Analysis of Superoxide Dismutase Activity

Joe M. McCord1

1 University of Colorado Health Science Center, Denver, Colorado
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.3
DOI:  10.1002/0471140856.tx0703s00
Online Posting Date:  May, 2001
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Abstract

Measuring the activity of superoxide dismutases (SODs), the enzymes responsible for maintaining the steady state level of hydrogen peroxide, is challenging because the substrate is unstable at physiological pH and it reacts with itself. Fortunately the rate of reaction with dismutase is far greater than the rate of self reaction. As described in this unit, this activity can be measured indirectly based on competition between SOD and an indicator molecule that reacts avidly with superoxide to produce a measurable change in absorption, thus it is possible to measure total SOD activity or that of CuZn‐SOD and MnSOD. The activity can also be measured by an activity stain applied to thin‐film agarose or native polyacrylamide gels.

     
 
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Table of Contents

  • Basic Protocol 1: Assay of Total SOD Activity by the Xanthine Oxidase/Xanthine/ Cytochrome c Method
  • Alternate Protocol 1: Assessment of Relative Contributions of Cu,Zn‐SOD and Mn‐SOD to Total SOD Activity in Cell Extracts
  • Basic Protocol 2: Quantitative Assay of SODs by Activity Staining of Electrophoretic Gels
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Assay of Total SOD Activity by the Xanthine Oxidase/Xanthine/ Cytochrome c Method

  Materials
  • SOD assay cocktail (see recipe)
  • SOD‐containing sample
  • Xanthine oxidase solution (see recipe)
  • Recording, visible‐wavelength spectrophotometer with thermostat set at 25°C
  • 3‐ml cuvettes

Alternate Protocol 1: Assessment of Relative Contributions of Cu,Zn‐SOD and Mn‐SOD to Total SOD Activity in Cell Extracts

  • SOD‐containing sample with known total activity (see protocol 1)
  • 25 mM sodium diethyldithiocarbamate
  • 25 mM H 2O 2

Basic Protocol 2: Quantitative Assay of SODs by Activity Staining of Electrophoretic Gels

  Materials
  • SOD‐containing sample with known total activity (see protocol 1)
  • Gel staining solutions A and B (see reciperecipes)
  • Thin‐film agarose gels (Universal Gel/8, Ciba‐Corning Diagnostics) or native polyacrylamide slab gels ( appendix 3A), 1‐mm thick
  • Small rectangular dish for soaking the gel in a minimal volume of staining solution
  • Fluorescent desk lamp for illuminating and developing the gel
  • Camera, densitometer, or other device(s) for recording band intensity
  • Additional reagents and equipment for native PAGE ( appendix 3A)
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Figures

  •   FigureFigure 7.3.1 (A) A thin‐film agarose gel (Universal Gel/8, Ciba‐Corning Diagnostics) loaded with samples containing from 0.2 to 0.5 standard units of recombinant human Cu,Zn‐SOD. The gel was electrophoresed in 0.02 M Tris/glycine buffer, pH 8.45, for 30 min at 200 V. The family of closely spaced bands reflects charge isomers, and is characteristic of the Cu,Zn‐SODs. The gel was stained as described in and the dried gel digitally scanned (not shown) and analyzed using Matrix software (QuantiVison Canada). (B) The integrated area under the curve from each lane is plotted versus the amount of SOD applied.
  •   FigureFigure 7.3.2 Calibration curves showing the relationship between units of SOD and percent inhibition of the standard assay. Purified bovine Cu,Zn‐SOD was assayed under standard assay conditions to obtain the data shown. (A) A rectangular hyperbolic calibrating curve. (B) A linear double‐reciprocal calibrating curve.

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Literature Cited

Literature Cited
   Crapo, J.D., McCord, J.M., and Fridovich, I. 1978. Superoxide dismutases: Preparation and assay. Methods Enzymol. 53:382‐393.
   Keele, B.B., Jr., McCord, J.M., and Fridovich, I. 1970. Superoxide dismutase from Escherichia coli B: A new manganese‐containing enzyme. J. Biol. Chem. 245:6176‐6181.
   Klug, D., Rabani, J., and Fridovich, I. 1972. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J. Biol. Chem. 247:4839‐4842.
   Marklund, S.L. 1982. Human copper‐containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. U.S.A. 79:7634‐7638.
   McCord, J.M. and Fridovich, I. 1969. Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). J. Biol.Chem. 244:6049‐6055.
   McCord, J.M., Gao, B., Leff, J., and Flores, S.C. 1994. Neutrophil‐generated free radicals: Possible mechanisms of injury in adult respiratory distress syndrome. Environ. Health. Perspect. 102:57‐60.
   Simic, M.G., Taub, I.A., Tocci, J., and Hurwitz, P.A. 1975. Free radical reduction of ferricytochrome‐C. Biochem. Biophys. Res. Commun. 62:161‐167.
   Waud, W.R., Brady, F.O., Wiley, R.D., and Rajagopalan, K.V. 1975. A new purification procedure for bovine milk xanthine oxidase: Effect of proteolysis on the subunit structure. Arch. Biochem.Biophys. 169:695‐701.
   Weisiger, R.A. and Fridovich, I. 1973. Superoxide dismutase. Organelle specificity. J. Biol. Chem. 248:3582‐3592.
   Yost, F.J., Jr. and Fridovich, I. 1973. An iron‐containing superoxide dismutase from Escherichia coli. J. Biol. Chem. 248:4905‐4908.
Key References
   Crapo et al., 1978. See above.
  A discussion of assay methodologies and variations.
   McCord and Fridovich, 1969. See above.
  Initial description of superoxide dismutase and the definition of the standard unit of activity.
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