Trypsin Inhibitor Activity Assay

The extraction and determination of trypsin inhibitor activity (TIA) was carried out using the method of Liu (37) with some modifications. Approximately 0.5 g dried powder of Tecticornia sp. was extracted with 25 mL NaOH. Then the mixture was shaken using a reciprocating shaker (RP1812, Paton Scientific) for 1 hr at 200 rpm. The mixture was allowed to settle for 10 min and the extract was carefully decanted without centrifugation for TIA estimation. For the TIA assay, an inhibitor assay buffer was prepared which contained 20 mM CaCl₂ and 50 mM Tris-HCl at pH 8.2. The N-benzoyl-D-L- arginine- p-nitroanilide (DL-BAPA) substrate (0.4 mg/mL) was prepared fresh on the same day in the assay buffer that contained 1% dimethyl sulfoxide solution and pre-warmed at 37°C. An aliquot (2.5 mL) of the DL-BAPA substrate was added to 1 mL of the diluted extract after which 1.0 mL bovine trypsin (20 μg/mL in 1 mM HCl solution containing 5 mM CaCl₂) was added and immediately mixed. The whole assay was conducted in a water bath at 37°C. Following incubation for 10 min at 37°C, the color reaction was terminated by addition of 0.5 mL of 30% acetic acid solution. The mixture was centrifuged at 3,000 × g for 10 min and the absorbance for the sample reading (A410S) at 410 nm was a measure of the trypsin activity in the presence of the sample inhibitors. The absorbance was read using a spectro-photo meter (Thermo Fisher Scientific Genesys 20, Melbourne, VIC, Australia) at 410 nm. Concurrently, the reaction was also run in the absence of inhibitors by replacing the sample extract with an equal amount of reverse osmosis (RO) water and reference reading was also recorded as A410R. Furthermore, sample blanks (A410SB) and reference blank (A410RB) were also run by adding the acetic acid solution before the trypsin solution. A trypsin unit is defined as an increase of 0.02 absorbance at 410 nm. The TIA is expressed in trypsin units inhibited (TUI) per mg sample and calculated as follows:

Where CS (corrected sample reading) = A410S-A410SB.

CR (corrected reference reading) = A410R-A410RB.