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).
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.
Keywords: Thiol oxidoreductases, Disulfide bonds, Oxidative folding
Materials and Reagents
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Flat-bottomed clear 96-well microplates (Optimum Line, catalog number: GP700 )
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Ribonuclease A from bovine pancreas (RNase A) (store -20 °C) (Sigma-Aldrich, catalog number: R6513-10 mg )
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Desalting columns-Biorad Econo-Pac 10DG Desalting Columns, 30 units (Bio-Rad Laboratories, catalog number: 7322010 )
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Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad Laboratories, catalog number: 7324614 )
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ENrichTM SEC 70 size exclusion columns (Bio-Rad Laboratories, catalog number: 7801070 )
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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 )
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Proteins of interest (purified to homogeneity proteins, concentration approx 7-8 mg/ml)
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Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-50TAB )
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L-Glutathione oxidized disodium salt (GSSG) (Sigma-Aldrich, catalog number: G4626-100 mg )
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L-Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G6529-1 g )
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DL-Dithiothreitol (DTT) (AppliChem GmbH, catalog number: 3483-12-3 ; Sigma-Aldrich, catalog number: 43815-1 G )
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Guanidine hydrochloride (Gdn-HCl) (AppliChem GmbH, catalog number: A11061000 )
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1 M Tris (pH 8.0) (Eurx, catalog number: E0273-01 )
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0.5 M EDTA (pH 8.0) (Eurx, catalog number: E240-01 )
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DTNB (Ellman’s Reagent) (5, 5-dithio-bis-(2-nitrobenzoic acid) (Thermo Fisher Scientific, catalog number: 22582 )
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Sodium phosphate dibasic (pH 8.0) (Sigma-Aldrich, catalog number: S3264-250 G )
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Cytidine 2’:3’-cyclic monophosphate monosodium salt (cCMP) (store -20 °C) (Sigma-Aldrich, catalog number: C9630-100 mg )
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2x Reaction buffer (see Recipes)
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Reduction buffer (see Recipes)
Equipment
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Plate reader (Tecan, Infinite®, model: 200 PRO series )
Procedure
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Preparation of proteins for assay (approx 1 week) (Chim et al., 2013)
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Purify proteins.
Note: We overexpressed proteins by autoinduction (Studier, 2005) and then purified by affinity chromatography using the NGC chromatography system (see Optional materials).
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To obtain higher purity, load your proteins onto size exclusion columns and elute with PBS.
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Determine the amount of protein by nanodrop.
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Oxidize proteins with 50 mM oxidized glutathione (GSSG) and incubate for 1 h at RT (5-7 mg in 1 ml).
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Exchange buffer on desalting columns according to manufacturer’s guidelines.
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Determine the amount of protein by nanodrop.
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Follow the Ellman assay to confirm proper redox state.
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Concentrate if necessary (using protein concentration columns). For this assay you need approx 3 mg/ml.
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Use fresh or in the next couple of days.
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Reduction and denaturation of RNase A (2 days) (Daniels et al., 2010)
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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.
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Incubate overnight at RT without shaking.
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Desalinate on columns and elute with PBS (according to manufacturer’s guidelines).
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Determine the amount of protein by nanodrop.
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If necessary, concentrate (for Ellman assay you need 2.5 mg/ml of RNase A).
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Follow the Ellman assay to confirm proper redox state.
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Use the same day.
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Oxidative folding of reduced RNase A (1 day) (Daniels et al., 2010)
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Prepare 2x reaction buffer.
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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.
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Add 100 μl of 2x reaction buffer to the mixture of protein and RNase A; mix gently by pipetting up and down.
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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
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Notes about microplate reader settings (Table 1).
Table 1. Universal settings for microplate reader

Recipes
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2x reaction buffer
200 mM Tris/HCl (pH 8.0)
4 mM EDTA
0.4 mM GSSG
2 mM GSH
9 mM cCMP
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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
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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.
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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.
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Greiner-Stoeffele, T., Grunow, M. and Hahn, U. (1996). A general ribonuclease assay using methylene blue. Anal Biochem 240(1): 24-28.
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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.
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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.
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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.
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Raines, R. T. (1998). Ribonuclease A. Chem Rev 98(3): 1045-1066.
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Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.