Objective

To measure the anti-trypsin activity of a plant extract or compound using UV-VIS spectroscopy, assessing its ability to inhibit the activity of trypsin on a substrate (such as BAPNA or casein).

Materials

• Trypsin enzyme: Bovine trypsin, typically from pancreas (lyophilized powder)
• Substrate: N-Benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) or casein
• Buffer: Tris-HCl (50 mM, pH 7.8) or Phosphate-buffered saline (PBS) (50 mM, pH 7.4)
• Plant extract/compound: Test sample with potential anti-trypsin activity
• Dimethyl sulfoxide (DMSO) or another suitable solvent (if required to dissolve plant extract)
• UV-VIS spectrophotometer
• Cuvettes: 1 cm path length
• Water bath/incubator: Set to 37°C
• Stop solution: Acetic acid or trichloroacetic acid (TCA, 10%)

Method

1. Preparation of Solutions

   • Trypsin Stock Solution: Dissolve trypsin in Tris-HCl buffer to a final concentration of 1 mg/mL. Prepare aliquots and store at −20°C.
   • BAPNA Substrate Solution: Dissolve BAPNA (1 mM) in Tris-HCl buffer. Alternatively, dissolve casein (1%) in PBS for casein assay.
   • Plant Extract Solution: Dissolve the plant extract in an appropriate solvent (such as DMSO) to a final concentration of 1 mg/mL. Prepare a range of dilutions for IC50 determination.

2. Control and Test Samples

   • Blank Control: Contains buffer and substrate but no trypsin or plant extract.
   • Positive Control: Contains trypsin and substrate without plant extract to measure maximum enzyme activity.
   • Test Sample: Contains trypsin, substrate, and various concentrations of plant extract to determine its inhibitory effect.

3. Incubation

   • In each cuvette, add the following components:
     • Blank Control: 900 µL buffer + 100 µL substrate
     • Positive Control: 800 µL buffer + 100 µL substrate + 100 µL trypsin
     • Test Sample: 700 µL buffer + 100 µL substrate + 100 µL trypsin + 100 µL plant extract solution (vary concentrations of plant extract for inhibition studies)
   • Mix gently and incubate at 37°C for 30 minutes.

4. Reaction Termination

   • After the incubation period, add 100 µL of stop solution (10% TCA) to terminate the reaction in each cuvette. This will precipitate the protein in casein assays or stop the release of p-nitroaniline from BAPNA.

5. UV-VIS Measurement

   • For BAPNA assay, measure the absorbance at 410 nm (corresponding to the yellow color of p-nitroaniline) using a UV-VIS spectrophotometer.
   • For casein assay, measure the absorbance at 280 nm to detect the amount of released tyrosine or other aromatic amino acids from casein digestion.
   • Record absorbance values for each sample.

Notes

• Ensure that the solvent used for the plant extract does not interfere with enzyme activity or UV-VIS measurements.
• Maintain all samples at 37°C during incubation for optimal enzyme activity.
• Replicate each sample in triplicate to ensure reliable results.

Results and Interpretation

• A decrease in absorbance at 410 nm (for BAPNA) or 280 nm (for casein) in the presence of plant extract indicates trypsin inhibition.
• The plant extract's ability to inhibit trypsin can be quantified by comparing it to the positive control.