Askim Hediye Sekmen, Ismail Turkan
This protocol outlines measurement of superoxide dismutase (SOD) activity in plant tissue by spectrophotomeric assay.
Background – Antioxidant enzymes
Plants, being aerobic organisms, utilize molecular O2as a terminal electron acceptor. As a reduction, highly reactive intermediates, reactive species (ROS), are produced. ROS such as singlet oxygen (O21), superoxide (O2-.) and hydrogen peroxide (H2O2) are normal products of metabolism and are produced in all cellular compartments within a variety of processes. In general, they are maintained at constant basal levels in healthy cells. However, they can destroy normal metabolism through oxidative damage of lipids, proteins, and nucleic acids when they are produced in excess as a result of oxidative stress (Gill and Tuteja, 2010). To overcome oxidative stress, together with non-enzymatic antioxidant molecules (ascorbate, glutathione, -tocopherol etc.), plants detoxify ROS by up-regulating antioxidative enzymes like superoxide dismutase (SOD; EC 184.108.40.206), catalase (CAT; E.C 220.127.116.11), peroxidase (POX; EC18.104.22.168), ascorbate peroxidase (APX; EC 22.214.171.124) and glutathione reductase (GR; EC 126.96.36.199) (Turkan and Demiral, 2009). SOD provide the first line of defense against the toxic effects of elevated levels of ROS. The SODs converts O2-.to H2O2. The hydrogen peroxide produced is then scavenged by catalase and a variety of peroxidases. Catalase dismutates H2O2into water and molecular oxygen, whereas POX decomposes H2O2by oxidation of co-substrates such as phenolic compounds and/or antioxidants. APX is involved in scavenging of H2O2in water-water and ASH-GSH cycles and utilizes ASH as the electron donor. GR is a potential enzyme of the ASH-GSH cycle and plays an essential role in defense system against ROS (Gill and Tuteja, 2010; Ahmad et al., 2010). This protocol is one of a number of ANTIOXIDANT ENZYME PROTOCOLS
|PROTOCOL: Catalase assay|
|PROTOCOL: Peroxidase assay|
|PROTOCOL: Ascorbate peroxidase assay|
|PROTOCOL: Glutathione reductase assay|
Background – superoxide dismutase
In this protocol, SOD (EC 188.8.131.52) activity was assayed by its ability to inhibit photochemical reduction of NBT at 560 nm (Beauchamp and Fridovich, 1971)
a) Chemical Materials
- NaH2PO4& Na2HPO2
- EDTA.Na2(372 g/mol)
- Polyvinylpolypyrrolidone (PVPP)
- Nitrotetrazolium bluechloride (NBT)
- L-methionine (149 g/mol)
- Riboflavin (376 g/mol)
- Liquid nitrogen
b) Apparatus and Equipments
- pH meter
- Mortar and pestle
- Various micropipettes
- Tubes with equal diameter
- Tube stand
- Polystrene cuvettes
- Flourescent light
Extraction Buffer 50 mM sodium phosphate buffer (pH 7.8) 1 mM EDTA – Na22% (w/v) PVPP Total volume:100 ml
- 50 mM Na-PO4buffer (pH 7.8), 100 ml
- 1 mM EDTA – Na2(372 g/mol), 100 ml
Weigh 0.037 g EDTA – Na2, dissolve in 100 ml 50 mM Na-PO4buffer (pH 7.8)
- 2% (w/v) PVPP, 100 ml
2 g PVPP is added in 100 ml, 50 mM Na-PO4buffer (pH 7.8)
- 50 mM Na-PO4buffer (pH 7.8)
- 1 mM NBT (817 g/mol), 100 ml
Weigh 0,088 g NBT, dissolve in 100 ml dIH2O.
- L. Methionine (149 g/mol), 100 ml
Weigh 0.745 g L-Methionine and dissolve in 100 ml dIH2O.
- 0.01 M EDTA. Na2(372 g/mol), 100 ml
Weigh 0.372 g EDTA.Na2and dissolve in 100 ml dIH2O.
- 0.2 mM Riboflavin (376 g/mol), 100 ml
Weigh 0.0075 g Riboflavin and dissolve in 100 ml dIH2O. Filtration is necessary.
Preparation of reaction mixture:
|Volumes (ml) for number of samples|
|Components||Concentration of the components in the reaction mixture||1||20||100|
|50 mM Na-P Buffer||50 mM||2.35||47||235|
|EDTA. Na2||0.66 mM||0.20||4||20|
|Total volume||3.0 ml||60.0 ml||300 ml|
Note: Reaction mixture is kept in the dark bottles. The Riboflavin is added before the use of the reaction mixture.
- Grind 0.5 g tissue in a cold mortar and pestle using liquid nitrogen and suspend in 1.5 ml of homogenization buffer solution.
- Centrifuge the suspension at 14000 rpm for 30 min at 4 0C
- Take the supernatant for the enzyme assay.Different volumes (50, 100, 150 and 200 l) of the extract are taken for analyses.
Assay Medium Blank 200 l extraction buffer+3 ml reaction mixture. To be kept in the dark! Control 200 μl extraction buffer+3 ml reaction mixture Samples
- A range of volumes of extract (50, 100, 150 and 200 μl) are placed in tubes with equal diameter. 150, 100 and 50 μl of the extraction buffer are added to the first three tubes (50, 100 and 150 μl), respectively (total volume=200 μl) and then 3 ml of reaction mixture is added to all tubes.
- For instance:
|Blank||Control||50 μl||100 μl||150 μl||200 μl|
|200 μl ext.buffer||200 μl ext.buffer||50 μl sample1||100 μl sample1||150 μl sample1||200 μsample1|
|3 ml reac. mix.||3 ml reac. mix.||150 μl extrac.buf.||100 μl extrac.buf.||50 μl extrac.buf.||0 μl ext.buf.|
|DARK||LIGHT||3 ml reac. mix.||3 ml reac. mix||3 ml reac. mix||3 ml reac.mix|
Note: During preparation of the samples and the control are placed in a darkened stand. Then, they are transffered to an ordinary stand, illuminated with luminescent lamps for 10 min. The extinction at 560 nm is read against blank. One enzyme unit of SOD is defined as that amount of protein (in mg) causing a 50% inhibition of the photoreduction.
Ahmad P., Jaleel CA, Salem MA, Nabi G, Sharma S, (2010), Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress, Critical Rev. Biotech. 30 (3): 161-175.
Beauchamp C., Fridovich I., Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 (1971) 276-287.
Singh Gill S, Tuteja N., (2010), Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiol and Biochem. 48: 909-930.
Turkan I, Demiral T., (2010), Recent developments in understanding salinity tolerance, Environ. Exp. Bot., 67 (1): 2-9.