Accurate measurement of enzyme activity requires that the 4-nitrophenol product is fully deprotonated, existing as 4-nitrophenolate, given the weak absorbance of 4-nitrophenol at 405 nm. Complete ionization of the alcohol functional group affects the conjugation of the pi bonds on the compound.
To accurately measure enzyme activity, it is essential that the 4-nitrophenol product is fully ionized as 4-nitrophenolate, due to its low absorbance at 405 nm. The deprotonation of the alcohol group also affects the conjugation of the pi bonds in the compound.