We are looking for a good assay to measure pepsin activity (not concentration, we already have that with ELISA) in samples. We need something that won't have overlap with other enzymes because our sample has lots of other enzymes in it and we will measure those separately. We need something really specific for pepsin! Any ideas? Any suggestions are greatly appreciated! :)
Hi David,
The most convenient assay for pepsin activity we’ve used (when rarely need) was that one described in Pharmacopeial Forum, Vol 30(1), 2004. Briefly: USP “Pepsin”, not less than 0.5 USP Pepsin Units per mg, dried basis. Hemoglobin substrate. Spectrophotometric assay. “One USP Unit of Pepsin activity is the activity that releases the equivalent of 1 μmole of tyrosine per minute under conditions of the Assay.” Identification - Fibrin blue: “The Identification test is based on the ability of Pepsin to cleave the chromophore from an insoluble protein-dye complex.” Preserve in tight containers, protected from light, and store between 20C and 80C.
Best wishes,
Ilya
Hi Ilya, thanks for your response. I heard about this assay but I thought that the hemoglobin would just be cleaved by all the proteases in my sample, not just pepsin. Is there something that makes it specific for pepsin?
I don’t know how to measure pepsin activity only, but maybe you can estimate that by crossing the concentration of pepsin in your sample (your ELISA test) and the specific activity of pure pepsin (U/mg of enzyme) measured in your specific assay conditions.
Kind regards,
Samira
Dear colleague David,
One has to face reality of the fact that pepsin has BROAD substrate specificity. From X-ray diffraction techniques determined the three-dimensional structure of pepsin through the mid-1970s thus allowing for a better understanding of the catalytic reaction (Fruton 2002), to recent renewed interest to pepsin due to the recognition of HIV-protease as a member of this aspartic protease family (Campos 2003) – all data evidenced that the enzyme has broad specificity with a preference for peptides containing linkages with aromatic or carboxylic L-amino acids. It preferentially cleaves C-terminal to Phe and Leu and to a lesser extent Glu linkages. For the protein to be active, one of the two aspartate residues in the catalytic site has to be protonated, and the other deprotonated. Even with its active site residues are precisely known (aspartic acid at D32 and D215), pepsin worldwide used to use as highly non-specific enzyme, in applications such as digestion of antibodies; preparation of collagen for cosmeceutical purposes; assessment of digestibility of proteins in food chemistry; subculture of viable mammary epithelial cells.
Sincerely,
Ilya
you can make your question to Mr Do Chau Minh Vinh Tho and Prof. Nguyen Trung Kien, they can help you to solve your issue!!
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