Analysis of Glutathione‐Related Enzymes

R.S. Esworthy1, F.‐F. Chu1, J.H. Doroshow1

1 City of Hope National Medical Center, Duarte, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.1
DOI:  10.1002/0471140856.tx0701s00
Online Posting Date:  May, 2001
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Glutathione peroxidase activity can be measured using a variety of substrates to define total glutathione peroxidase activity or the activity of subsets of the family of enzymes. The assays for these activities are spectrophotometric assays, and the specificity depends on the choice of substrate and/or prior treatment of the samples by chromatography or immunoprecipitation. This unit provides the assays and support protocols for synthesizing substrates and calculating specific activity.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Measurement of Glutathione Peroxidase Activity
  • Alternate Protocol 1: Assay of GSTs with GPX Activity and SeGPX with Cumene Hydroperoxide
  • Alternate Protocol 2: Assay of GPX4 (PHGPX) with Phosphatidyl Choline Hydroperoxide
  • Support Protocol 1: Synthesis of Phosphatidyl Choline Hydroperoxide (PLOOH) for the Assay of PHGPX
  • Alternate Protocol 3: Inclusive GPX Assay with Universal Substrates
  • Support Protocol 2: Synthesis and Isolation of Linoleic Acid Hydroperoxide for the Assay of GPX
  • Alternate Protocol 4: Assay of SeGPX and GST in Mixtures (Dual Assays)
  • Alternate Protocol 5: Assay of GPX1 and GPX4
  • Alternate Protocol 6: Chromatographic Separation and Assay of GPX Isoenzymes
  • Alternate Protocol 7: Assay of GPX1 and GPX2 After Selective Immunoprecipitation
  • Alternate Protocol 8: The Ferricyanate Method of Hydroperoxide Determination to Assay Glutathione Peroxidase
  • Support Protocol 3: Calculations of Specific Activity
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Measurement of Glutathione Peroxidase Activity

  • Samples for analysis of glutathione peroxidase (GPX) activity
  • GPX‐positive control (for first‐time investigations; see recipe)
  • 10 mM GSH (freshly prepared; see recipe)
  • 2 mM β‐NADPH (freshly prepared; see recipe)
  • 5 mM H 2O 2 (see recipe)
  • 100 U/ml glutathione reductase (see recipe)
  • Assay buffer: 50 mM sodium phosphate, pH 7.0 ( appendix 2A)
  • 1.125 M sodium azide (see recipe)
  • Recording spectrophotometer with lamp and filters for 340‐nm operation
  • 1‐ml near‐uv‐rated plastic cuvettes with 1‐cm path (the assay can be easily scaled to smaller or larger volumes, if desired)

Alternate Protocol 1: Assay of GSTs with GPX Activity and SeGPX with Cumene Hydroperoxide

  • 30 mM cumene hydroperoxide (see recipe)

Alternate Protocol 2: Assay of GPX4 (PHGPX) with Phosphatidyl Choline Hydroperoxide

  • Assay mix for GPX4 (see recipe)
  • 100 U/µl glutathione reductase (see recipe) in ice‐cold 0.4 M Tris⋅Cl, pH 7.4
  • 10% (v/v) peroxide‐free Triton X‐100 (Sigma, Aldrich, Boehringer Mannheim) in H 2O (keep on ice)
  • Phosphatidyl hydroperoxide (PLOOH) substrate in methanol (see protocol 4; concentration such that the substrate can be added to a final concentration of 20 to 30 µM in the assay mix by adding a 20‐ to 25‐µl aliquot of the methanol solution)
  • GPX4 positive control (see recipe)
  • Negative control: GPX1 standard free of GPX4 (optional, but advisable for the novice; see recipe)
  • 3‐ to 4‐ml near‐UV‐rated plastic cuvettes with 1‐cm path for spectrophotometer

Support Protocol 1: Synthesis of Phosphatidyl Choline Hydroperoxide (PLOOH) for the Assay of PHGPX

  • Phosphatidyl choline (PL; Avanti Polar Lipids, Sigma, or Aldrich)
  • Source of nitrogen gas
  • 0.2 M sodium borate, pH 9 (or other buffer for pH 9 to 10 range)
  • 0.05 M sodium deoxycholate
  • Soybean lipoxygenase (Sigma Type IV or equivalent)
  • Absolute methanol
  • GPX4 positive control sample (see recipe)
  • 1‐ml Sep‐Pak C18 cartridge column, or equivalent
  • Additional reagents and equipment for assaying GPX4 (see protocol 3)

Alternate Protocol 3: Inclusive GPX Assay with Universal Substrates

  • Linoleic acid (Cayman Chemical)
  • 0.5 M sodium borate, pH 9
  • Petroleum ether
  • Ethyl acetate
  • 70:40:1 acetonitrile/H 2O/acetic acid
  • Rotary evaporator or full H‐type nitrogen or argon cylinder
  • Compressed air or oxygen tank to facilitate oxygenation of substrate
  • 3‐ml and 1‐ml Sep‐Pak C18 cartridge columns, or equivalent
  • Separatory funnels or other glassware for extractions

Support Protocol 2: Synthesis and Isolation of Linoleic Acid Hydroperoxide for the Assay of GPX

  • Column buffer with reducing agent (see recipe)
  • Column standards: any set of proteins with native sizes of 15,000 to 100,000 Da
  • Sample to be fractionated
  • Columns: 50 cm length × 1.5 cm diameter to 100 cm length × 2 cm diameter (see Hagel, )
  • Tubing to connect the column and fraction collector
  • Sephadex G‐150 to 200 (see Hagel, )
  • Fraction collector with a capacity for up to 100 tubes (5‐ to 10‐ml volume)
  • Additional reagents and equipment for assays of GPX activities (see protocol 1, protocol 2, and protocol 5)
NOTE: Perform all steps at 4°C to 10°C using a cold room or other suitable means.

Alternate Protocol 4: Assay of SeGPX and GST in Mixtures (Dual Assays)

  • GPX‐depleted immune (IgG) fraction of rabbit plasma (Esworthy et al., ): preimmune and anti‐GPX1
  • Immunoprecipitation buffer (see recipe)
  • 10% (w/v) suspension of washed Staphylococcus aureus protein A (see recipe)
  • Additional reagents and equipment for assay of GPX4 (see protocol 3)

Alternate Protocol 5: Assay of GPX1 and GPX4

  • 50% or 100% trichloroacetic acid (TCA)
  • 10 mM ferrous ammonium sulfate
  • 2.5 M potassium thiocyanate
  • Spectrophotometer
NOTE: The first step is either to carefully plan the assay so that some quantitative data can be obtained right away or to perform the assay on a trial basis using GSH as the reducing substrate so that real planning is possible. The time course of the reaction for a given sample activity can be roughly estimated from the parameters of the protocol 1 or protocol NaN, or protocol 7. GSH is usually the best substrate for GPX, so that reactions with alternative reducing substrates should run more slowly.
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Literature Cited

Literature Cited
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Key References
   Esworthy et al., 1993. See above.
  Presents basic information on GPX3 and the assay of this enzyme activity, as well as the assay of GPX4 by several means.
   Esworthy et al., 1998. See above.
  Two methods for the detection of GPX2 activity are demonstrated in this paper, one utilizing Gpx1 gene knockout mice and the other anti‐GPX1 antibody.
   Maiorino et al., 1990 See above.
  Discusses finding and methods for studying GPX4, at length. Comparative hydroperoxide rate constants for GPX4 versus GPX1 are listed.
   Paglia and Valentine, See above.
  These two references provide much basic information about glutathione peroxidases and the assay variations for GPX1 and GSTs.
   Lawrence and Burk, See above
  Basic information about Aop2 is presented in these two papers and the references within.
   Peshenko et al., See above
   Singh and Shichi, See above
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