Purification of paraoxonase 1 isoenzyme from human serum and investigation effects of some plant extract on enzyme activity

Paraoxonase 1 (E.C.3.1.8.1; PON1) is a 2 Ca2+ -studded HDL dependant metaloenzyme that acting as a crucial organophosphate hydrolyzer in metabolism. Enzyme prevents HDL from oxidation. Morever, enzyme protects LDL and has a vital role of inhibition of atherosclerosis legions. As a part of thesis, antioxidant activity of extracts from Crataegus tanacetifolia, Rumex acetosella, Thymus sibthorpii ve Asphodeline taurica were determined via ferrous reduction, FRAP, CUPRAC, ABTS, DPPH, DMPD, bipyridyl, superoxide anion radical scavenging, total phenolic compounds and total antioxidant activity methods. Then, major compounds of plant extracts were identified via LC/MS-IT-TOF and possible interactions with PON1 isoenzyme were demonstrated in silico studies. Furthermore, PON1 isoenyzme was purified with 5.49% yield from human serum using a method in order to ammonium sulphate precipitation, DEAE-Sephadex A-50 ion exchange and Sephadex G-100 gel filtration chromatography. Purification factor and specific activity were calculated as 79.54 and 2612.22 EU/mg protein, respectively. Purity of PON1 isoenzyme was tested using SDS-PAGE and also subunit molecular weight was determined as 43 kDa. In vitro effects of extracts to paraoxonase activity was presented in Activity (%) – [Extract] graphs. The highest activity value was attained from ethanol extact of Rumex acetosella, and on the contrary the lowest activity value from aqueous extract of Asphodeline taurica. In conclusion, cytotoxicity of extracts was investigated with Mitochondrial Activity Test (MTT). Keywords: Paraoxonase 1, Enzyme purification, enzyme activity, docking, antioxidant activity, MTT