α-Chymotrypsin Assay

The modified method of Rehman et al was adopted for α-chymotrypsin enzyme inhibition activity. The contents of Tris-HCl buffer of 60 μl, 0.9 units (15 μl) purified chymotrypsin enzyme and 10 μl test compound was taken in a 100 μl total volume assay mixture. The assay mixture was incubated at 37°C for 10min and visualized at 410nm wavelength. The 15 μl (1.3 mM) of N-succinyl phenyl-alanine-P-nitroanilide (substrate) was added to start the reaction and check its absorbance change. The Chymostatin (0.5 mM/well) was considered as standard. Each reactions was done for three times and inhibition (%) was calculated by the following formula.

Inhibition (%) = (Abs. of Control- Abs. of Test / Abs. of Control) × 100

IC50 values (concentration at which there is 50% enzyme catalyzed reaction occur) compounds were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).