Measurement of Peroxiredoxin Activity

Kimberly J. Nelson1, Derek Parsonage1

1 Department of Biochemistry, Wake Forest University School of Medicine, Winston‐Salem, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.10
DOI:  10.1002/0471140856.tx0710s49
Online Posting Date:  August, 2011
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Abstract

Peroxiredoxins are cysteine‐dependent peroxidases that react with hydrogen peroxide, larger hydroperoxide substrates, and peroxynitrite. Protocols are provided to measure Prx activity with peroxide by (1) a coupled reaction with NADPH, thioredoxin reductase, and thioredoxin, (2) the direct monitoring of thioredoxin oxidation, (3) competition with horseradish peroxidase, and (4) peroxide consumption using the FOX assay. Curr. Protoc. Toxicol. 49:7.10.1‐7.10.28. © 2011 by John Wiley & Sons, Inc.

Keywords: antioxidants; antioxidant enzymes; sulfenic acids; hydrogen peroxide; hydroperoxides; thiol peroxidase; Prx; PRDX; cysteine oxidation; redox catalysis; HRP; FOX

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measurement of NADPH‐Dependent Peroxiredoxin Activity in Presence of Trx, TrxR, and Peroxide
  • Alternate Protocol 1: Measuring Peroxidase Activity with AhpF as Reductant
  • Basic Protocol 2: Measurement of Peroxide‐Dependent Peroxiredoxin Activity with Excess Trx (by Monitoring Trx Oxidation)
  • Basic Protocol 3: Measurement of Second‐Order Rate Constant with Hydrogen Peroxide Using Horseradish Peroxidase (HRP) Competition Assay
  • Alternate Protocol 2: Measurement of the Catalytic Cysteine pKa Using the HRP Competition Assay
  • Basic Protocol 4: Measurement of Peroxidase Activity Using FOX Assay to Measure Peroxide Disappearance
  • Alternate Protocol 3: Measurement of Peroxidase Activity In Vivo Using FOX Assay
  • Support Protocol 1: Prereduction of Prx or Trx Proteins
  • Support Protocol 2: Measurement of Thiol Content by Reaction with DTNB
  • Support Protocol 3: Measurement of Peroxide Concentration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of NADPH‐Dependent Peroxiredoxin Activity in Presence of Trx, TrxR, and Peroxide

  Materials
  • Prx reaction buffer (see recipe)
  • Purified Trx (500 µM diluted in Prx reaction buffer); E. coli and human purified Trx and are commercially available from multiple sources or can be overexpressed and purified from E. coli (Wollman et al., ; Lennon and Williams, )—following this protocol, 1 mg Trx will be sufficient for ∼30 1‐ml assays
  • Purified E. coli TrxR (50 µM diluted in Prx reaction buffer); E. coli and mammalian purified TrxR are commercially available from multiple sources or can be overexpressed and purified from E. coli (see Poole et al., , for E. coli, and Rengby et al., , for rat)—following this protocol, 0.1 mg TrxR will be sufficient for ∼30 1‐ml assays
  • 10 mM peroxide solution: hydrogen peroxide (see recipe), cumene hydroperoxide (see recipe), or t‐butyl hydroperoxide (see recipe)
  • 15 mM NADPH (dissolved in 10 mM Tris⋅SO 4, pH 8.5)
  • Peroxiredoxin (Prx; 50 µM diluted into Prx reaction buffer); in order to obtain sufficient quantities for these assays, the Prx of interest will typically be recombinantly expressed in and purified from E. coli (details of expression and purification will have to be determined separately for each protein prior to starting the assays)
  • Spectrophotometer with kinetic capabilities (required) and temperature regulation (preferred; i.e., with an attached water bath)
  • 1‐ml semi‐micro quartz cuvettes

Alternate Protocol 1: Measuring Peroxidase Activity with AhpF as Reductant

  Materials
  • Purified E. coli Trx
  • Dithiothreitol (DTT)
  • Prx reaction buffer (see recipe)
  • Purified peroxiredoxin (Prx; typically stored at ∼10 mg/ml)
  • 10 mM peroxide substrate: hydrogen peroxide (see recipe), cumene hydroperoxide (see recipe), or t‐butyl hydroperoxide (see recipe)
  • Syringe‐tip filters, 0.22‐ or 0.45‐µm
  • Spectrophotometer capable of measuring UV absorbance
  • Stopped‐flow spectrophotometer, capable of measuring fluorescence with excitation at 280 nm and emission monitored at >320 nm
  • Software program with multifunction nonlinear regression capabilities (e.g., Sigmaplot from Systat Software, http://www.systat.com)
  • Additional reagents and equipment for prereduction of Trx ( protocol 8), protein assay ( appendix 3I)

Basic Protocol 2: Measurement of Peroxide‐Dependent Peroxiredoxin Activity with Excess Trx (by Monitoring Trx Oxidation)

  Materials
  • 10 mM hydrogen peroxide (see recipe)
  • HRP solution (see recipe)
  • Standard Prx buffer (see recipe)
  • Prereduced Prx (see protocol 8)
  • 100‐mm Petri dishes
  • 96‐well UV‐transparent half area plate (no lid required; ∼200 µl well volume)
  • Plate reader spectrophotometer

Basic Protocol 3: Measurement of Second‐Order Rate Constant with Hydrogen Peroxide Using Horseradish Peroxidase (HRP) Competition Assay

  Materials
  • FOX Reagent A (see recipe)
  • FOX Reagent B (see recipe)
  • 10 mM peroxide substrate: hydrogen peroxide (see recipe), cumene hydroperoxide (see recipe), or tert‐butyl hydroperoxide (see recipe)
  • Standard Prx buffer (see recipe)
  • Pure Prx protein
  • 100 mM DTT (see recipe)
  • Spectrophotometer

Alternate Protocol 2: Measurement of the Catalytic Cysteine pKa Using the HRP Competition Assay

  Materials
  • Bacterial expression vector containing Prx gene
  • Bacterial growth medium: LB (e.g., unit 9.9) or other medium of choice)
  • FOX Reagent A (see recipe)
  • FOX Reagent B (see recipe)
  • 10 mM peroxide substrate: cumene hydroperoxide (see recipe), t‐butyl hydroperoxide (see recipe), or linoleic acid hydroperoxide (see recipe)
  • Spectrophotometer
  • Flasks for bacterial growth
  • Additional reagents and equipment for transformation of bacteria with plasmids (Seidman et al., )

Basic Protocol 4: Measurement of Peroxidase Activity Using FOX Assay to Measure Peroxide Disappearance

  Materials
  • Purified Prx or Trx protein (∼10 mg/ml)
  • 100 mM DTT stock
  • PD‐10 desalting column (GE Healthcare)
  • Suitable buffer for desired assay: Prx reaction buffer (see recipe) or standard Prx buffer (see recipe)
  • Spectrophotometer

Alternate Protocol 3: Measurement of Peroxidase Activity In Vivo Using FOX Assay

  Materials
  • Guanidine buffer (see recipe)
  • 30 mM 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) in DMSO
  • Prereduced Prx protein with DTT removed (see protocol 8)

Support Protocol 1: Prereduction of Prx or Trx Proteins

  Materials
  • Prx reaction buffer (see recipe)
  • Purified E. coli Trx (500 µM diluted into Prx reaction buffer)
  • Purified E. coli TrxR (50 µM diluted into Prx reaction buffer)
  • Peroxiredoxin (50 µM diluted into Prx reaction buffer)
  • 15 mM NADPH (dissolved in 10 mM Tris⋅SO 4 pH 8.5)
  • 10 mM peroxide solution to be measured: cumene hydroperoxide (see recipe) or t‐butyl hydroperoxide (see recipe)
  • Spectrophotometer
  • 1‐ml semi‐micro quartz cuvettes
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Figures

Videos

Literature Cited

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Key References
   Fourquet, S., Huang, M.E., D'Autreaux, B. and Toledano, M.B. 2008. The dual functions of thiol‐based peroxidases in H2O2 scavenging and signaling. Antioxid. Redox Signal. 10:1565‐1576.
  Review of biochemical and kinetic properties of peroxiredoxins and the related family of glutathione peroxidases.
   Hall, A., Nelson, K.J., Poole, L.B., Karplus, P.A. 2010. Structure‐based insights into the catalytic power and conformational dexterity of peroxiredoxins. Antioxid. Redox Signal. 402:194‐209.
  Review of Prx function and subfamily with an emphasis on structural characteristics.
Internet Resources
   http://www.csb.wfu.edu/prex/
  PREX is a searchable database containing >6,000 Prx protein sequences unambiguously classified into one of six distinct subclasses. Subfamily classifications use information around the active sites of structurally characterized subfamily members to search for sequences with conserved functionally‐relevant motifs (Nelson et al., , Soito et al., ).
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