A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates

Abstract

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in thioredoxin-reductase activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes, and quantification of thioredoxin-activity can therefore be a valuable indicator of clinical conditions. Here we describe a new, direct assay (termed the SC-TR assay) to determine the activity of TR based upon the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as is in older methods, the SC-TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC-TR assay can be used in a cuvette using traditional spectrophotometry, or as a 96-well plate based format using a plate reader. In addition, the SC-TR assay is compatible with the use of non-ionic detergents, making it more versatile than other methods using cell lysates.

Hiding in Plain Sight: Nebivolol Exhibits Compelling Tocolytic Properties

“…The selenocystamine‐thioredoxin reductase (SC‐TR) assay was performed as previously described with the exception that selenocystamine was used in lieu of selenocystine as the subtrate. HEK293 cells were grown to 90% confluence and lysed with ice‐cold TE‐buffer (pH 7.5).…”

Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling

“…The activity of recombinant rat thioredoxin reductase 1 (TrxR1) obtained from IMCO (Stockholm, Sweden) was analyzed by SeͲcystineͲdependent NADPH oxidation as described by Cunniff et al [18].…”

A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1

“…Similar TrxR1 activity with selenocystine (SeC) has previously been shown [19,28,29]. We assume that a similar kind of reaction might happen with the MSC metabolite, i.e., MS.…”

“…The assay mixture without TrxR1 was used as blank. Under this condition, the molar extinction coefficient was 6220 M −1 cm −1 [19,20] for NADPH. T max represents the time taken to consume all the available NADPH in the assay mixture.…”