Hi there,

pKa of BES is around 7.1 then BES buffer has to be used between pH 6.1 and 8.1. To observe PNP coloration, pH has to be slightly basic in order to get the phenolate form which is actually the colored one (pKa of PNP is around 7.1 too). So pH between 7.5 and 8 should be OK (the higher pH the better but if running continuous assay the pH has to ensure enzyme stability too)

Dear Neeta

pKa of BES is near about 7.0.

Dear Neeta,

I agree w/ the answers provided by Dominique Liger and Sayyed IIiyas. One question: What is the pH optimum of the amylase you are using, and are you running a continuous assay where you monitor pNP release over time, or a fixed-time assay where you incubate for a pre-determined time, then stop the reaction and read the absorbance of the released pNP? If the pH optimum of the amylase is neutral or slightly alkaline, you can do the continuous assay without a problem. However, if the pH optimum is acidic, e.g., ~pH 4-5, then you probably won't be able to do a continuous assay, as pNP is ~colorless in this pH range. In that case, you will have to do the assay for a fixed time, after which you can stop the reaction w/ base, e.g., NaOH, to bring the pH into the alkaline range so you can read the absorbance of the pNP.

I hope this helps you.

Bill Colonna, Center for Crops Utilization Research, Iowa State University, Ames, IA, USA.