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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Abstract

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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Materials

Basic Protocol 1: Discontinuous Spectrophotometric Ferrous Oxidation Assay for H2O2

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
   Bai, J., Rodriguez, A.M., Melendez, J.A., and Cederbaum, A.I. 1999. Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J. Biol. Chem. 274:26217‐26224.
   Barja, G. 2002. The quantitative measurement of H2O2 generation in isolated mitochondria. J. Bioenerg. Biomembr. 34:227‐233.
   Boutin, J.A., Kass, G.E., and Moldeus, P. 1989. Drug‐induced hydrogen peroxide production in isolated rat hepatocytes. Toxicology 54:129‐137.
   Cohen, G., Kim, M., and Ogwu, V. 1996. A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures. J. Neurosci. Methods 67:53‐56.
   Del Rio, L.A., Ortega, M.G., Lopez, A.L., and Gorge, J.L. 1977. A more sensitive modification of the catalase assay with the Clark oxygen electrode. Application to the kinetic study of the pea leaf enzyme. Anal. Biochem. 80:409‐415.
   Goth, L. 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin. Chim. Acta. 196:143‐151.
   Halliwell, B., Long, L.H., Lee, T.P., Lim, S., and Kelly, R. 2004. Establishing biomarkers of oxidative stress: The measurement of hydrogen peroxide in human urine. Curr. Med. Chem.11:1085‐1092.
   Hosono, M., Iwamoto, N., Hayashi, H., Takeuchi, N., and Iwamura, J. 1996. Measurement of serum catalase activity and its clinical significance. Rinsho Byori 44:444‐448.
   Ibrahim, W., Lee, U.S., Yen, H.C., St. Clair, D.K., and Chow, C.K. 2003. Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice. Free Radic. Biol. Med. 28:397‐402.
   Jiang, Z.Y., Woollard, A.C., and Wolff, S.P. 1990. Hydrogen peroxide production during experimental protein glycation. FEBS Lett. 268:69‐71.
   Kyle, M.E., Miccadei, S., Nakae, D., and Farber, J.L. 1987. Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxi city of acetaminophen. Biochem. Biophys. Res. Commun. 149:889‐896.
   Michaels, H.B. and Hunt, J.W. 1978. Determination of peroxides and hydroperoxides in irradiated solutions of nucleic acid constituents and DNA. Anal. Biochem. 87:135‐140.
   Mueller, S., Riedel, H.D., and Stremmel, W. 1997. Determination of catalase activity at physiological hydrogen peroxide concentrations. Anal. Biochem. 245:55‐60.
   Ohmori, T., Takamoto, K., and Ogata, M. 1991. The role of catalase in protecting erythrocytes against methemoglobin formation. Acta Med. Okayama. 45:321‐324.
   Ou, P. and Wolff, S.P. 1996. A discontinuous method for catalase determination at “near physiological” concentrations of H2O2 and its application to the study of H2O2 fluxes within cells. J. Biochem. Biophys. Methods. 31:59‐67.
   Siraki, A.G., Pourahmad, J., Chan, T.S., Kahn, S., and O'Brien, P.J. 2002. Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes. Free Radic. Biol Med. 32:2‐10.
   Slaughter, M.R. and O'Brien, P.J. 2000. Fully‐automated spectrophotometric method for measurement of antioxidant activity of catalase. Clin. Biochem. 33:525‐534.
   Tarasova, N.S. 1998. Combination of low catalase activity with lipid peroxidation activation in serum of chronic alcoholics with kidney diseases. Klin. Med. (Mosk). 76:43‐44.
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   Wang, D.H., Ishii, K., Zhen, L.X., and Taketa, K. 1996. Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride. Arch. Toxicol. 70:189‐194.
   Wolfbeis, O.S., Durkop, A., Wu, M., and Lin, Z. 2002. A europium‐ion‐based luminescent sensing probe for hydrogen peroxide. Angew. Chem. Int. Ed. Engl. 41:4495‐4498.
   Wozniak, A., Drewa, G., Wozniak, B., and Schachtschabel, D.O., 2004. Activity of antioxidant enzymes and concentration of lipid peroxidation products in selected tissues of mice of different ages, both healthy and melanoma‐bearing. Z. Gerontol. Geriatr. 37:184‐189.
   Wu, M., Lin, Z., and Wolfbeis, O.S. 2003. Determination of the activity of catalase using a europium(III)‐tetracycline‐derived fluorescent substrate. Anal. Biochem. 320:129‐135.
   Zigman, S., Schultz, J.B., and Schultz, M. 1998. Measurement of oxygen production by in vitro human and animal lenses with an oxygen electrode. Curr. Eye Res. 17:115‐119.
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