I am doing a carbonic anhydrase activity assay using p-nitrophenyl acetate.
reaction conditions: 37 °C for 10 minutes
Blank: 40ul substrate + 160 ul 50mM Tris buffer pH 7.4
crude: 40 ul substarte+ + 60 ul 50 mM Tris buffer pH 7.4 + 100ul carbonic anhydrase crude
After 10 minutes of reactions, I noticed a yellow color in all the reaction tubes except the blank, which remained colorless. To stop the enzyme reaction, I added 800 ul of 2M Na2CO3 to all the tubes. Surprisingly, even the blank changed color to yellow. As a next step, I examined the absorbance at 400 nm. However, the results obtained turned out to be inaccurate. Upon rechecking the values, I observed either lower values or, at times, even negative values. I am consistently receiving a lot of unreliable results. What steps should I take to address this issue?
The pH value of your sodium carbonate solution is likely around 11.5 and I would expect your p-nitrophenyl acetate to hydrolyze rapidly at this pH value to give yellow ionized p-nitrophenol.