Measurement of Protein Glutathionylation

Aleksandra Filipovska1, Michael P. Murphy1

1 Medical Research Council, Dunn Human Nutrition Unit, Cambridge
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.11
DOI:  10.1002/0471140856.tx0611s28
Online Posting Date:  June, 2006
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Abstract

Proteins contain free, exposed thiols that can be glutathionylated in the native state as a result of thiol‐disulfide exchange reactions with glutathione disulfide, catalyzed by glutaredoxin. A number of other reactions can also lead to protein glutathionylation. The modification of proteins by glutathionylation is important in oxidative damage and may be an important post‐translational modification to proteins involved in redox signaling. This unit describes methods for the identification of glutathionylated proteins and quantification of the extent of glutathionylation. The protocols described use isolated mitochondrial protein complexes, mitochondrial membranes, and intact mitochondria, but can be easily adapted to other systems.

Keywords: protein glutathionylation; glutathione; protein thiols; oxidative stress; redox regulation

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Qualitative Labeling of Exposed Protein Thiols Using [35S]GSSG
  • Alternate Protocol 1: Qualitative Detection of Glutathionylated Protein Thiols Using a Monoclonal Antibody
  • Alternate Protocol 2: Qualitative Labeling of Proteins and Respiratory Complexes in Intact Mitochondria Using [35S]GSH
  • Basic Protocol 2: Quantitation of Glutathione Binding to Exposed Protein Thiols Using [35S]GSSG
  • Support Protocol 1: Preparation of 12% Tris‐Glycine Page Gel for Bio‐Rad Protean III System
  • Support Protocol 2: Preparation of 5%–12% Blue Native Linear Gradient Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Qualitative Labeling of Exposed Protein Thiols Using [35S]GSSG

  Materials
  • 2 mCi/ml [35S]GSH (Perkin‐Elmer; sp. act. 700 to 1000 Ci/mmol)
  • KP i buffer (see recipe), pH 6.0 and 8.0
  • Chloroform
  • 100 µM glutathione disulfide (GSSG)
  • Argon source
  • Experimental sample: bovine heart mitochondrial membranes isolated as in Walker et al. ( ) or complex I isolated from bovine heart mitochondria as in Sazanov et al. ( )
  • 100 mM N‐ethylmaleimide (NEM) in KP i buffer, pH 6
  • Acetone, ice cold
  • Nonreducing SDS‐PAGE loading buffer (see recipe) containing 50 mM NEM
  • Nonreducing SDS‐PAGE loading buffer (see recipe) containing 100 mM DTT
  • 12.5% Tris‐glycine polyacrylamide gels ( protocol 5)
  • Tris‐glycine reservoir buffer (see recipe)
  • Coomassie stain (see recipe)
  • Destaining solution: 7.5% (v/v) acetic acid/20% (v/v) methanol
  • Dehydration solution: 2% (v/v) glycol/50% (v/v) methanol
  • Amplify fluor solution (Amersham Biosciences)
  • Boiling water bath
  • BioRad Mini Protean III PAGE system
  • Fuji Medical X‐ray film
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3F) and fluorography ( appendix 3D)

Alternate Protocol 1: Qualitative Detection of Glutathionylated Protein Thiols Using a Monoclonal Antibody

  • 5 mM glutathione disulfide (GSSG)
  • Transfer buffer (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 2% (w/v) nonfat milk powder
  • Primary antibody: anti–glutathionylated protein mouse monoclonal antibody (αGSH; ViroGen)
  • Horseradish peroxidase (HRP)–conjugated secondary antibody against mouse IgG
  • Enhanced chemiluminescence reagent (ECL; Amersham Biosciences)
  • 0.45‐µM PVDF Immobilon transfer membrane (Millipore)
  • Bio‐Rad Mini Protean Transfer Cell
  • Film cassette

Alternate Protocol 2: Qualitative Labeling of Proteins and Respiratory Complexes in Intact Mitochondria Using [35S]GSH

  Materials
  • Source of mitochondria
  • KCl buffer (see recipe)
  • 4 mg/ml rotenone (Sigma)
  • 2 mCi/ml [35S]GSH (Perkin‐Elmer; sp. act. 700 to 1000 mCi/mmol)
  • 1 M potassium succinate
  • 100 mM diamide (N,N,N′,N′‐tetramethylazodicarboxamide; Sigma) in KCl buffer
  • 100 mM N‐ethylmaleimide (NEM) in KCl buffer, pH 6.0
  • Extraction buffer (see recipe) containing 10 mM N‐ethylmaleimide (NEM)
  • 10% (w/v) dodecyl‐β‐D‐maltoside (DDM; Sigma)
  • Serva Blue loading buffer (see recipe) with and without Serva Blue
  • 100 mg/ml ferritin from equine spleen (Sigma)
  • 5%–12% blue native polyacrylamide gradient gels ( protocol 6)
  • Denaturing buffer: 0.5 M Tris⋅Cl, pH 6.8 ( appendix 2A) containing 1% (w/v) SDS (store up to 6 months at room temperature)
  • 12.5% Tris‐glycine polyacrylamide gels (see protocol 5)
  • Blue native electrophoresis buffers (see recipe)
  • Stacking gel (see protocol 5)
  • Nonreducing SDS‐PAGE loading buffer (see recipe)
  • Coomassie stain (see recipe)
  • Destaining solution: 7.5% (v/v) acetic acid/20% (v/v) methanol
  • Dehydration solution: 2% (w/v) polyethylene glycol/50% (v/v) methanol
  • Amplify fluor solution (Amersham Biosciences)
  • 30°C water bath or incubator
  • Beckman Airfuge centrifuge
  • BioRad Mini Protean III PAGE system
  • Wide gel combs (5‐well)
  • Fuji Medical X‐ray film
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3F) and fluorography ( appendix 3D)

Basic Protocol 2: Quantitation of Glutathione Binding to Exposed Protein Thiols Using [35S]GSSG

  Materials
  • 2 mCi/ml [35S]GSH (Perkin‐Elmer; sp. act. 700 to 1000 Ci/mmol)
  • KP i buffer (see recipe), pH 6.0 and 8.0
  • Chloroform
  • Samples, e.g., mitochondria, mitochondrial membrane, or complex I
  • 100 µM glutathione disulfide (GSSG)
  • Argon source
  • 100 mM N‐ethylmaleimide (NEM) in KP i buffer, pH 6
  • Acetone, ice cold
  • 1% (v/v) Triton X‐100 in H 2O
  • Scintillant (e.g., Scintran FluoranSafe 2, BDH)
  • Scintillation vials
  • Additional reagents and equipment for bicinchoninic acid protein assay ( appendix 3I)

Support Protocol 1: Preparation of 12% Tris‐Glycine Page Gel for Bio‐Rad Protean III System

  Materials
  • 37.5:1 acrylamide/bisacrylamide (store at 4°C)
  • 3 M Tris⋅Cl, pH 8.8 ( appendix 2A; filter and store up to 3 months at room temperature)
  • 1.5% (w/v) ammonium persulfate (store frozen in aliquots; thawed aliquots should be stable for several days at 4°C)
  • TEMED (e.g., Sigma or Invitrogen; store at 4°C)
  • 10% (w/v) SDS
  • n‐butanol
  • 0.5 M Tris⋅Cl, pH 6.8 ( appendix 2A; filter and store up to 3 months at room temperature)
  • Bio‐Rad Mini Protean III system or appropriate spacers, plates, well combs, casting frame, and casting stand
  • 15‐ml conical polypropylene centrifuge tubes
  • Filter paper

Support Protocol 2: Preparation of 5%–12% Blue Native Linear Gradient Gel

  Materials
  • Acrylamide mix: combine 48 g acrylamide and 1.5 g bisacrylamide in 100 ml H 2O (store up to 3 months at 4°C)
  • 3× gel buffer: 1.5 M aminocaproic acid in 150 mM Bis‐Tris, pH 7.0 (adjust pH of Bis‐Tris buffer at 4°C with HCl)
  • Glycerol
  • 10% (w/v) ammonium persulfate
  • TEMED (e.g., Invitrogen or Sigma)
  • Butanol
  • Bio‐Rad Mini Protean III system with 1‐mm gel spacers
  • Gradient mixer
  • Filter paper
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Figures

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Literature Cited

Literature Cited
   Beer, S.M., Taylor, E.R., Brown, S.E., Dahm, C.C., Costa, N.J., Runswick, M.J., and Murphy, M.P. 2004. Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: Implications for mitochondrial redox regulation and antioxidant defense. J. Biol. Chem. 279:47939‐47951.
   Brookes, P.S., Pinner, A., Ramachandran., A., Coward, L., Barnes, S., Kim, H., and Darley‐Usmar, V.M. 2002. High throughput two‐dimensional blue‐native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2:969‐977.
   Chappell, J.B. and Hansford, R.G. 1972. Preparation of mitochondria from animal tissues and yeasts. In Subcellular Components: Preparation and Fractionation (G.D. Birnie, ed.) pp. 77‐91. Butterworths, London.
   Demasi, M., Shringarpure, R., and Davies, K.J. 2001. Glutathiolation of the proteasome is enhanced by proteolytic inhibitors. Arch. Biochem. Biophys. 389:254‐263.
   Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Meng ozzi, M., Duffieux. F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. 2002. Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 99:3505‐3510.
   Fratelli, M., Demol, H., Puype, M., Casagrande, S., Villa, P., Eberini, I., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. 2003. Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells. Proteomics 3:1154‐1161.
   Hurd, T.R., Costa, N.J., Dahm, C.C., Beer, S.M., Brown, S.E., Filipovska, A., and Murphy, M.P. 2005. Glutathionylation of mitochondrial proteins. Antioxid. Redox. Signal. 7:999‐1010.
   Sazanov, L.A., Peak‐Chew, S.Y., Fearnley, I.M., and Walker, J.E. 2000. Resolution of the membrane domain of bovine complex I into subcomplexes: Implications for the structural organization of the enzyme. Biochemistry 39:7229‐35.
   Scarlett, J.L., Packer, M.A., Porteous, C.M., and Murphy, M.P. 1996. Alterations to glutathione and nicotinamide nucleotides during the mitochondrial permeability transition induced by peroxymitrite. Biochem. Pharmacol. 52:1047‐1055.
   Schagger, H. 1995. Native electrophoresis for isolation of mitochondrial oxidative phosphorylation complexes. Methods Enzymol. 260:190‐202.
   Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fu jimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76‐85.
   Sullivan, D.M., Wehr, N.B., Fergusson, M.M., Levine, R.L., and Finkel, T. 2000. Identification of oxidant‐sensitive proteins: TNF‐alpha induces protein glutathiolation. Biochemistry 39:11121‐11128.
   Taylor, E.R., Hurrell, F., Shannon, R.J., Lin, T.K., Hirst, J., and Murphy, M.P. 2003. Reversible glutathionylation of complex I increases mitochondrial superoxide formation. J. Biol. Chem. 278:19603‐19610.
   Tyler, D.D. and Gonze, J. 1967. The preparation of heart mitochondria from laboratory animals. Meth. Enzymol. 10:75‐78.
   Walker, J.E., Skehel, J.M., and Buchanan, S.K. 1995. Structural analysis of NADH: Ubiquinone oxidoreductase from bovine heart mitochondria. Methods Enzymol. 260:14‐34.
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