UV-VIS Anti-Trypsic Assay

  • Product Code: 125265
Colorimetric assay measuring inhibition of porcine trypsin hydrolysis of BAPNA substrate by absorbance at 410 nm. BAPNA is a manufactured protein substrate comprising of a color covalently bound to an amino acid. BAPNA has an amino acid with a dye molecule attached to it; enzymatic activity by trypsin breaks the bond between the amino acid and the dye molecule, coloring the clear solution yellow.
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label Sample Names

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assignment Instructions

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description Sample Requirement

Liquid extract or solution 2 mL

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timeline Service Steps

Step Procedure Expected Result
1

Add 40...
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enzyme
2

Add 40...
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sample
3

Incubate for...
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pre-incubation of sample and enzyme
4

Add 100...
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start reaction
5

Incubate for...
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incubation time for reaction
6

Add 20...
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stop reaction
7

Read the...
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reading absorbance
8

Use distilled...
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ensure test reaction
9

The enzyme...
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% inhibition of assay

UV-VIS Anti-Trypsic Assay

UV-VIS Anti-Trypsic Assay

UV-VIS Anti-Trypsic Assay

Materials and Reagents

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
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Service Steps
Step Procedure Expected Result
1

Add 40...

enzyme
2

Add 40...

sample
3

Incubate for...

pre-incubation of sample and enzyme
4

Add 100...

start reaction
5

Incubate for...

incubation time for reaction
6

Add 20...

stop reaction
7

Read the...

reading absorbance
8

Use distilled...

ensure test reaction
9

The enzyme...

% inhibition of assay