Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity

Abstract

The thioredoxin system, found in all living creatures, consists of thioredoxin protein (Trx), thioredoxin reductase enzyme (TrxR), and NADPH. In this study, turkey liver mitochondrial TrxR enzyme with 3.07 EU x mg-1 specific activity was purified 990-fold in a yield of 2.05% using 2′,5′-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was measured and the molecular weight of its subunits was determined to be 45.5 kDa by SDS-PAGE. The molecular mass of the enzyme's natural state was found to be 88 kDa using Sephadex-G 150 gel filtration chromatography. In addition, characteristic and kinetic properties of the enzyme were determined. Then the inhibitory effects of some heavy metal ions (Ag+, Fe3+, Cd2+, Cu2+, Zn2+, Pb2+, Ni2+, and Co2+) on the activity of TrxR enzyme were examined under in vitro conditions. IC50 values were found with the heavy metal concentration with which 50% of the activity of the TrxR enzyme was inhibited. Finally, Ki values for these substances were calculated from the Lineweaver-Burk plots. It was determined that Ag+, Fe3+, Cd2+, Cu2+, and Zn2+ ions inhibited TrxR enzyme, Pb2+ ion increased enzyme activity, and Ni2+ and Co2+ ions had no effect on enzyme activity. © TÜBİTAK.