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.
Accurate measurement of enzyme activity requires that the 4-nitrophenol product is fully ionized as 4-nitrophenolate, which is necessary due to its low absorbance at 405 nm. The deprotonation of the alcohol functional group also impacts the conjugation of the pi bonds in the compound.