Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue

Abstract

Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24-48 h to complete.

Figure 1. Antioxidant enzyme schematic

There are three major types of primary intracellular antioxidant enzymes in mammalian cells - SOD, catalase, and peroxidase, of which glutathione peroxidase (GPx) is the most prominent. The SODs convert O₂^•- into H₂O₂, while the catalases and peroxidases convert H₂O₂ into water. If H₂O₂-removal is inhibited, then there is direct toxicity resulting from H₂O₂-mediated damage. GPx requires several secondary enzymes including glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PD) and cofactors including glutathione (GSH), NADPH, and glucose 6-phosphate to function at high efficiency. If GR is inhibited, cells cannot remove H₂O₂ via the glutathione peroxidase system and increasing the levels of glutathione disulfide (GSSG). If glutathione synthesis is inhibited, either by inhibiting gluthatione synthetase (GS) or γ-glutamyl cysteine synthetase (γ-GCS), glutathione will be depleted and GPx will not be able to remove H₂O₂. If catalase is inhibited, cells also cannot remove H₂O₂. Finally, if glucose uptake is inhibited creating a chemically induced state of glucose deprivation, hydroperoxide detoxification will also be inhibited.

Figure 2. MnSOD and CuZnSOD activity gels

To determine a change in activity of the antioxidant proteins CuZnSOD and MnSOD in MCF-10A immortalized breast epithelial cells after infection with the AdEmpty, AdCuZnSOD or AdMnSOD (50 or 100 multiplicity of infectivity, MOI) adenoviral vector constructs, an activity gel was performed. Protein (100 μg) was loaded and the gels were electrophoresed at 4 °C. SOD expression was visualized by first soaking gels in NBT. MnSOD expression alone was visualized by the addition of NaCN which inhibits CuZnSOD activity. The adenoviral vector AdCuZnSOD increased CuZnSOD activity while the AdMnSOD vector increased MnSOD activity.

Figure 3. Catalase and GPx activity gels

MCF 10A cells transduced with increasing MOI of adenoviral catalase (Adcatalase) were subjected to native gel electrophoresis (100 μg protein/well) and stained as described (A). An empty adenovirus and untreated control lystates were utilized as baseline levels of catalase expression. Achromatic (clear) bands represent areas of catalase enzyme activity. MiaPaCa pancreatic cancer cells were grown in the presence of increasing MOI AdGPx (B). Samples were harvested and the protein lysates (250 μg) were loaded onto gels (8%) and subjected to GPx native gel activity analysis. Following the GPx staining protocol, the resulting bands show increasing GPx activity with increasing GPx infection. Bovine GPx was utilized as a positive control (lower band). The upper band represents GST contamination within this standard.

Figure 4. Antioxidant immunofluorescent staining of cultured cells

Robust MnSOD immunofluorescent signal (green, 1:100 MnSOD 1°Ab; Cy2 goat-anti-rabbit 2°Ab) appears as punctate staining within MCF 10A cells following AdMnSOD (300 MOI) transduction for 48 h suggesting subcellular localization in the mitochondria (A). The nuclei are counterstained with DAPI (blue). Localization of CuZnSOD protein expression is visualized following AdCuZnSOD transduction (300 MOI) as robust red staining throughout the cytoplasm (1:100 MnSOD 1°Ab; Cy2 goat-anti-rabbit 2°Ab) and the nuclei appear blue following DAPI counterstain (B). 400x, bar = 50 μm.

Figure 5. Immunohistochemistry for MnSOD

Loss of MnSOD expression in pancreatic ductal cells from pancreatic resections of adenocarcinoma is shown. Immunohistochemistry for MnSOD expression using the avidin-biotin peroxidase complex method was performed on pancreatic specimens previously fixed in formalin and embedded in paraffin. A quantitative digital imaging methodology was used to examine MnSOD staining in the pancreatic tissue. Cytoplasmic regions of pancreatic ductal cells were identified and digitized. Mean gray-level pixel values were then obtained (A). Strong staining is seen in the cytoplasm in cells from normal pancreas. Staining is nearly undetectable in cells from pancreatic cancer resections with a marked decrease in the mean gray level value when compared to normal pancreas (B). All experiments using animals or human samples should be reviewed and approved by the Institutional animal care and use committee. Bar = 50 μm.

Figure 6. Sub-cellular localization of MnSOD by immunogold

MIA PaCa-2 human pancreatic cancer cells were infected with adenoviral vectors containing the cDNA for MnSOD. Ultrastructural examination was performed by Dr. Terry Oberley at the University of Wisconsin. Sections were treated with primary MnSOD antibody overnight, washed, and treated with gold-conjugated goat anti-rabbit immunoglobulin, fixed and stained. Labeling of MnSOD was extremely light in cells treated with the AdEmpty vector (A). MIA PaCa-2 cells treated with AdMnSOD demonstrated increases in labeling (arrow) in the mitochondria (bottom panel) (B). All experiments using animals or human samples should be reviewed and approved by the Institutional animal care and use committee. Bar = 5 μm

Figure 7. Glutathione peroxidase activity assay

The GPx assay is an indirect, coupled assay for glutathione peroxidase. This assay takes advantage of glutathione disulfide (GSSG) formed by the enzymatic action of GPx and is regenerated by excess glutathione reductase (GR) in the assay. The action of GR is monitored by following the disappearance of the co-substrate NADPH.