by three alpha-helices and two loops. Each monomer can separately bind a molecule of thioredoxin. Mammalian Mammalian TrxR structure is similar to E. coli. It contains a FAD and NADPH binding domain, and an interface between two monomer subunits. In mammalian ThxR there is an insertion in the FAD binding domain between two alpha helices which forms a small pair of beta strands. The active disulfide in the enzyme is located on one of these helices and thus the active disulfide bond is located in the FAD domain and not the NADPH domain as in E. coli and other prokaryotes. Mechanism E. coli In E. coli ThxR the spatial orientation of the FAD and NADPH domains are such that the redox-active rings of FAD and NADPH are not in close proximity to each other. When the FAD domain of E. coli is rotated 66 degrees with the NADPH domain remaining fixed the two prosthetic groups move into close contact allowing electrons to pass from NADPH to FAD and then to the active site disulfide bond. The conserved active site residues in E. coli are -Cys-Ala-Thr-Cys-. Mammalian Mammalian TrxRs have a much higher sequence homology with glutathione reductase than E. coli. The active-site Cys residues in the FAD domain and bound NADPH domain are in close proximity removing the necessity for a 66 degree rotation for electron transfer found in E. coli. An additional feature of the mammalian mechanism is the presence of a selenocysteine residue at the C-terminal end of the protein which is required for catalytic activity. The conserved residues in mammalian active site are -Cys-Val-Asn-Val-Gly-Cys-. Detection methods Thioredoxin reductase can be quantified by various methods such as the DTNB assay using Ellman's reagent. The disulfide-based TRFS series of fluorescent probes have shown selective detection of TrxR. Mafireyi synthesized the first diselenide probe that was applied in the detection of TrxR. Other detection methods include immunological techniques and the selenocystine-thioredoxin reductase assay (SC-TR assay). Clinical significance Cancer treatment Since the activity of this enzyme is essential for cell growth and survival, it is a good target for anti-tumor therapy. Furthermore, the enzyme is upregulated in several types of cancer, including malignant mesothelioma. For example, motexafin gadolinium (MGd) is a new chemotherapeutic agent that selectively targets tumor cells, leading to cell death and apoptosis via inhibition of thioredoxin reductase and ribonucleotide reductase. Cardiomyopathy Dilated cardiomyopathy (DCM) is a common diagnosis