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

Abstract

DOI: 10.5152/IstanbulJPharm.2018.382627

Thioredoxin reductase (TrxR, EC 1.6.4.5) is a ubiquitous flavoenzyme that is present from Archaea to humans, and it is the only 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 the literature 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 of its kinetic properties were examined. Molar mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed only a single protein band corresponding to a molecular weight of 35 kDa. Optimum pH and temperature were found 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 enzyme application as an oxidative stress biomarker in mussels and fishes for monitoring contamination in coastal environments.

Keywords

• Enzyme purification,kinetic properties,Mytilus galloprovincialis Lam.

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