Hi there,

What is actually your assay? Lipase will have no activity on pNP alone but on pNP esters. So the substrate for such assay has to be a pNP ester of a fatty acid.

Hello Dominique,

Thanks for pointing that out! I just realized that I had made a mistake in my prompt. I am using 4-nitrophenyl laurate as my substrate and not p-nitrophenol. I also corrected my post to clear up any further confusion.

12/16/21

Dear Aidan,

This is probably NOT the reason why you are getting the results you describe, but at what pH are you conducting the lipase assay? Also, how do you terminate the enzymolysis? As you know, the lipase releases pNP from pNP-laurate. The expected yellow color is not due to pNP, but to the p-nitrophenolate anion. To see this, the final pH needs to be above 5.9. At a lower pH, pNP is protonated and therefore colorless. Only after de-protonation to the anionic form is the yellow color visible. In enzyme assays where pNP is released (e.g., phosphatase assays using p-nitro-pheny-PO4), the reaction is stopped by adding NaOH to deprotonate pNP and visualize the yellow color. My guess is that you already know this, but I mention it only as a possibility.

I hope you can get the lipase assay to work.

Bill Colonna Ames, Iowa, USA [email protected]