Antioxidant enzyme activity assays

0 downloads 0 Views 110KB Size Report
Antioxidant enzyme activity assays. Enzyme extraction (Ruley et al. 2004). 1. Harvest and homogenize the seedlings submitted to the different treatments with ...
Antioxidant enzyme activity assays

Enzyme extraction (Ruley et al. 2004) 1. 2. 3. 4.

Harvest and homogenize the seedlings submitted to the different treatments with liquid nitrogen. Add 1 mL of phosphate buffer per 100 mg plant (fresh weight). Centrifuge the homogenates at 4000 × g at 4 °C for 10 min to remove gross particulate plant debris. Unless stated otherwise, dilute the supernatant two-fold in phosphate buffer.

Ascorbate peroxidase (EPA, 1994) 1. To 100 μl of the initial supernatant add 450 μl of 17 mM H2O2 and 450 μl of 25mM ascorbate. 2. Read absorbances for 3 min at 290 nm. One unit of enzyme activity is defined as a decrease in absorbance of 0.001/min at 290 nm.

Guaiacol peroxidase (EPA, 1994) 1. 2.

To 100 μl of the initial supernatant add 450 μl of 17 mM H2O2 and 450 μl of 2% guaiacol. Read absorbances for 3 min at 510 nm

One unit of enzyme activity was defined as an increase in absorbance of 0.001/min at 510 nm.

Catalase (Aebi, 1983) 1. 2.

To 666 μl of the initial supernatant add 334 μl of 73 mM H2O2. Read absorbances for 3 min at 240 nm.

One unit of enzyme activity was defined as a decrease in absorbance of 0.001/min at 240 nm

Lipid peroxidation (Li, 2000) 1. 2. 3. 4. 5.

Homogenize plant material with 3 ml of 0.5% thiobarbituric acid in 20% trichloroacetic acid (w/v). Incubate the homogenate at 100 ºC for 30 min and stop the reaction in ice or cooled water. Centrifuge the samples at 10,000xg for 10 min Measure absorbances spectrophotometrically at 450, 532 and 600 nm Determine MDA concentration by the following formula:

Superoxide dismutase (Tang and Newton, 2004) 1. Homogenize 1 g of sample under ice-cold conditions in 3ml of extraction buffer, containing 50mM phosphate buffer (pH 7.4), 1mM EDTA, 1 g PVP, and 0.5% (v/v) Triton X-100 at 4 ◦C. 2. Centrifuge at 10,000 × g for 20 min. Use the supernatant fraction for the assays. 3. To 50–150 µl of enzyme extract add 3.5 ml of O2-generating solution containing 14.3mM methionine, 82.5 µM NBT, and 2.2 µM riboflavin. 4. Adjust the extracts to a final volume of 0.3 mL with 50mM K-phosphate (pH 7.8) and 0.1mM Na2EDTA. Allow the reaction to run for 10 min and stop it by switching the light off. 5. The change in NBT is determined by reading absorbance at 560 nm. One unit of SOD is defined as the amount of enzyme that produced a 50% inhibition of NBT reduction under the assay conditions.

Glutathione reductase (Groppa et al., 2001) 1. Homogenize 0.3 g of leaves under ice cold conditions in 3ml of extraction buffer containing 50mM Tris–HCl buffer (pH 7.6) and 1mM EDTA. 2. To 100 µL of the previous mixture add 1 mL of a reaction mixture containing 1mM EDTA, 0.5 mM GSSG, 0.15 mM NADPH, 50 mM Tris–HCl buffer (pH 7.5) and 3 mM MgCl2. 3. GR activity is measured by following the decrease in absorbance at 340 nm due to NADPH oxidation, during 1 min.

References Aebi, H.E. Catalase, in: H.U. Bergemeyer (Ed.), Methods of Enzymatic Analysis, third ed, Academic Press, NewYork, 1983, pp. 273–286. Li, H.S. Principles and techniques of plant physiological biochemical experiment. Higher Education Press, Beijing, 2000, (In Chinese). Groppa, M.D., Tomaro, M.L., Benavides, M.P. Polyamines as protectors against cadmium or copperinduced oxidative damage in sunflower leaf discs. Plant Sci. 161 (2001) 481-488. Ruley, A.T., Sharma, N.C., Sahi, S.V. Antioxidant defense in a lead accumulating plant, Sesbania drummondii. Plant Physiol. Bioch. 42 (2004) 899–906. United States Environmental Protection Agency, Plant peroxidise activity determination, SOP# 2035, 11/28/94. Xing, W., Li, D., Liu G. Antioxidative responses of Elodea nuttallii (Planch.) H. St. John to short-term iron exposure. Plant Physiol. Bioch. 48 (2010) 873-878.