodium salt (GSSG) (Sigma-Aldrich, catalog number: G4626-100 mg )
- L-Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G6529-1 g )
- DL-Dithiothreitol (DTT) (AppliChem GmbH, catalog number: 3483-12-3 ; Sigma-Aldrich, catalog number: 43815-1 G )
- Guanidine hydrochloride (Gdn-HCl) (AppliChem GmbH, catalog number: A11061000 )
- 1 M Tris (pH 8.0) (Eurx, catalog number: E0273-01 )
- 0.5 M EDTA (pH 8.0) (Eurx, catalog number: E240-01 )
- DTNB (Ellman’s Reagent) (5, 5-dithio-bis-(2-nitrobenzoic acid) (Thermo Fisher Scientific, catalog number: 22582 )
- Sodium phosphate dibasic (pH 8.0) (Sigma-Aldrich, catalog number: S3264-250 G )
- Cytidine 2’:3’-cyclic monophosphate monosodium salt (cCMP) (store -20 °C) (Sigma-Aldrich, catalog number: C9630-100 mg )
- 2x Reaction buffer (see Recipes)
- Reduction buffer (see Recipes)
Equipment
- Plate reader (Tecan, Infinite®, model: 200 PRO series )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 活性
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