2.9. Extraction and Measurement of Phenylalanine Ammonia Lyase (PAL) Activity

Leaf samples (150 mg fresh weight) were extracted in 2 mL of buffer (100 mM Tris pH 8.8, 14 mM 2-mercaptoethanol and 3% w/v polyvinylpolypyrrolidone) and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected, and the total protein concentration was determined using the Bradford assay [21]. PAL activity was measured according to the procedure described by El-Shora et al. [22]. The reaction mixture contained 1.9 mL of 100 mM Tris–HCl buffer (pH 8.8), 1 mL of 15 mM l-phenylalanine and 100 µL of enzyme extract. After incubating at 30 °C for 15 min, the reaction was stopped by adding 200 µL of 6 M HCl, and A₂₉₀ was measured. One unit of PAL activity was defined as the number of enzymes leading to the conversion of 1 mmol of l-phenylalanine into trans-cinnamic acid per minute. The trans-cinnamic acid synthesized was calculated using its molar extinction coefficient (9630/M/cm) and expressed as nmol trans-cinnamic acid/min/mg of protein.