Measurement of Glutathione Conjugates

Michael A. Shultz1, Dexter Morin1, Katherine Watt Chan1, Alan R. Buckpitt1

1 University of California, Davis, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.8
DOI:  10.1002/0471140856.tx0608s14
Online Posting Date:  February, 2003
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Abstract

The involvement of reactive metabolites in cancer and cellular necrosis has been well established. The nucleophile, glutathione, provides a major mechanism of intracellular protection from electrophilic metabolites. Conjugation with glutathione to generate stable, water‐soluble metabolites has been utilized to determine the nature and rates of formation of precursor reactive metabolites. In addition, because activities of the glutathione transferases may play a key role in tissue/cellular susceptibilities to electrophilic compounds, measurement of catalytic activities of these proteins can play an important role in discerning the underlying mechanisms of cell‐selective toxicities. This unit outlines HPLC methods found to provide good separation of glutathione conjugates and includes two additional procedures that can be utilized in experiments where high throughput assays are needed for measuring transferase activities.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Glutathione Conjugates by HPLC
  • Alternate Protocol 1: Radiometric Assay of Glutathione Conjugate Formation
  • Alternate Protocol 2: Spectrophotometric Assay for Glutathione S‐Transerases
  • Support Protocol 1: Microsomal Incubations
  • Support Protocol 2: Preparation of Microsomal and Cytosolic Fractions
  • Support Protocol 3: Purification of Glutathione S‐Transferases
  • Support Protocol 4: Preparative Separation and Isolation of Glutathione Conjugates for Characterization by Mass Spectrometry and Proton NMR Spectroscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Glutathione Conjugates by HPLC

  Materials
  • Sample derived from microsomal incubation (see protocol 4)
  • 100% HPLC‐grade acetonitrile
  • Solvent A: 0.06% triethylamine, pH 3.1 (see recipe)
  • Solvent B: 60% acetonitrile in 0.06% triethylamine (see recipe)
  • Scintillation cocktail appropriate for aqueous samples
  • Gradient HPLC:
    • 0.46 × 20–cm C18 ODS‐2 Spherisorb HPLC column with 5‐µm packing material and matching precolumn (Waters) or equivalent
    • Injector
    • Gradient pump
    • UV (variable wavelength or photodiode array preferred)
    • Fraction collector (e.g., Gilson model 202)
    • Waste collector
    • Data collection and storage device (e.g., Millennium integration and control software; Waters)
  • Scintillation vials
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by the local radiation safety officer (also see appendix 1A).CAUTION: The electrophilic metabolites generated for glutathione trapping are generally more toxic than the parent compounds and appropriate precautions should be taken to ensure the safety of investigators. Many of these electrophilic metabolites are potent mutagens/carcinogens and/or cytotoxicants. Appropriate protective clothing, gloves, and bench paper should be used while handling all samples.

Alternate Protocol 1: Radiometric Assay of Glutathione Conjugate Formation

  Materials
  • 1‐Hexanol
  • Glass culture tube with Teflon‐lined screw cap (for volatile substrates)
  • Scintillation fluid
  • Scintillation vials
  • Additional reagents and equipment for microsomal or cytosolic incubations (see protocol 4)
CAUTION: Radiolabeled probes are hazardous; see appendix 1A for guidelines on handling, storage, and disposal.

Alternate Protocol 2: Spectrophotometric Assay for Glutathione S‐Transerases

  Materials
  • 0.1 M sodium phosphate buffer, pH 6.5 ( appendix 2A)
  • 3.3 mM glutathione (see recipe)
  • 33 mM CDNB (see recipe)
  • Enzyme preparation (e.g., cytosol; see protocol 5)
  • VIS spectrophotometer (e.g., Beckman DU‐70) with thermostatting device

Support Protocol 1: Microsomal Incubations

  Materials
  • Microsomes from tissue of interest (0.5 to 5 mg microsomal protein/ml; see protocol 5) or recombinant cytochrome P450/P450 reductase
  • 0.5 mM 5000 dpm/nmole [3H]glutathione (see recipe)
  • 50 to 100 CDNB U/ml glutathione S‐transferases (see protocol 6)
  • 1 M MgCl 2
  • 12.5 mM NADPH or NADPH regeneration system (see reciperecipes)
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • 50 mM 1‐nitronapthalene (see recipe)
  • Methanol, ice cold
  • 1‐ml glass vials with caps
  • 37°C shaking water bath
    Table 6.8.2   Materials   Basic Components of Incubations to Isolate Glutathione Conjugates   Basic Components of Incubations to Isolate Glutathione Conjugates

    Incubation no. Label 5 mg/ml microsomes 0.5 mM [3H]glutathione 50 U/ml glutathione S‐transferase NADPH regenerating system 1 M MgCl 2 50 mM 1‐nitronaphthalene 0.1 M phosphate buffer, pH 7.4 Total volume
    1 Complete 100 50 50 20 2.5 2.5 25 250
    2 No nitronaphthalene 100 50 50 20 2.5 0 27.5 250
    3 No glutathione 100 0 50 20 2.5 2.5 75 250
    4 No NADPH regenerating system 100 50 50 0 2.5 2.5 45 250

     aAll volumes are given in microliters.
     bAll concentrations in the table refer to concentrations of the substance to be added. The final concentrations of various incubation components are specified earlier.

Support Protocol 2: Preparation of Microsomal and Cytosolic Fractions

  Materials
  • Mouse livers
  • Homogenization buffer (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Motor‐driven Potter‐Elvehjem Teflon‐glass homogenizers
  • Refrigerated ultracentrifuge (capable of 100,000 × g) and appropriate tubes
  • Cotton‐tipped applicator
  • Hand‐held glass‐glass homogenizer
  • Additional reagents and equipment for Bradford assay of protein concentration ( appendix 3G)
CAUTION: Glass homogenizers should be placed in an ice bucket and held at the top of the vessel only. The high rotating force created during the homogenization processes can occasionally cause the homogenization vessels to shatter, resulting in a significant hazard to the operator. The use of puncture resistant gloves is strongly recommended.CAUTION: Samples for the ultracentrifuge must be balanced within 100 mg. All rotor gaskets and the speed disk should be checked prior to use of the ultracentrifuge.NOTE: The following procedures should be conducted at 0° to 4°C.

Support Protocol 3: Purification of Glutathione S‐Transferases

  Materials
  • 175‐ to 520‐µm coarse Sephadex G‐25 resin
  • Resuspension buffer (see recipe) with and without 0.5 M NaCl
  • Sample (100,000 × g supernatant; see Support Protocol protocol 5, step )
  • Glutathione affinity resin—i.e., glutathione‐agarose (Sigma) or equivalent
  • Glutathione elution buffer (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Glass columns
  • Buffer resevoir
  • Column monitor (e.g., ISCO UA5; optional)
  • Amicon PM‐10 membrane ultraconcentrator (10,000 MWCO) or equivalent
  • 12,000‐ to 14,000‐MWCO dialysis membrane

Support Protocol 4: Preparative Separation and Isolation of Glutathione Conjugates for Characterization by Mass Spectrometry and Proton NMR Spectroscopy

  Materials
  • Substrate
  • Methanol, ice cold
  • SM‐16 resin (macroporous polystyrene divinyl benzene; BioRad) or equivalent
  • Methanol, HPLC grade
  • 1% acetic acid or equivalent (e.g., 0.5% formic acid, 0.05% trifluoroacetic acid)
  • 50% glacial acetic acid or equivalent (e.g., 50% concentrated formic acid, trifluoroacetic acid)
  • 75% acetonitrile
  • 12.4 M pyridine (99%)
  • Rotary evaporator and flask or large‐volume centrifugal evaporator
  • Disposable low‐pressure column
  • Teflon or silanized‐glass containers (optional)
  • HPLC system:
    • Semipreparative 8‐ to 30‐mm‐diameter column packed with material  identical to that used in the analytical separation (see protocol 1)
    • Gradient HPLC pump capable of delivering flow rates that vary from 5 to 40 ml/min
  • Lyophilizer
  • Additional reagents and equipment for sample preparation (see protocol 4, steps to )
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Figures

Videos

Literature Cited

Literature Cited
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   Buckpitt, A.R., Castagnoli, N., Nelson, S.D., Jones, A.D., and Bahnson, L.S. 1987. Stereoselectivity of naphthalene epoxidation by mouse, rat and hamster pulmonary, hepatic and renal microsomal enzymes. Drug Metab. Dispo. 15:491‐498.
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