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
 Chia-Lin Chung
R. Nelson Lab
Cornell University

Enzyme Activities Assay by Chia-Lin Chung

1. 2 wk seedlings are inoculated by spraying 2.5 ml of 10 4/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 0.4 ml 2M monobasic 1.6 ml 2M monobasic
(pH 7.5) (NaH2PO4 H2O; MW (NaH2PO4 H2O; MW
137.99 ) 137.99 )
2.1 ml 2M dibasic 8.4 ml 2M dibasic
(Na2HPO4, anhydrous (Na2HPO4, anhydrous
MW 141.96) MW 141.96)
1 mM polyethyleneglycol (M.W. 0.4 g 0.8 g 3.2 g
8000)
1 mM phenylmethylsulfonyl 500 ul 0.1 M 1 ml 0.1 M PMSF 4 ml 0.1 M PMSF stock
fluoride (M.W. 174.19) PMSF stock stock (add right before use)
- 0.1 M PMSF stock: 0.87 g (add right (add right before use)
PMSF + 50 ml isopropanol, before use)
 Chia-Lin Chung
R. Nelson Lab
Cornell University

store at 4oC)
8 % (w/v) polyvinylpyrolydone 4g 8g 32 g
(M.W. 40000)
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- 80 ml 1M Tris-
HCl HCl
0.25 mM ascorbic acid (M.W. 176.1) 250 ul 50 mM 500 ul 50 mM 2 ml 50 mM
- 50 mM ascorbic acid stock: 0.088 g ascorbic acid ascorbic acid ascorbic acid
ascorbic acid + 10 ml H2O, store at stock stock stock
4oC, only for 1 wk) (can be
oxidized in the air in 1 day)
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- 80 ml 1M Tris-
HCl HCl
3 mM EDTA 0.6 μl 0.5M EDTA 2.4 μl 0.5M EDTA
0.2 mM NADPH (M.W. 833) 1.19 ml 8.4 mM 2.38 ml 8.4 mM
- 8.4 mM NADPH stock: NADPH stock NADPH stock
0.0294 g NADPH + 4.2 ml
H2O, aliquot then store at -20
o
C)
0.5 mM oxidized glutathione 0.5 ml 50 mM 1 ml 50 mM
(M.W. 612.7) GSSG stock GSSG stock
- 50 mM GSSG stock: 0.306 g
oxidized glutathione + 10 ml
H2O, aliquot then store at -20
o
C)

Glutathione-S-Transferase (GST)
Absorbance: 340 nm
 Chia-Lin Chung
R. Nelson Lab
Cornell University

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

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 1.64 g sodium acetate
(M.W. 82.03) (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