Dear Sanaullah, you may use the following methods for the determination of enzymatic activities of leaf samples.

Fresh leaf material (0.25 g) was homogenized in 5 mL (50 mM) prechilled

potassium phosphate buffer (7.8 pH) with pre-chilled mortar

and pestle in an ice bath and centrifuged the mixture at 12,000 × g for

20 min at 4 °C. The supernatant was separated and stored at −20 °C for

the determination of the following antioxidant enzymes.

Superoxide dismustase (SOD)

The Van Rossum et al. (1997) protocol was employed to determine

the activity of SOD. Following it, 400 μL distilled water, 250 μL

(50 mM) potassium phosphate buffer (pH 7.8), 100 μL L-methionine,

100 μL tritron-X, 50 μL nitrobluetetrazolium (NBT), 50 μL riboflavin

and 50 μL leaf sample were added to plastic cuvettes. The plastic cuvettes

were illuminated under the light for 5 min and absorbance recorded

at 560 nm using a spectrophotometer.

Peroxidase (POD)

The POD activity was measured using the method proposed by

Chance and Maehly (1955). According to this procedure, 1.8 mL

(50 mM) potassium phosphate buffer (7.8 pH), 100 μL sample, 100 μL

(20 mM) guaiacol and 100 μL (40 mM) H2O2 were added to a quartz

cuvette, respectively and a change in absorbance was observed at

470 nm after every 20 s for 3 min.

Catalase (CAT)

CAT activity was appraised using the procedure of Chance and

Maehly (1955). The CAT activity was determined by adding 1.9 mL

potassium phosphate buffer (50 mM, pH 7.8), 100 μL sample and 1 mL

of 5.9 mM H2O2 in a quartz cuvette and a change in OD was recorded at

240 nm after every 20 sec. for 2 min.

See the attached PDFs. Hopefully these will solve your problem.