Phenol → 4-Nitrophenol

Participants

Reaction Steps

A convenient assay of AAD-13 was devised using 2-(2-chloro,4-nitrophenoxy)propionate (CNPP) as substrate. Cleavage of CNPP by AAD-13 will release 2-chloro,4-nitrophenol. This phenol has a bright yellow absorbance at 410 nm at pH 7 enabling the reaction to be followed continuously or by endpoint analysis. The presence of AAD-13 activity can be monitored visually without the need for addition of further reagents. Microplate-based spectrophotometric assays were performed in a total volume of 0.2 ml 20 mM MOPS pH 6.75 containing 200 μM NH4FeSO4, 200 μM sodium ascorbate, 1 mM α-ketoglutarate, the appropriate amount of CNPP (added from a 10 mM stock made up in DMSO), and enzyme. Assays are initiated by addition of CNPP, enzyme, or α-ketoglutarate at time zero and the increase in absorbance followed for 10 min at 410 nm in a microplate reader. The first 2 min of the reaction will be used to determine initial rates. A calibration curve performed under standard assay conditions (200 μl final assay volume) indicated that an absorbance at 410 nm of 0.1 was obtained from 25.1 μM 2-chloro, 4-nitrophenol. Using this assay, the kinetic constants for CNPP as a substrate were determined to be Km=31±5.5 μM and kcat=16.2±0.79 min−1.

Provenance & Evidence