Investigating physiological and biochemical

Superoxide dismutase (SOD) activity was determined by the nitro blue 
tetrazolium (NBT) method (Giannopolitis and Ries, 1977) by measuring the 
photoreduction of NBT at 560 nm. The reaction mixture (3 ml) contained 50 mM 
sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 10 µM EDTA, 2 
mM riboflavin and enzyme extract (100µl). Reaction was started by placing tubes 
below two 15 W fluorescent lamps for 10 min, then stopped by switching off the light. 
Non-illuminated and illuminated reactions without supernatant served as calibration 
standards. The absorbance was measured at 560 nm. One unit of SOD was defined as 
the quantity of enzyme that produced 50% inhibition of NBT reduction under the 
experimental conditions. 
Catalase (CAT) activity was determined according to Aebi (1974). The assay 
mixture (3 ml) consisted of 100µl enzyme extract, 100µl H
2
O
2
(300 mM) and 2.8 ml 
50mM phosphate buffer with 2 mM EDTA (pH 7.0). The CAT activity was assayed 
by monitoring the decrease in the absorbance at 240 nm as a consequence of H
2
O
2
disappearance (molar extinction coefficient (ε)= 39.4 mM cm
-1
).
Peroxidase (POD) activity was measured according to the method of Amako et 
al. (1994) with some modification. The reaction mixture (3 ml) consisted of 100µl 
enzyme extract, 100µl guaiacol, 100µl H
2
O
2
(300 mM) and 2.7 ml 25 mM potassium 
phosphate buffer with 2 mM EDTA (pH 7.0). Increase in the absorbance due to 
oxidation of guaiacol was measured spectrophotometrically at 470 nm (ε= 26.6 mM 
cm
-1
).
Ascorbate peroxidase (APX) activity was assayed according to the method of 
Nakano and Asada (1981). The reaction mixture consisted of 100µl enzyme extract, 
100µl ascorbate (7.5 mM), 100µl H
2
O
2
(300 mM) and 2.7 ml 25 mM potassium 
phosphate buffer with 2 mM EDTA (pH 7.0). The oxidation of ascorbate was 
determined by the change in absorbance at 290 nm (ε= 2.8 mM cm
-1
)