Immuno‐Spin Trapping: Detection of Protein‐Centered Radicals

Dario C. Ramirez1, Ronald P. Mason1

1 National Institute of Environmental Health Science, National Institutes of Health, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.7
DOI:  10.1002/0471140856.tx1707s24
Online Posting Date:  June, 2005
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Protein‐centered radicals are involved in biological oxidative damage induced by drugs, environmental hazards, and cellular reactive oxygen species. Presently, the technique most widely used to study protein‐centered radicals is electron spin resonance (ESR; also known as electron paramagnetic resonance, EPR); used either directly or in combination with the spin‐trapping technique. Protein‐centered radicals may be trapped with the nitrone spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) forming DMPO‐radical adducts. However, after a few minutes these adducts decay, often by oxidation to DMPO‐protein radical–derived nitrone adducts, which are ESR‐silent species. Because nitrone adducts are not free radicals and their formation involves the creation of a covalent linkage, they are stable long after the ESR signal decays. In the new alternative technique of immuno‐spin trapping, nitrone adducts are detected by using an antibody, i.e., anti‐DMPO, that recognizes their nitrone moiety. Immuno‐spin trapping is a simple, reliable, affordable, sensitive, and specific approach to detecting protein‐centered radicals, and its development brings the power of immunoassays to bear on the field of toxicology of free radical–mediated biological damage.

Keywords: oxidative damage; protein‐centered radical; DMPO‐protein radical–derived nitrone adduct; immuno‐spin trapping

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Production and Detection of Hemoprotein‐Centered Radicals by Immuno‐Spin Trapping Elisa
  • Basic Protocol 2: Preparation and Immunoblot Analysis of DMPO‐Protein Radical–Derived Nitrone Adducts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Production and Detection of Hemoprotein‐Centered Radicals by Immuno‐Spin Trapping Elisa

  Materials
  • Metmyoglobin (metMb; USB)
  • Oxyhemoglobin (oxyHb; Apex Biochemicals)
  • H 2O 2 (Alfa Aesar)
  • Chelex‐treated sodium phosphate buffer, pH 7.7 (see recipe)
  • 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO; Alexis Biochemicals)
  • Chelex‐treated sodium phosphate buffer, pH 7.4, containing 1 mM diethylenetriaminepentaacetic acid (DTPA)
  • PD‐10 desalting columns (Amersham Biosciences)
  • Catalase solution (100 or 500 IU, Roche Applied Bioscience)
  • Coating buffer (see recipe)
  • Wash buffer (see recipe)
  • Blocking buffer (see recipe)
  • Primary antibody solution (see recipe)
  • Secondary antibody solution (see recipe)
  • Chemiluminescence substrate for ELISA (see recipe)
  • 37°C incubator with agitator
  • 96‐well white microtiter plates (e.g., Greiner Bio‐One, PGC)
  • Automated microtiter plate washer (optional)
  • Multichannel pipettor
  • Luminescence reader for microtiter plates or strips (optional)

Basic Protocol 2: Preparation and Immunoblot Analysis of DMPO‐Protein Radical–Derived Nitrone Adducts

  Materials
  • Metmyoglobin (metMb; USB)
  • H 2O 2 (Alfa Aesar)
  • Oxyhemoglobin (oxyHb; Apex Biochemicals)
  • 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO; Alexis Biochemicals)
  • Chelex‐treated sodium phosphate buffer, pH 7.4, containing 1 mM diethylenetriaminepentaacetic acid (DTPA)
  • PD‐10 desalting columns (Amersham Biosciences)
  • Catalase solution (100 or 500 IU, Roche Applied Bioscience)
  • 4× NuPage LDS sample buffer (Invitrogen)
  • 10× NuPAGE sample reducing agent (Invitrogen)
  • 4% to 12% NuPAGE Novex Bis‐Tris gel (1.0‐mm, 10‐well; Invitrogen)
  • Running buffer (see recipe)
  • Nitrocellulose membranes (0.45‐µm pore size, Invitrogen)
  • Transfer buffer (see recipe), cold
  • 0.1% (w/v) Ponceau S red in 4% (v/v) acetic acid
  • Blocking buffer (see recipe)
  • Wash buffer (see recipe)
  • Primary antibody solution (see recipe)
  • Secondary antibody solution (see recipe)
  • TBS, pH 9.6 (see recipe)
  • Chemiluminescence substrate for westernblots (see recipe)
  • 1‐Step NBT/BCIP reagent (Pierce)
  • 37°C incubator with agitator
  • 70° to 80°C water bath
  • Mini electrophoresis system (e.g., XCell SureLock Mini‐Cell system, Invitrogen)
  • Mini‐gel blot module (e.g., XCell II Blot Module, Invitrogen)
  • X‐ray film (e.g., CL‐XPosure film, Pierce)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Berlett, B.S. and Stadtman, E.R. 1997. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313‐20316.
   Chen, Y.‐R., Chen, C.‐L., Liu, X., Li, H., Zweier, J.L., and Mason, R.P. 2004. Involvement of protein radical, protein aggregation, and effects on NO metabolism in the hypochlorite‐mediated oxidation of mitochondrial cytochrome c. Free Radic. Biol. Med. 37:1591‐1603.
   Clement, J.‐L., Gilbert, B.C., Rockenbauer, A., and Tordo, P. 2001. Radical damage to proteins studied by EPR spin trapping techniques. J. Chem. Soc. Perkin Trans. 9:1463‐1470.
   Davies, M.J. and Hawkins, C.L. 2004. EPR spin trapping of protein radicals. Free Radic. Biol. Med. 36:1072‐1086.
   Dean, R.T., Fu, S., Stocker, R., and Davies, M.J. 1997. Biochemistry and pathology of radical‐mediated protein oxidation. Biochem. J. 324:1‐18.
   Deterding, L.J., Ramirez, D.C., Dubin, J.R., Mason, R.P., and Tomer, K.B. 2004. Identification of free radicals on hemoglobin from its self‐peroxidation using mass spectrometry and immuno‐spin trapping. J. Biol. Chem. 279:11600‐11607.
   Detweiler, C.D., Deterding, L.J., Tomer, K.B., Chignell, C.F., Germolec, D., and Mason, R.P. 2002. Immunological identification of the heart myoglobin radical formed by hydrogen peroxide. Free Radic. Biol. Med. 33:364‐369.
   Guo, Q., Detweiler, C.D., and Mason, R.P. 2004. Protein radical formation during lactoperoxidase‐mediated oxidation of the suicide substrate glutathione. Immunochemical detection of a lactoperoxidase radical‐derived 5,5‐dimethyl‐1‐pyrroline N‐oxide. J. Biol. Chem. 279:13272‐13283.
   Hawkins, C.L. and Davies, M.J. 2001. Generation and propagation of radical reactions on proteins. Biochim. Biophys. Acta 1504:196‐219.
   He, Y.Y., Ramirez, D.C., Detweiler, C.D., Mason, R.P., and Chignell, C.F. 2003. UVA‐ketoprofen‐induced hemoglobin radicals detected by immuno‐spin trapping. Photochem. Photobiol. 77:585‐591.
   Mason, R.P. 2000. In vivo spin trapping‐from chemistry to toxicology. In Toxicology of the Human Environment. The Critical Role of Free Radicals (C.J. Rhodes, ed.) pp. 49‐70. Taylor and Francis, London.
   Mason, R.P. 2004. Using anti‐5,5‐dimethyl‐1‐pyrroline N‐oxide (anti‐DMPO) to detect trapped protein radicals in time and space with immuno‐spin trapping. Free Radic. Biol. Med. 36:1214‐1223.
   Ramirez, D.C., Chen, Y.R., and Mason, R.P. 2003. Immunochemical detection of hemoglobin‐derived radicals formed by reaction with hydrogen peroxide: Involvement of a protein‐tyrosyl radical. Free Radic. Biol. Med. 34:830‐839.
   Ramirez, D.C., Gomez‐Mejiba, S.E., and Mason, R.P. 2005. Mechanism of hydrogen peroxide–induced Cu,Zn‐superoxide dismutase‐centered radical formation as explored by immuno‐spin trapping. The role of copper‐ and carbonate radical anion‐mediated oxidations. Free Radic. Biol. Med. 38:201‐214.
   Romero, N., Radi, R., Linares, E., Augusto, O., Detweiler, C.D., Mason, R.P., and Denicola, A. 2003. Reaction of human hemoglobin with peroxinitrite: Isomerization to nitrate and secondary formation of protein radicals. J. Biol. Chem. 278:44049‐44057.
   Tijssen, P. 1985. Practice and theory of enzyme immunoassays. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 15 (R.H. Burdon and P.H. van Knippenberg, eds.). Elsevier, New York.
Key References
   Detweiler et al., 2002. See above.
  Describes the production and validation of the anti‐DMPO antiserum and its application to the detection of DMPO‐myoglobin radical‐derived nitrone adducts.
   Mason, 2004. See above.
  Reviews all published literature relevant to immuno‐spin trapping.
   Ramirez et al., 2003. See above.
  This report describes immuno‐spin trapping and its validation for the detection of hemoglobin radical adducts in red blood cells.
Internet Resources
   http://epr.niehs.nih.gov
  Laboratory where the anti‐DMPO antiserum was developed
   https://catalog.invitrogen.com/
  Commercial supplier of gels and electrophoresis equipment
   http://www.piercenet.com
  Commercial supplier for protein staining, dialysis cassettes, and anti‐rabbit (IgG)–alkaline phosphatase conjugate
   http://www.alexis‐corp.com
  Commercial suppliers of the rabbit anti‐DMPO antiserum
   http://www.oxfordbiomed.com
   http://www.caymanchem.com
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library