Hydrolysis of the NHS ester competes with the primary amine reaction.
The rate of hydrolysis increases with buffer pH and contributes to less-efficient crosslinking in less-concentrated protein solutions. The half-life of hydrolysis for NHS-ester compounds is 4 to 5 h at pH 7.0 and 0°C. This half-life decreases to 10 mins at pH 8.6 and 4°C. The extent of NHS-ester hydrolysis in aqueous solutions free of primary amines can be measured at 260 to 280 nm, because the NHS byproduct absorbs in that range.