Measurement of Thioredoxin and Thioredoxin Reductase

Elias S.J. Arnér1, Arne Holmgren1

1 Karolinska Institutet, Stockholm, Sweden
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.4
DOI:  10.1002/0471140856.tx0704s05
Online Posting Date:  May, 2001
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Abstract

The thioredoxin system is ubiquitous, providing reducing equivalents to essential biosynthetic enzymes like ribonucleotide reductase. It is essential for cellular redox regulation, control of oxidative stress, and protection against oxidative damage. This unit includes protocols for measuring thioredoxin or thioredoxin reductase in biological preparations or as purified enzymes.

     
 
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Table of Contents

  • Basic Protocol 1: Spectrophotometric Insulin Assay for Measurement of Enzymatic Activity of Purified Thioredoxins or Thioredoxin Reductases
  • Alternate Protocol 1: End‐Point Insulin Assay for Measurement of Thioredoxin or Thioredoxin Reductase Enzymatic Activity in Biological Samples
  • Basic Protocol 2: Measuring Mammalian‐Type Thioredoxin Reductase
  • Basic Protocol 3: Thioredoxin‐Catalyzed Insulin Reduction by DTT
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spectrophotometric Insulin Assay for Measurement of Enzymatic Activity of Purified Thioredoxins or Thioredoxin Reductases

  Materials
  • 1.6 mM insulin (see recipe) in Tris⋅Cl, pH 7.5
  • 50 mM NADPH (see recipe)
  • TE buffer, pH 7.5: 50 mM Tris⋅Cl, pH 7.5 ( appendix 2A) containing 2 mM EDTA
  • Pure control thioredoxin reductase (TrxR; IMCO)
  • Pure control thioredoxin (Trx; IMCO)
  • Protein sample to be assayed for Trx or TrxR activity
  • Double‐beam spectrophotometer with semimicro quartz cuvettes
NOTE: Wild‐type human Trx has a pronounced tendency to loose activity and aggregate due to oxidation of structural cysteine residues upon storage and freezing‐thawing in air‐containing buffers. This affects the assay and can be overcome by reducing the Trx sample with DTT. The E. coli and mutant human C62S/C73S Trx are however preferred for the assay since these thioredoxins do not have the tendency to aggregate due to oxidation and are stable upon storage.

Alternate Protocol 1: End‐Point Insulin Assay for Measurement of Thioredoxin or Thioredoxin Reductase Enzymatic Activity in Biological Samples

  • 200 mM HEPES buffer, pH 7.6
  • 0.2 M EDTA ( appendix 2A)
  • Protein sample to be analyzed, e.g., from cell or tissue extracts
  • DTNB/guanidine solution (see recipe)

Basic Protocol 2: Measuring Mammalian‐Type Thioredoxin Reductase

  Materials
  • 62.5 mM (25 mg/ml) DTNB in spectroscopic grade 99% ethanol
  • 50 mM NADPH (see recipe)
  • PE buffer, pH 7.0: 100 mM potassium phosphate, pH 7.0 ( appendix 2A) containing 2 mM EDTA
  • 20 mg/ml bovine serum albumin (BSA) in water
  • Pure control mammalian TrxR
  • TrxR sample to be analyzed
  • Double‐beam spectrophotometer with semimicro quartz cuvettes
NOTE: Inclusion of albumin in the assay protects TrxR from absorbing to the plastic surface of the vial; the disulfides of albumin are buried internally and are not reduced by the enzyme, and thereby do not interfere with the assay. The standard assay is performed at room temperature (20°C).

Basic Protocol 3: Thioredoxin‐Catalyzed Insulin Reduction by DTT

  Materials
  • 1.6 mM insulin (see recipe) in 100 mM potassium phosphate buffer, pH 6.5
  • PE buffer, pH 6.5: 100 mM potassium phosphate, pH 6.5 containing 2 mM EDTA
  • 100 mM dithiothreitol (DTT; appendix 2A), freshly made in water
  • Sample: purified Trx to be analyzed
  • Double‐beam spectrophotometer with semimicro quartz cuvettes
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Figures

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Literature Cited

Literature Cited
   Arnér, E.S.J., Zhong, L., and Holmgren, A. 1999a. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. Methods Enzymol. 300:226‐239.
   Arnér, E.S.J., Sarioglu, H., Lottspeich, F., Holmgren, A., and Böck, A. 1999b. High‐level expression in Escherichia coli of selenocysteine‐containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial‐type SECIS elements and co‐expression with the selA, selB and selC genes. J. Mol. Biol. 292:1003‐1016.
   Böck, A., Forchhammer, K., Heider, J., Leinfelder, W., Sawers, G., Veprek, B., and Zinoni, F. 1991. Selenocysteine: The 21st amino acid. Mol. Microbiol. 5:515‐520.
   Dai, S., Saarinen, M., Ramaswamy, S., Meyer, Y., Jacquot, J.P., and. Eklund, H. 1996. Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 Å resolution. J. Mol. Biol. 264:1044‐1057.
   Gasdaska, P.Y., Gasdaska, J.R., Cochran, S., and Powis, G. 1995. Cloning and sequencing of a human thioredoxin reductase. FEBS Lett. 375:5‐9
   Gladyshev, V.N., Jeang, K.‐T., and Stadtman, T.C. 1996. Selenocysteine, identified as the penultimate C‐terminal residue in human T‐cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. U.S.A. 93:6146‐6151.
   Gladyshev, V. N., Krause, K., Xu, X.‐M., Korotkov, K.V., Kryukov, G.V., Sun, Q.‐A., Lee, B.J., Wootton, J.C., and Hatfield, D.L. 1999. Selenocysteine‐containing thioredoxin reductase in C. elegans. Biochem. Biophys. Res. Commun. 259:244‐249
   Hill, K.E., McCollum, G.W., and Burk, R.F. 1997. Determination of thioredoxin reductase activity in rat liver supernatant. Anal. Biochem. 253:123‐125
   Holmgren, A. 1977. Bovine thioredoxin system: Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. J. Biol. Chem. 252:4600‐4006.
   Holmgren, A. 1979a. Reduction of disulfides by thioredoxin: Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J. Biol. Chem. 254:9113‐9119.
   Holmgren, A. 1979b. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J. Biol. Chem. 254:9627‐9632.
   Holmgren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54:237‐271.
   Holmgren, A. 1989. Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264:13963‐13966.
   Holmgren, A. 1995. Thioredoxin structure and mechanism: Conformational changes on oxidation of the active‐site sulfhydryls to a disulfide. Structure 3:239‐243.
   Holmgren, A. and Björnstedt, M. 1995. Thioredoxin and thioredoxin reductase. Methods Enzymol. 252:199‐208.
   Kuriyan, J., Krishna, T.S.R., Wong, L., Guenther, B., Pahler, A., Williams, C.H., Jr., and Model, P. 1991. Convergent evolution of similar function in two structurally divergent enzymes. Nature 352:172‐174.
   Low, S.C. and Berry, M.J. 1996. Knowing when not to stop: Selenocysteine incorporation in eukaryotes. Trends Biochem. Sci. 21:203‐208.
   Luthman, M. and Holmgren, A. 1982. Rat liver thioredoxin and thioredoxin reductase: Purification and characterization. Biochemistry 21:6628‐6633.
   Miranda‐Vizuete, A., Damdimopoulos, A.E., Pedrajas, J.R., Gustafsson, J.A., and Spyrou, G. 1999. Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur. J. Biochem. 261:405‐412.
   Nordberg, J., Zhong, L., Holmgren, A., and Arnér, E.S.J. 1998. Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue. J. Biol. Chem. 273:10835‐10842.
   Pedrajas, J.R., Kosmidou, E., Miranda‐Vizuete, A., Gustafsson, J.Å, Wright, A.P., and Spyrou, G. 1999. Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J. Biol. Chem. 274:6366‐6373.
   Powis, G., Briehl, M., and Oblong, J. 1995. Redox signalling and the control of cell growth and death. Pharmacol. Ther. 68:149‐173.
   Sun, Q.A., Wu, Y., Zappacosta, F., Jeang, K.T., Lee, B.J., Hatfield, D.L., and Gladyshev, V.N. 1999. Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J. Biol. Chem. 274:24522‐24530.
   Tamura, T. and Stadtman, T.C. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity. Proc. Natl. Acad. Sci. U.S.A. 93:1006‐1011.
   Tormay, P. and Böck, A. 1997. Barriers to heterologous expression of a selenoprotein gene in bacteria. J. Bacteriol. 179:576‐582.
   Waksman, G., Krishna, T.S., Williams, C.H., Jr., and Kuriyan, J. 1994. Crystal structure of Escherichia coli thioredoxin reductase refined at 2 Å resolution: Implications for a large conformational change during catalysis. J. Mol. Biol. 236:800‐816.
   Watabe, S., Makino, Y., Ogawa, K., Hiroi, T., Yamamoto, Y., and Takahashi, S.Y. 1999. Mitochondrial thioredoxin reductase in bovine adrenal cortex: Its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine. Eur. J. Biochem. 264:74‐84.
   Williams, C.H., Jr. 1992. Lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and mercuric ion reductase—a family of flavoenzyme transhydrogenases. In Chemistry and Biochemistry of Flavoenzymes (F. Müller ed.) pp. 121‐211. CRC Press, Boca Raton, Fla.
   Yodoi, J. and Tursz, T. 1991. ADF, a growth‐promoting factor derived from adult T cell leukemia and homologous to thioredoxin: Involvement in lymphocyte immortalization by HTLV‐I and EBV. Adv. Cancer Res. 57:381‐411.
   Zhong, L., Arnér, E.S.J., Ljung, J., Åslund, F., and Holmgren, A. 1998. Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl‐terminal elongation containing a conserved catalytically active penultimate selenocysteine residue. J. Biol. Chem. 273:8581‐8591.
Key References
   Arnér et al., 1999b. See above.
  Describes the purification procedure for mammalian thioredoxin and thioredoxin reductase and lists known substrates and inhibitors of mammalian thioredoxin reductase; also describes how to assay for novel substances interacting with mammalian TrxR.
   Holmgren 1977, See above.
  These papers describe in detail the first characterizations of the reduction of insulin by Trx, both coupled to TrxR and to DTT, and the reduction of DTNB by mammalian TrxR.
Internet Resources
   http://www.imcocorp.se
  The web site of IMCO, Corporation Ltd. AB, Sweden, a vendor of pure control bacterial and mammalian Trx and TrxR.
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