Enzymatic p-Nitroaniline Hydrolysis

From a 100 mM p-NA stock solution (prepared in acetonitrile, CH₃CN), a 1 mM p-NA solution in PB was freshly prepared (pH = 7.2, 1 vol% CH₃CN). This substrate solution was passed through the filter holder at 3 μL min⁻¹ for 180 min by using a syringe pump (AL1000 from World Precision Instruments). Every 3 min, 2 μL were sampled from the outflow with a Gilson Pipetman P10, and the absorption spectrum was measured with the NanoDrop One instrument (l = 0.1 cm, background: PB). Values of A₄₀₅ were taken as a measure of the formed p-nitrophenolate. The total concentration of p-nitrophenolate + p-nitrophenol in the outflow was calculated on the basis of ε₄₀₅ (p-nitrophenolate) = 10 510 M⁻¹ cm⁻¹,^{61 (link)} and [p-nitrophenolate]/[p-nitrophenol] = 1.15 at pH 7.2 using pK_a (p-nitrophenol) = 7.14.^{52 (link)} Since p-NA undergoes significant autohydrolysis, reference measurements were carried out with “empty” filters (PB only). Data for this non-enzymatic p-NA hydrolysis were subtracted from the data obtained for the filter with immobilised BCA.