Protocol

Authors

Askim Hediye Sekmen, Ismail Turkan

Overview

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 1.15.1.1), catalase (CAT; E.C 1.11.1.6), peroxidase (POX; EC1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) (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 1.15.1.1) activity was assayed by its ability to inhibit photochemical reduction of NBT at 560 nm (Beauchamp and Fridovich, 1971)

Materials/Equipment

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
• Eppendorph
• Tubes with equal diameter
• Tube stand
• Spectrophotometer
• Centrifuge
• Polystrene cuvettes
• Flourescent light

Procedure

a) Solutions

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)

Assay Solutions

• 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
L-Methionine	10 mM	0.30	6.0	30
NBT	33 M	0.10	2.0	10
Riboflavin	0.0033 mM	0.05	1.0	5
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.

b) Method

Extraction

• 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.

Literature references

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.