Catalase Activity Assays

Nandita Shangari1, Peter J. O'Brien1

1 University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.7
DOI:  10.1002/0471140856.tx0707s27
Online Posting Date:  March, 2006
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Catalase (hydrogen peroxide/hydrogen peroxide oxidoreductase) is an important cellular antioxidant enzyme that defends against oxidative stress. It is found in the peroxisomes of most aerobic cells. It serves to protect the cell from toxic effects of high concentrations of hydrogen peroxide (H2O2) by catalyzing its decomposition into molecular oxygen and water, without the production of free radicals. It is important to measure catalase levels because oxidative stress is inherent in pathological conditions such as cancer, diabetes, cataracts, atherosclerosis, neurodegenerative disease, aging, and nutritional deficiencies. This unit provides methods for catalase activity measurements.

Keywords: catalase; catalytic activity; hydrogen peroxide; xylenol orange

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Table of Contents

  • Basic Protocol 1: Discontinuous Spectrophotometric Ferrous Oxidation Assay for H2O2
  • Alternate Protocol 1: Measurement of Catalase Activity in Red Blood Cells
  • Basic Protocol 2: Spectrophotometric Molybdate Assay for H2O2
  • Basic Protocol 3: Europium Fluorescence One‐Step Kinetic Assay for H2O2
  • Basic Protocol 4: Clark Oxygen Electrode Assay for Measuring the Oxygen Product of Catalase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Discontinuous Spectrophotometric Ferrous Oxidation Assay for H2O2

  • 30 % (v/v) H 2O 2 (Sigma‐Aldrich; store at 2°C to 8°C)
  • Tissue samples (e.g., rat liver), primary cultures, or cell cultures (e.g., hepatocytes)
  • Homogenization buffer (see recipe)
  • Bradford protein determination kit (Bio‐Rad; or see appendix 3H)
  • Hypotonic cell lysis buffer (see recipe)
  • 0.1 M phosphate buffer (either sodium or potassium phosphate; appendix 2A) with or without 0.1% (v/v) Triton X‐100, optional
  • BSA (Sigma‐Aldrich; store at 2°C to 8°C), optional
  • 2.2 mM H 2O 2
  • FOX 1 reagent (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Timer
  • Microcentrifuge
  • 1‐ml quartz cuvettes
  • Spectrophotometer with UV and visible light source

Alternate Protocol 1: Measurement of Catalase Activity in Red Blood Cells

  • 0.5 ml blood samples, collected in heparnized microcuvettes or tubes
  • Phosphate‐buffered saline: 15 mM potassium phosphate/150 mM NaCl, pH 7.4, ice cold
  • 10 mM potassium phosphate, pH 7.2: prepared by diluting 0.1 M phosphate buffer ( appendix 2A)

Basic Protocol 2: Spectrophotometric Molybdate Assay for H2O2

  • Tissue samples (e.g., rat liver)
  • 0.1 M sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • 65 µmol/ml H 2O 2 in 6.0 mM sodium‐potassium phosphate buffer, pH 7.4 (buffer concentrate; Sigma‐Aldrich)
  • 32.4 mM ammonium molybdate (Sigma‐Aldrich)
  • Balance accurate to ±0.01 g
  • Dounce homogenizer
  • Spectrophotometer

Basic Protocol 3: Europium Fluorescence One‐Step Kinetic Assay for H2O2

  • Europium‐tetracycline (EuTc) solution (see recipe)
  • 5 mM H 2O 2, prepared fresh
  • 10 mM MOPS (see recipe)
  • Tissue, primary cultures, or cell cultures (for preparation see protocol 1, steps and )
  • 11.2 mM 3‐amino‐1,2,4‐triazole (AMT; Sigma‐Aldrich)
  • 100 U/ml catalase control and 5 to 50 U/ml catalase standards: prepared by diluting catalase from bovine liver (Sigma; 1,277,500 U/ml) with 10 mM MOPS (see recipe)
  • 96‐well microtiter plate with lid
  • 30°C incubator
  • Multichannel pipettor
  • Microtiter plate fluorometer with 405‐nm excitation and filters for collection of emissions at 612 or 620 nm

Basic Protocol 4: Clark Oxygen Electrode Assay for Measuring the Oxygen Product of Catalase

  • 50 mM phosphate buffer, pH 7, air saturated: prepared by diluting 0.1 M phosphate buffer ( appendix 2A; either sodium or potassium phosphate) with Milli‐Q‐purified water and bubbling with air for ∼10 min
  • Nitrogen gas tank with pressure regulator
  • 0.08 U/ml catalase: prepared fresh by dissolving beef liver catalase (Sigma‐Aldrich; 40,000 to 60,000 U/mg protein) in 50 mM phosphate buffer, pH 7.0
  • Tissue homogenates or cell culture lysates (see protocol 1, steps and )
  • 33.5 mM H 2O 2: prepared fresh by diluting 30% H 2O 2 (Sigma‐Aldrich) with water
  • 10% bleach
  • Triton X‐100 (optional)
  • Oxygen electrode (e.g., Fisher) with thermostated jacket, magnetic stirrer, and XY recorder (e.g., Pharmacia)
  • 3‐ml syringe
  • Spectrophotometer
  • 20 × 30–cm sheets for the XY recorder
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  •   FigureFigure 7.7.1 Chemical reactions relating to hydrogen peroxide (H2O2) metabolism in cells. (A) Toxic hydroxyl radicals are formed in the reaction of H2O2 with ferrous iron. (B) Catalase causes decomposition of H2O2 to H2O and O2. (C) Superoxide dismutase (SOD) produces H2O2 from superoxide radicals generated by the mitochondrial respiratory chain and cytochrome P450 electron transport. (D) Xanthine oxidase generates H2O2. (E) Glutathione (GSH) peroxidase reacts with H2O2 to produce oxidized GSH (GSSG).
  •   FigureFigure 7.7.2 Standard curve for hydrogen peroxide measured with FOX 1 reagent.
  •   FigureFigure 7.7.3 A diagrammatic representation of an oxygen electrode. The electrode measures the concentration of oxygen in a solution contained in the chamber of the apparatus. The base of the chamber is formed by a Teflon membrane permeable to oxygen. Underneath this membrane is a shallow compartment containing a saturated KCl solution and two electrodes, a platinum cathode, and a silver anode. A fixed polarizing voltage is applied between the electrodes and the resulting tiny current is proportional to oxygen concentration. The current is determined using a chart recorder. The method is essentially polarographic, oxygen being reduced at the platinum cathode. The saturated KCl allows the current to flow and silver chloride is formed on the silver anode. The current depends upon the rate of diffusion of oxygen to the cathode, and this rate is proportional to the dissolved oxygen concentration. To ensure that the oxygen concentration in the electrode compartment follows that in the main chamber, it is essential to stir the contents of the main chamber continuously. A closely fitting stopper is used in experiments when it is necessary to exclude atmospheric oxygen from the system. Its position may be carefully adjusted so that the liquid exactly fills the space under it. (Adapted from
  •   FigureFigure 7.7.4 A Clark oxygen electrode recorder trace of catalase determination (adapted from Del Rio et al., ). The reaction is carried out at 37°C in 50 mM phosphate buffer, pH 7.0, with a final volume of 3.10 ml. The electrode reading is initially set at 100% deflection in air‐saturated buffer, and the medium is flushed with nitrogen. H2O2 is added followed by sample or catalase standards. The arrows indicate the additions to the system. The broken lines are tangents drawn for measurement of initial velocity (a) and for correction of spontaneous decomposition of H2O2 (b).


Literature Cited

Literature Cited
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