Purification and characterization of mitochondrial thioredoxin reductase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the in vitro effects of some metal ions on the enzyme

Abstract

Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is an enzyme belonging to the avoprotein family of pyridine nucleotide-disulfide oxidoreductases. In this study, mitochondrial TrxR enzyme was purified from rainbow trout mitochondria. Thanks to the 2 consecutive procedures (preparation of homogenate and 2′,5′-ADP Sepharose 4B affinity chromatography), the enzyme, having the specific activity of 11.9 EU mg protein-1, was purified with a yield of 2.38% and 672-fold. The purity of the enzyme was monitored and the molecular weight of its subunits was calculated as 70 kDa by SDS-PAGE. The native molecular mass of the enzyme was found to be approximately 151 kDa by gel filtration chromatography. Characteristic and kinetic properties of the enzyme were also determined. Furthermore, Se4+, Cu2+, Co2+, Ni2+, Fe3+, and Al3+ metal ions' in vitro effects on mitochondrial TrxR purified from rainbow trout was investigated. While Se4+ ion increased the enzyme activity, all of the other metal ions used in this study showed an inhibitory effect.