Measurement of MnSOD and CuZnSOD Activity in Mammalian Tissue Homogenates

Douglas R. Spitz1, Larry W. Oberley1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.5
DOI:  10.1002/0471140856.tx0705s08
Online Posting Date:  August, 2001
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Three basic forms of mammalian SODs exist and they are distinguished by their sizes and locations. SOD enzyme activity is not easily monitored by direct measurement because the substrate disappearance is very rapid at physiological pH. Activity can be measured as described in this unit by a number of indirect competitive inhibition assays based on the principle that the superoxide anion radical will reduce an inhibitory substrate [such as nitroblue tetrazolium (NBT) or cytochrome c] and SOD activity will reduce the rate of reduction in a competitive fashion. The SODÔÇÉmediated inhibition of the indicator substrate reduction can then be quantitated and plotted as a function of the quantity of protein added to the reaction to construct an inhibition curve.

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Table of Contents

  • Basic Protocol 1: NBT‐BCS Superoxide Dismutase Activity Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: NBT‐BCS Superoxide Dismutase Activity Assay

  • 3 × 107 cells or tissue
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1.34 mM DETAPAC buffer (see recipe)
  • BSA in DETAPAC buffer (see recipe)
  • 40 U/ml catalase (Sigma‐Aldrich) in 0.05 M potassium phosphate buffer (see recipe); store up to 1 month at 4°C as a 4000 U/ml solution
  • 2.24 mM nitroblue tetrazolium (NBT; see recipe)
  • 1.18 mM xanthine (see recipe)
  • 0.05 M potassium phosphate buffer, pH 7.8 (see recipe)
  • 0.33 M NaCN (see recipe)
  • BCS (see recipe)
  • 1 M HCl or 1 M KOH (optional)
  • 5 µg/ml bovine CuZn superoxide dismutase (CuZnSOD; OXIS International): store up to 5 years at −20°C (avoid multiple freeze‐thaw cycles)
  • 13.2 U/ml xanthine oxidase (XO; Sigma‐Aldrich), on ice
  • 15‐ml centrifuge tubes
  • 400‐W microtip sonicator (nonfragile cell culture or tissue)
  • Brinkman homogenizer with microtip (tissue)
  • 10 × 75–mm borosilicate glass test tubes
  • Beckman 650 spectrophotometer or equivalent reading at 560 nm with 6 position automatic sample changer and enzyme kinetics software package
  • 1‐ml polystyrene cuvettes
NOTE: All solutions, unless otherwise specified, are prepared in 0.05 M potassium phosphate buffer, pH 7.8, stored at 4°C, and warmed to room temperature prior to assay. The water is double‐distilled or Nanopure 18.3‐megaohm purified (Barnstead/Thermolyne).
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Literature Cited

   Beauchamp, C. and Fridovich, I. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276‐287.
   Brown, K.F., Kinter, M.T., Oberley, T.D., Freeman, M.L., Frierson, H.F., Ridnour, L.A., Tao, Y., Oberley, L.W., and Spitz, D.R. 1998. Enhanced gamma glutamyl transpeptidase expression and selective loss of CuZn superoxide dismutase in hepatic iron overload. Free Radic. Biol. Med. 24:545‐555.
   Fridovich, I. 1978. The biology of oxygen radicals. Science. 201:873‐880.
   Halliwell, B. and Gutteridge, J.M.C. 1999. Free Radicals in Biology and Medicine: Third Edition. Oxford University Press, Oxford.
   Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement using the folin phenol reagent. J. Biol. Chem. 193:265‐275.
   Marklund, S.L. 1985. Direct assay with potassium superoxide. In Handbook of Methods for Oxygen Radical Research, (R. Greenwald, ed.), p. 249. CRC Press, Boca Raton, Fla.
   Oberley, L.W., McCormick, M.L., Sierra‐Rivera, E., and Kasemset‐St. Clair, D. 1989. Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts. Free Radic. Biol. Med. 6:379‐384.
   Spitz, D.R. and Oberley, L.W. 1989. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem. 179:8‐18.
   Spitz, D.R., Elwell, J.H., Sun, Y., Oberley, L.W., Oberley, T.D., Sullivan, S.J., and Roberts, R.J. 1990. Oxygen toxicity in control and H2O2‐resistant Chinese hamster fibroblast cell lines. Arch. Biochem. Biophys. 279:249‐260.
   Wiess, R.H., Flickinger, A.G., Rivers, W.J., Hardy, M.M., Aston, K.W., Ryan, U.S., and Riley, D.P. 1993. Evaluation of activity of putative superoxide dismutase mimics: Direct analysis by stopped‐flow kinetics. J. Biol. Chem. 268:23049‐23054.
Key References
   Beauchamp and Fridovich, 1971. See above.
  First report on NBT‐based SOD activity assays.
   Spitz and Oberley, 1989. See above.
  Original description of the NBT‐BCS activity assay for use in mammalian tissue homogenates.
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