A simple method to determine trypsin and chymotrypsin inhibitory activity

Area of Science:

• Biochemistry
• Enzymology
• Protein Expression Analysis

Background:

• Serine proteases are crucial enzymes with diverse biological roles.
• Accurate quantification of protease inhibitors is essential for research and development.
• Existing methods like gel activity assays can lack quantification accuracy or sensitivity.

Purpose of the Study:

• To modify and validate a colorimetric assay for serine protease inhibition.
• To quantify recombinant Bowman-Birk Inhibitor (rBBI) expression in Pichia pastoris.
• To compare the modified assay's performance against existing methods.

Main Methods:

• A colorimetric method using N-Acetyl-DL-Phenylalanine beta-Naphthylester (APNE) and o-Dianisidine tetrazotized (oD) was optimized.
• Standard curves for trypsin, chymotrypsin, and Bowman-Birk Inhibitor (BBI) were generated.
• Recombinant BBI (rBBI) expression levels in Pichia pastoris were quantified using the modified assay.

Main Results:

• The modified assay demonstrated strong linearity and sensitivity for trypsin and chymotrypsin.
• IC50 values for trypsin and chymotrypsin inhibition by BBI were determined.
• Quantification of rBBI expression yielded levels of 50-56 ng/microl, consistent with Western blot analysis.

Conclusions:

• The modified colorimetric assay provides a sensitive and reproducible method for quantifying serine protease inhibitors.
• This assay effectively quantifies rBBI expression, overcoming limitations of the gel activity method.
• The developed method combines the sensitivity of gel assays with the quantification capabilities of Western blots.