Purification of PON1 enzyme, immobilization of pon1 enzyme on magnetic chitosan nanoparticles, preparation of metal hybrid nano structures and investigation of effects of some drugs on enzyme systems as in vitro

In our work; the paraoxonase enzyme was purified from human serum according to the triple phase separation technique (TPP). Purification was carried out in three steps: Firstly, the human serum was exposed to 60-80% ammonium sulfate saturation. Secondly, the obtained precipitate was treated with 1.0: 0.5, 1.0: 1.0, 1.0: 1.5, 1.0: 2 ratio at constant n-butanol concentration after dissolving in 3 mM glycine buffer (pH: 8.0) and the highest activity detected at 1.0: 0.5 serum:N-butanol. In the third phase; after the precipitate obtained from the previous step was dissolved in the same buffer, it was precipitated again at 20%, 25%, 30%, 35%, 40%, 45%, 50% of the ammonium sulfate ratios with the determined N-butanol ratio, and the highest activity was determined at 20% ammonium sulphate concentration. The paraoxonase (PON1) enzyme was purified 304.5 fold with 76.4% yield from human serum. Then, Iron-Magnetic Chitosan nanoparticles were prepared and the enzyme paraoxonase was covalently immobilized on these nanoparticles. In this method, first, the surface of chitosan was magnetized by coating with Fe3O4 nanoparticles. Then, the glutaraldehyde intermediate was covalently bound to the magnetic chitosan molecules. In the last stage; PON1 were immobilized on the Fe3O4-chitosan NPs throught the PON1 enzyme L-glutaraldehyde intermediate. The percent binding of the immobilized enzyme was determined to be 82.68% and the increase in catalytic activity to be 2.30%. Subsequently, the nano flower PON1-Ca2+ hybrid structure was synthesized. The hybrid PON1 nanoflower was prepared in a one-step procedure. The binding percentage of hybrid enzyme was determined as 88.49% and the increase of catalytic activity as 26.4%. The Zebrafish was used to determine the toxicity of theobtained nano-flower hybrid structure and it was determined that the hybrid construct did not exhibit any toxicity against zebra fish. In addition, SEM, TEM, EDX, FT-IR ve XRD analyzes were performed separately for the characterization of immobilized and nano-flower PON1 hybrid structures. Then, the characterization of free, immobilized and hybrid human PON1 enzymes (optimum pH, stable pH, optimum temperature, stable temperature, Vmax, KM etc) were carried out and their catalytic activities were compared with each other. In the last stage of the study; the effects of some antilipid drugs (Valeric acid, phenoxy – isobutyric acid, N-desmethyl rosuvastatin) and some antioxidant compounds (Gallic acid, Quercitin, Pyrogallol, Ascorbic acid) on the activities of immobilized and nano flower hybrid PON1 enzymes were investigated. Inhibition kinetics (IC50 and Ki values) were determined.