Enzyme Activities Assay

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