Purification and partial characterization of thioredoxin reductase from the hepatopancreas of the mollusc Mytilus galloprovincialis Lam.

Abstract

Thioredoxin reductase (TrxR, EC 1.6.4.5) is a ubiquitous flavoenzyme that is present from Archaea to humans, and it is theonly enzyme capable of catalyzing the reduction of thioredoxin (Trx) by nicotinamide adenine dinucleotide phosphate (NADPH).Although TrxR has been purified and characterized from different bacteria, plants, and mammalian organisms, a survey of theliterature revealed no studies on the purification and characterization of TrxR from the mussel Mytilus galloprovincialis Lam.In this study, TrxR was purified to homogeneity from the hepatopancreatic tissue of M. galloprovincialis Lam. by extraction,ammonium sulfate precipitation, and DEAE-Sepharose CL-6B anion and 2’,5‘-ADP-agarose chromatographies, and some ofits kinetic properties were examined. Molar mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresisrevealed only a single protein band corresponding to a molecular weight of 35 kDa. Optimum pH and temperature werefound to be 7.0 and 60°C, respectively. Km and Vmax values for NADPH were found to be 85 µmol and 4.82 µmol/min/mg,respectively. For 5,5ʹ-dithiobis (2-nitrobenzoic) acid (DTNB), the Km and Vmax values were 193 µmol and 1.32 µmol/min/mg,respectively. Increasing the knowledge on the kinetic properties of TrxR will significantly increase the prospects of enzymeapplication as an oxidative stress biomarker in mussels and fishes for monitoring contamination in coastal environments.