Phenylalanine Ammonia Lyase (PAL) Assay

The PAL activity was assessed following the procedure outlined by Zucker (1965) (link). The homogenate was prepared in a chilled pestle and mortar by crushing 0.5 g of lead sample with 5 ml of sodium borate buffer (0.1 M) pH 8.5 solution. The slurry was centrifuged in a 2-ml tube at 10,000–12,000 rpm for 10 min. The supernatant was transferred to another 2-ml tube. 62.5 µl enzyme extract, 800 µl of sodium borate buffer, and 700 µl of (12 mM) phenylalanine were added in each test tube and incubated at 40°C in a water bath for 1 h. The reaction was halted by adding 200 µl of 5 N HCl, followed by the addition of 0.5 ml of 0.1 M Trans-cinnamic acid (TCA). The 62.5 µl enzyme extract and 800 µl of sodium borate buffer were added in each test tube, along with 700 µl of (12 mM) phenylalanine, and tubes were incubated at 40°C in a water bath for 1 h. 200 µl of 5 N HCl was added to stop the reaction. Then, 0.5 ml of trans-cinnamic acid (TCA) (1 M) was added, and the light absorbance was estimated at 290 nm. The absorbance reading was measured at 290 nm.