Measurement of Glutathione Reductase Activity

Bengt Mannervik1

1 Uppsala University, Uppsala, Sweden
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.2
DOI:  10.1002/0471140856.tx0702s00
Online Posting Date:  May, 2001
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Reduced glutathione, a thiol, is essential to the survival of the cells of most aerobic organisms. It is present intracellularly and provides protection from hydroperoxides and free radicals. This unit describes a continuous spectrophotometric assay for reductase activity: it follows the reduction of glutathione disulfide to reduced glutathione by monitoring the oxidation of NADPH as visualized by a decrease in absorbance at 340 nm.

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Table of Contents

  • Basic Protocol 1: Continuous Spectrophotometric Assay of Glutathione Reductase
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Continuous Spectrophotometric Assay of Glutathione Reductase

  • Assay buffer: 0.2 M potassium phosphate, pH 7.0/2 mM EDTA (see appendix 2A for preparation of potassium phosphate and EDTA stocks)
  • 20 mM GSSG (Boehringer or Sigma) in water, prepared fresh each day
  • 2 mM NADPH (Boehringer or Sigma) in 10 mM Tris⋅Cl, pH 7.0, prepared fresh each day and kept on ice
  • Sample to be tested for glutathione reductase activity (see )
  • Spectrophotometer (preferably a recording instrument) with thermostat
  • Spectrophotometer cuvette (transparent at 340 nm, quartz or disposable) with 1‐cm pathlength
  • Water bath (or other temperature‐controlling device)
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Literature Cited

Literature Cited
   Boggaram, V., Brobjer, T., Larson, K., and Mannervik, B. 1979. Purification of glutathione reductase from porcine erythrocytes by the use of affinity chromatography on 2′,5′‐ADP‐Sepharose 4B and crystallization of the enzyme. Anal. Biochem. 95:335‐340.
   Carlberg, I. and Mannervik, B. 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250:5475‐5480.
   Carlberg, I. and Mannervik, B. 1985. Glutathione reductase. Methods Enzymol. 113:484‐490.
   Carlberg, I. and Mannervik, B. 1986. Reduction of 2,4,6‐trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer. J. Biol. Chem. 261:1629‐1635.
   Carlberg, I., DePierre, J.W., Mannervik, B. 1981. Effect of inducers of drug‐metabolizing enzymes on gutathione reductase and glutathione peroxidase in rat liver. Biochem. Biophys. Acta. 677:140‐145.
   Josephy, P.D., Mannervik, B., and Ortiz de Montellano, P. 1997. Molecular Toxicology Oxford University Press, New York.
   Mannervik, B. and Axelsson, K. 1975. Reduction of disulphide bonds in proteins and protein mixed disulphides catalysed by a thioltransferase in rat liver cytosol. Biochem. J. 149:785‐788.
   Mannervik, B., Carlberg, I., and Larson, K. 1989. Glutathione: General review of mechanism of action. In Coenzymes and Cofactors, Vol. 3A (D. Dolphin, R. Poulson, and O. Avramovic, eds.) pp. 475‐516. John Wiley & Sons, New York.
   Williams, C.H. Jr. 1991. Lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and mercuric ion reductase—a family of flavoenzyme transhydrogenases. In Chemistry and Biochemistry of Flavoenzymes, Vol. III (F. Müller, ed.) 121‐211. CRC Press, Boca Raton, Fla.
   Worthington, D.J. and Rosemeyer, M.A. 1974. Human glutathione reductase: Purification of the crystalline enzyme from erythrocytes. Eur. J. Biochem. 48:167‐177.
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