Enzyme Activities Assay
Reference: Venisse, J.-S., Gullner, G., and Brisset, M.-N. 2001. Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora. Plant Physiology 125: 2164-2172.
Enzyme Extraction:
Buffer:
50 mM sodium phosphate buffer (pH 7.5)
1 mM polyethyleneglycol
1 mM phenylmethylsulfonyl fluoride
8 % (w/v) polyvinylpyrolydone
0.01 % (v/v) Triton X-100
Enzyme Assay:
Ascorbate Peroxidase (AsPOX)
Absorbance: 290 nm, oxidation of ascorbic acid
Reaction Mixture: 10 μl leaf extract + 1 ml reaction mix
0.2 M Tris/HCL buffer (pH 7.8)
0.25 mM ascorbic acid
0.5 mM H2O2
Glutathione Reductase (GR)
Absorbance: 340 nm, oxidation of NADPH
Reaction Mixture: 50 μl leaf extract + 1 ml reaction mix
0.2 M Tris/HCL buffer (pH 7.8)
3 mM EDTA
0.2 mM NADPH
0.5 mM oxidized glutathione
Glutathione-S-Transferase (GST)
Absorbance: 340 nm
Reaction Mixture: 50 μl leaf extract + 1 ml reaction mix
0.1 M potassium phosphate (pH 6.5)
3.6 mM reduced glutathione
1 mM 1-chloro-2,4-dinitrobenzene
Peroxidase (POX)
Absorbance: 470 nm, formation of tetraguaiacol
Reaction Mixture: 50 μl leaf extract (5X or 10X dilution) + 2 ml reaction mix
50 mM sodium acetate buffer (pH 7)
25 mM guaiacol
25 mM H2O2
Enzyme Activities Assay by Chia-Lin Chung
1. 2 wk seedlings are inoculated by spraying 2.5 ml of 104/ml spore suspension (0.02% Tween 20) on leaf surface
2. 2 or 4 days after inoculation, move the plants from GH to the lab.
3. Cut one infected leaf (inoculated area) from the plant. Immediately weight and grind 0.25 g of leaf tissue with liquid nitrogen. Use a spatula to collect all the ground powder into a 1.5 ml or 2 ml microtube, add 1 ml extraction buffer, then vortex for a few seconds.
4. Centrifuge at 18000 rpm for 20min at 4oC. (Move the centrifuge into the cold room for at least overnight.)
5. Transfer the supernatant to a new microtube, and keep the crude extract on ice. Analyze the crude extract right away. The activity drops down if the sample is freeze-thawed.
6. Depends on the target enzyme, pipette 1 ml reaction mixture into 1.5 ml microtubes. Add 1~50 ul of the crude extract to the reaction mixture, vortex, and record the start point of time.
7. After certain amount of time (eg. 20 min for GST, 5 min for peroxidase), transfer the reaction samples to the cuvettes. To prevent the oxidation in the air, cover the opening of cuvettes with parafilm.
8. Use the spectrometer to read the absorbance at the specific wavelength. Record exact time of reading. Time course study is needed, because
Enzyme activity = [(A2 – A1) / (T2 – T1)] / mg protein = change of absorbance / per mg protein per min. (Determine protein concentration of the samples using Commassie (Bradford) Protein Assay Kit.)
