UV-VIS Anti-Trypsic Assay

UV-VIS Anti-Trypsic Assay

Materials and Reagents

• Plant extract with antitryptic activity
• Trypsin enzyme solution
• Substrate (e.g., Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA))
• Tris-HCl buffer (50 mM, pH 8.0)
• Dimethyl sulfoxide (DMSO) or another suitable solvent for dissolving BAPNA
• UV-VIS spectrophotometer
• Quartz cuvettes
• Distilled water

Preparation of Solutions

1. Trypsin Solution: Dissolve trypsin in Tris-HCl buffer to a concentration of 0.01 mg/mL.
2. Substrate Solution (BAPNA): Dissolve BAPNA in DMSO to make a 5 mg/mL stock solution. Dilute this stock solution with Tris-HCl buffer to a final concentration of 0.1 mM.
3. Plant Extract Solution: Dissolve the plant extract in Tris-HCl buffer. Prepare different concentrations for testing (e.g., 0.1 mg/mL, 0.2 mg/mL, etc.).

Assay Procedure

1. Blank Preparation: Mix 2.9 mL of Tris-HCl buffer with 0.1 mL of the substrate solution. This serves as a blank to zero the spectrophotometer.
2. Control Reaction: Mix 2.8 mL of Tris-HCl buffer, 0.1 mL of substrate solution, and 0.1 mL of trypsin solution. This will measure the activity of trypsin without inhibition.
3. Test Reaction: Mix 2.7 mL of Tris-HCl buffer, 0.1 mL of substrate solution, 0.1 mL of trypsin solution, and 0.1 mL of plant extract solution. Prepare separate test reactions for each concentration of plant extract.

Measurement

1. Incubation: Incubate all reaction mixtures at 37°C for 10 minutes.
2. Measurement: Measure the absorbance at 410 nm using the UV-VIS spectrophotometer. The increase in absorbance is due to the release of p-nitroaniline from the substrate by trypsin action.

Calculation of Antitryptic Activity

1. Determine the Change in Absorbance:

   • ΔAcontrol\Delta A_{\text{control}}ΔAcontrol​ = Absorbance of control reaction - Absorbance of blank
   • ΔAtest\Delta A_{\text{test}}ΔAtest​ = Absorbance of test reaction - Absorbance of blank

2. Inhibition Percentage:

   Inhibition (%)=(ΔAcontrol−ΔAtestΔAcontrol)×100\text{Inhibition (\%)} = \left( \frac{\Delta A_{\text{control}} - \Delta A_{\text{test}}}{\Delta A_{\text{control}}} \right) \times 100Inhibition (%)=(ΔAcontrol​ΔAcontrol​−ΔAtest​​)×100