Purification of paraoxonase enzyme from pumpkin seeds (Cucurbitae peponis semen) and effect of some chemicals and pesticides on enzyme activity

Abstract

An organophosphate-degrading enzyme known as paraoxonase is connected to high-density lipoprotein (HDL) and has a physiological role, such as primarily protecting low-density lipoprotein (LDL) from oxidation. The aim of this study is to purify the paraoxonase enzyme (EC: 3.1.8.1.) from pumpkin seeds and investigate the effect of some chemicals and pesticides on enzyme activity. Paraoxonase enzyme from pumpkin seeds was purified 158.09 fold with a yield of 77.55% using sepharose 4B-L-tyrosine-1-naphthylamine affinity chromatography. The purity of the purified paraoxonase enzyme was checked by SDS-PAGE electrophoresis. The molecular weight of the purified paraoxonase enzyme was determined as 12942 Da and 20421 Da by gel filtration chromatography. K_m and V_max values were calculated as 3.475 mM and 2.935 µmol L^-1.min^-1 for paraoxon substrate, 3.067 mM and 2.647 µmol L^-1.min^-1 for phenyl acetate substrate. Mg²⁺, Cu²⁺, Mn²⁺ and Fe³⁺ cations, ascorbic acid, EDTA, β-mercaptoethanol and SDS compounds, diazinon, methyl parathion, ethyl parathion, dimethoate, dichlorvos, fention, acetamiprid and methamidophos pesticides strongly inhibited the enzyme. The analysis results showed that β-mercaptoethanol (IC₅₀: 0.90 mM) was the most potent inhibitor among cations and compounds, dichlorvos (IC₅₀: 0.91 mM) among pesticides.