HPLC‐Based Assays for Enzymes of Glutathione Biosynthesis

Collin C. White1, Cecile J. Krejsa1, David L. Eaton1, Terrance J. Kavanagh1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.5
DOI:  10.1002/0471140856.tx0605s00
Online Posting Date:  May, 2001
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Abstract

Glutamate cysteine ligase and gluthione synthase carry out the two‐step synthesis of glutathione. The fluorescent thiol‐reactive compound monobromobimane is used to derivatize reaction products in an HPLC‐based assay with fluorescence detection. The assay described in this unit can be adapted for tissue homogenates or cultured cells.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Glutamate Cysteine Ligase (GLCL) Activity in Tissues
  • Alternate Protocol 1: Analysis of GLCL Activity in Cultured Cells
  • Basic Protocol 2: Analysis of Glutathione Synthetase (GS) Activity in Tissues
  • Alternate Protocol 2: Analysis of GS Activity in Cultured Cells
  • Support Protocol 1: Measurement of γ‐GC and GSH by HPLC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Glutamate Cysteine Ligase (GLCL) Activity in Tissues

  Materials
  • Tissue of interest
  • TES/SB homogenization buffer (see recipe), ice cold
  • GLCL reaction cocktail (see recipe), ice cold
  • 200 mM 5‐sulfosalicylic acid (SSA; Sigma), ice cold
  • 5 mM L‐cysteine (Sigma), prepared fresh
  • γ‐Glutamylcysteine (γ‐GC; Sigma)
  • Glutathione (GSH; Sigma)
  • 0.2 M N‐ethylmorpholine (NEM; Sigma)/0.02 M KOH
  • 25 mM monobromobimane (MBB; Thiolyte reagent, Calbiochem) in acetonitrile
  • 15‐ml Corex tubes
  • Polytron tissue homogenizer (Kinematic AG)
  • Additional reagents and equipment for HPLC (see protocol 5)
NOTE: Cysteine, γ‐GC, and GSH solutions must be made immediately prior to use, or stored on ice if made in advance. Solutions should not be made more than one hour prior to use in this assay. Cysteine should be at room temperature when it is added to the incubation tubes.

Alternate Protocol 1: Analysis of GLCL Activity in Cultured Cells

  • Cell cultures of interest
  • PBS/SB: PBS ( appendix 2A) containing 20 mM boric acid and 1 mM serine, pH 7.4
  • Probe sonicator

Basic Protocol 2: Analysis of Glutathione Synthetase (GS) Activity in Tissues

  Materials
  • Tissue of interest
  • TES/SB homogenization buffer (see recipe), ice cold
  • GS reaction cocktail (see recipe), ice cold
  • 200 mM 5‐sulfosalicylic acid (SSA; Sigma), ice cold
  • 5 mM γ‐glutamylcysteine (γ‐GC; Sigma), prepared fresh
  • Glutathione (GSH; Sigma)
  • 0.2 M N‐ethylmorpholine (NEM: Sigma)/0.02 M KOH
  • 25 mM monobromobimane (MBB; Thiolyte reagent, Calbiochem) in acetonitrile
  • 15‐ml Corex tubes
  • Polytron tissue homogenizer (Kinematic AG)
  • Flow Microdialysis System (Life Technologies)
  • Additional reagents and equipment for HPLC (see protocol 5)
NOTE: γ‐GC and GSH solutions must be made immediately prior to use, or stored on ice if made in advance. Solutions should not be made more than one hour prior to use in this assay. γ‐GC should be at room temperature when it is added to the incubation tubes.

Alternate Protocol 2: Analysis of GS Activity in Cultured Cells

  • Cell cultures of interest
  • PBS/SB: PBS ( appendix 2A) containing 20 mM boric acid and 1 mM serine, pH 7.4
  • Probe sonicator

Support Protocol 1: Measurement of γ‐GC and GSH by HPLC

  Materials
  • Derivatized samples and standards for analysis (see protocol 1 and protocol 3 and protocol 2 and protocol 4)
  • 1 mM tetrabutylammonium phosphate (TBAP; Regis), pH adjusted to 3.0 with 10% (v/v) phosphoric acid
  • HPLC‐grade methanol
  • Shimadzu SIL‐6B HPLC system with autosampler, CR5A integrator, and RF‐535 fluorescence detector (or equivalent)
  • Pellicular C18 guard column (Alltech)
  • 150‐mm reversed‐phase C18 HPLC column (Alltech)
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Figures

Videos

Literature Cited

Literature Cited
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  This work has been supported by NIH grants ES04696, ES07033, ES07032, AG01751, and DOE Cooperative Agreement #DE‐FCO1‐95EW55084. This support does not constitute an endorsement by DOE of the views expressed herein.
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