I’m determining esterase activity with pNPB as substrate, using Tris-HCl buffer. In optimal pH assays (ranging from 2-12), I’m observing substrate hydrolysis in pH 7 and higher; the intensity of color development increases with pH, in mixtures containing only substrate and buffer. What could be the reason for this?
The reason is that the substrate, which is an ester, is unstable in water, with the rate of spontaneous hydrolysis being pH-dependent. The more acidic or basic the pH, the faster the rate of spontaneous hydrolysis. This spontaneous rate must be subtracted from the enzyme-catalyzed rate at each condition.
Hi there,
There is an other issue : pNP yellow color is dependent on pH as it is actually a tautomeric form of the phenolate ion which is absorbing (pK is around 7.2). For an accurate measurement of pNP formation, you should first incubate the mixture at a given pH and then dilute it into a given volume of NaOH (in order to get the same final basic pH around 9) and then measure the OD.
At the moment you can't conclude if the color is due to actual hydrolysis and/or the presence of pNP in the substrate solution (unless you have run kinetics and seen the increase in color intensity).
Apart from above one thing I would likes to add that preparing fresh substrate solution will reduce spontaneous hydrolysis. The substrate solution has to be stored at 4 degree Celsius.
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