Purification of Paraoxonase Enzyme from Pumpkin Seeds (Cucurbitae peponis semen) and Effectof Some Chemicals and Pesticides on Enzyme Activity

Year 2024, Volume: 8 Issue: 1, 12 - 17, 15.07.2024

Elife Kaya , Nazan Demir

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 chemicalsand 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. Km and Vmaxvalues were calculated as 3.475 mM and 2.935 μmol L-1.min-1for paraoxon substrate, 3.067 mM and 2.647 μmol L-1.min-1for 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.

Keywords

Paraoxonase, Enzymes, Purification, Inhibitors, Pesticides

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