What is the role of BAPNA in the assay?

• A. A substrate for pepsin that releases a detectable product
• B. A buffer
• C. A protein substrate
• D. A color indicator

Answer: (A)

BAPNA serves as a convenient substrate for pepsin in this assay. It’s a synthetic molecule (N-benzoyl-D-arginine-p-nitroanilide) that pepsin cleaves, and when the peptide bond is hydrolyzed, a colored product, p-nitroaniline, is released. That product has a bright yellow color and can be measured with a spectrophotometer, so the amount of color produced directly reflects the enzyme’s activity. So the role is to provide a defined substrate that yields a detectable signal upon enzymatic cleavage. It isn’t simply a buffer, a protein substrate, or a standalone color indicator.