Chia-Lin Chung

R. Nelson Lab

Cornell University

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.)

Preparation of the Reagents

Enzyme Extraction:

Extraction Buffer (freshly prepared)

50 ml / 100 ml / 400 ml
50 mM sodium phosphate buffer (pH 7.5) / 0.4 ml 2M monobasic (NaH2PO4 H2O; MW 137.99 )
2.1 ml 2M dibasic (Na2HPO4, anhydrous MW 141.96) / 1.6 ml 2M monobasic (NaH2PO4 H2O; MW 137.99 )
8.4 ml 2M dibasic (Na2HPO4, anhydrous MW 141.96)
1 mM polyethyleneglycol (M.W. 8000) / 0.4 g / 0.8 g / 3.2 g
1 mM phenylmethylsulfonyl fluoride (M.W. 174.19)
- 0.1 M PMSF stock: 0.87 g PMSF + 50 ml isopropanol, store at 4oC) / 500 ul 0.1 M PMSF stock
(add right before use) / 1 ml 0.1 M PMSF stock
(add right before use) / 4 ml 0.1 M PMSF stock
(add right before use)
8 % (w/v) polyvinylpyrolydone (M.W. 40000) / 4 g / 8 g / 32 g
0.01 % (v/v) Triton X-100 / 50 μl / 100 μl / 400 μl

Enzyme Assay:

Ascorbate Peroxidase (AsPOX)

Absorbance: 290 nm, oxidation of ascorbic acid (decrease of absorbance at 290 nm)

Reaction Mixture: 10 μl leaf extract + 1 ml reaction mix

Reaction Mix

50 ml / 100 ml / 400 ml
0.2 M Tris/HCL buffer (pH 7.8) / 20 ml 1M Tris-HCl / 80 ml 1M Tris-HCl
0.25 mM ascorbic acid (M.W. 176.1)
- 50 mM ascorbic acid stock: 0.088 g ascorbic acid + 10 ml H2O, store at 4oC, only for 1 wk) (can be oxidized in the air in 1 day) / 250 ul 50 mM ascorbic acid stock / 500 ul 50 mM ascorbic acid stock / 2 ml 50 mM ascorbic acid stock
0.5 mM H2O2 (30%, M.W. 34) / 2.85 μl / 5.7 μl

Glutathione Reductase (GR)

Absorbance: 340 nm, oxidation of NADPH

NADPH + H+ + GSSR → 2GSH + NADP+; detect the oxidation of NADPH (decrease of absorbance at 340 nm)

Reaction Mixture: 50 μl leaf extract + 1 ml reaction mix

Reaction Mix

50 ml / 100 ml / 400 ml
0.2 M Tris/HCL buffer (pH 7.8) / 20 ml 1M Tris-HCl / 80 ml 1M Tris-HCl
3 mM EDTA / 0.6 μl 0.5M EDTA / 2.4 μl 0.5M EDTA
0.2 mM NADPH (M.W. 833)
- 8.4 mM NADPH stock: 0.0294 g NADPH + 4.2 ml H2O, aliquot then store at -20 oC) / 1.19 ml 8.4 mM NADPH stock / 2.38 ml 8.4 mM NADPH stock
0.5 mM oxidized glutathione (M.W. 612.7)
- 50 mM GSSG stock: 0.306 g oxidized glutathione + 10 ml H2O, aliquot then store at -20 oC) / 0.5 ml 50 mM GSSG stock / 1 ml 50 mM GSSG stock

Glutathione-S-Transferase (GST)

Absorbance: 340 nm

GSH + CDNB → GS-DNB conjugate + HCl, detect the formation of GS-DNB conjugate (increase of absorbance at 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

Reaction Mix

50 ml / 100 ml / 400 ml
0.1 M potassium phosphate (pH 6.5) / 6.7 ml 2M monobasic (KH2PO4)
3.295 ml 2M dibasic (K2HPO4) / 26.8 ml 2M monobasic (KH2PO4)
13.18 ml 2M dibasic (K2HPO4)
3.6 mM reduced glutathione (M.W. 307.3)
- 360 mM stock: 1.106 g reduced glutathione + 10 ml H2O, aliquot then store at -20 oC) / 500 ul 360 mM reduced glutathione stock / 1 ml 360 mM reduced glutathione stock
1 mM 1-chloro-2,4-dinitrobenzene (M.W. 202.55)
- 100 mM stock: 0.405 g CDNB + 20 ml EtOH, store at -20 oC) / 500 ul 100 mM CDNB stock / 1 ml 100 mM CDNB stock

Peroxidase (POX): Guaiacol smells really bad. The whole process has to be conducted IN THE HOOD.

Absorbance: 470 nm, formation of tetraguaiacol

4 guaiacol + 2 H2O2 → tetraguaiacol + 8 H2O

formation of tetraguaiacol (increase of absorbance at 470 nm)

Reaction Mixture: 1 μl leaf extract + 1 ml reaction mix

50 mM sodium acetate buffer (pH 7)

25 mM guaiacol

25 mM H2O2

Reaction Mix

50 ml / 100 ml / 400 ml
50 mM sodium acetate buffer (pH 7) / 0.41 g sodium acetate (M.W. 82.03) / 1.64 g sodium acetate (M.W. 82.03)
25 mM guaiacol (M.W. 124.14) / 155 μl / 310 μl
25 mM H2O2 (30%, M.W. 34) / 142.5 μl / 285 μl