Thioredoxin reductase assay kit

Thioredoxin reductase activity was measured using the Thioredoxin Reductase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, 10 μl of cytosolic lysate was added to wells in the 96 well plate and then 190 μl of mixture containing 180 μl of working buffer (100 mM potassium phosphate, 10 mM EDTA, and 0.24 mM NADPH), 6 μl of 100 mM DTNB, and 4 μl of either 1 x assay buffer (100 mM potassium phosphate, pH 7.0, 10 mM EDTA) or thioredoxin reductase inhibitor was added to the wells. The Absorbance was read at 412 nm every 10 s for 2 min in a spectrometer (Bio-Tek) to calculate the activity. All samples were run in duplicate.

The activity of TXNRD was measured in cytosolic or mitochondrial fractions using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich, St. Louis, MO), according to the manufacturer’s instructions. The absorbance was read at 412 nm every 10 s for 2 min in a microplate reader (Molecular Devices, Sunnyvale, CA) to calculate the activity. All samples were run in duplicate.

TrxR1 activity was assessed using the Thioredoxin Reductase Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, Milan, Italy). TrxR1 activity was spectrophotometrically assayed at 412 nm absorbance using a microplate reader (Tecan Trading AG, Switzerland). The assay is based on the NADPH-dependent reduction of the substrate, reacting with 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB). Different doses of Au3BC, Au4BC, and auranofin (positive control inhibitor) were incubated with TrxR1 extracted from rat liver. Furthermore, 10 μg of protein extracted from Huh7 cells was used for determination of TrxR1 activity in cell lysates.

Recombinant Human TrxR1 (Sigma-Aldrich SRP6081, St. Louis, MO, USA) was incubated in a 96 well plate with different concentration of Au complex previously dissolved in 25 μL of PBS at pH 7.4. The cell solution was incubated in shake motion for 5 min at 37 °C. After incubation time, procedure in Thioredoxin Reductase Assay Kit (Sigma-Aldrich CS0170, St. Louis, MO, USA) was followed. Finally, reaction was started by adding 6 μL DNTB and absorbance at 405 nm was recorded every 30 s for 22 min as a measure of Thioredoxin Reductase Activity.

Cells were seeded in 10 cm dishes at a density of 2 × 10⁵ cells/mL (10 mL volume of medium) for 24 h and subjected to defined treatments. Cells were washed, harvested and lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined by the Pierce BCA Protein Assay Kit, and an equal amount of proteins was used for each condition. The TrxR activity was determined with a Thioredoxin Reductase Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol using a reaction scheme for a 96-well plate assay. The reduction of 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB) was followed by measuring optical density (OD) at 412 nm using an enzymatic kinetic program with a delay of 5 min and 20 measurements during 20 min. Reactions were carried out at 25 °C. Since in the crude biological sample, other enzymes, such as glutathione reductase and glutathione peroxidase, could also reduce DTNB, the DTNB reduction by the sample in the presence of the TrxR specific inhibitor provided in the kit was used to determine TrxR-specific activity. The difference between the two reads is the DTNB reduction by TrxR for a given sample. TrxR activity is reported and plotted with the first reading at 412 nm for each sample set to be 0.

The activity of thioredoxin reductase was measured using the Thioredoxin Reductase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, cytosolic fractions were added to 96 well plate and then mixed with working buffer, 100 mm DTNB. Either 1× assay buffer or thioredoxin reductase inhibitor was added to the wells. Enzymatic activity was determined by subtracting the time-dependent increase from total activity in absorbance at 412 nm every 10 s for 2 min in a spectrometer in the presence of the thioredoxin reductase inhibitor. All samples were run in duplicate.