Preparation and assay of mammalian thioredoxin and thioredoxin reductase

This chapter describes the preparation and assay of mammalian thioredoxin and thioredoxin reductase (TrxR). The amino acid sequences of mammalian TrxR revealed a strikingly high homology to glutathione reductase. 14,19 The conserved features of all the structural components of glutathione reductase are preserved in mammalian TrxR, including a redox active disulfide motif in the N-terminal FAD region, the NADPH binding region, and the carboxyterminal interface region that governs the association of the two subunits in the homodimeric holoenzyme. Mammalian thioredoxin reductase has gained increased interest due to its wide reductive capacity, the discovery of selenium in the enzyme, and its lipid hydroperoxide reductase activity. As thioredoxin shows a growing number of new roles in redox regulation of cellular processes and as an extracellular cytokine, the interest in TrxR in these contexts naturally follows. Future studies of mammalian thioredoxin systems should be an exciting area of research, yielding results required for a deeper understanding of thiol redox control and mechanisms protecting against oxidative stress.