Purification of Paraoxonase (PON1) from Olive (Olea europaea L.) and Effect of Some Chemicals on Paraoxonase Activity in vitro

Abstract

Paraoxonase was purified from olive (Olea europaea L.) using sepharose 4B-L-tyrosine-1- naphthylamine affinity chromatography. This enzyme was purified 173.4-fold. SDS-polyacrylamide electrophoresis of the enzyme indicates a single protein staining band with an apparent Mr of 45 kDa. The kinetic properties of the purified enzyme were determined. The enzyme exhibited high activity at broad pH (pH 5.0-9.0) and temperature (40 and 70 °C). The purified enzyme remained stable at 4 °C for more than 1 year. Paraoxonase was mostly stable at 40 ºC. Its' activity decreased in 55 % for 1 h at 60 ºC and 20 % for 4 h at 50 ºC. Using paraoxon as a substrate, the K_m and V_max values for the purified enzyme were estimated to be 3.76 mM and 131.5 μmol L dak^-1, respectively. The activities were strongly inhibited by Hg²⁺ and Fe³⁺, while Cu²⁺, b-mercaptoethanol, dithioerythritol and SDS slightly activated the enzyme. As judged by catalytic efficiencies, paraoxon is the preferred substrate.