Purification of paraoxonase enzyme by hydrophobic interaction technique and investigation of its affinity to certain compounds

Paraoxonase 1 (PON1) is an isoenzyme synthesized in the liver and functions in association with certain lipoproteins. This enzyme, present in all mammals, exhibits antiatherosclerotic properties due to its ability to inhibit the oxidation of low-density lipoprotein (LDL). Additionally, it is known to exhibit hydrolytic properties towards various compounds, including organophosphate insecticides and homocysteine thiolactones. Therefore, extensive research has been conducted on factors that can modulate serum PON1 activity. However, literature research has revealed that studies on animal PON1 enzymes are extremely limited, particularly on sheep and cattle. This study, conducted as a doctoral thesis, can be divided into two parts. In the first part, hsPON1 was purified from bovine serum using a hydrophobic gel with a chemical structure of Sepharose 4B-L-tyrosine-naphthylamine, previously synthesized by our research group. The effect of drugs, namely Furosemide, Prednisolone, Metamizole, Enrofloxacin, Meloxicam, Symphytum, and Bacterial, on pure hsPON1 enzyme was determined. All drugs were found to inhibit the hsPON1 enzyme at different levels, with Enrofloxacin identified as the strongest inhibitor. In the second part, a new hydrophobic gel was synthesized using a different ligand (4-iodoaniline). Using this newly synthesized gel, kPON1 was purified from sheep serum at a yield of 88.3 % and 73.4-fold purification. Furthermore, the effects of the aforementioned drugs were investigated on pure kPON1 enzyme. Among the compounds tested, Meloxicam was identified as the strongest inhibitor of kPON1 (IC50 = 1.56 mM).