Oxidized Thioredoxin Reductase Assay Kit (Spectrophotometry)

Thioredoxin reductase (TrxR) catalyzes the reduction of DTNB by NADPH to generate TNB and NADP⁺. TNB exhibits a characteristic absorption peak at 412 nm. However, reduced glutathione (GSH) can also react with DTNB to produce TNB. Therefore, 2-vinylpyridine is used to inhibit endogenous reduced glutathione in the sample. The activity of TrxR can then be calculated by measuring the rate of increase in TNB absorbance at 412 nm.

Taking 50T/48S packing for example:

Prior to the formal determination, a preliminary assay should be conducted using 2-3 samples with large expected differences.

I. Preparation of Lab Instruments

Visible spectrophotometer, 1 mL glass cuvette, water bath, balance, centrifuge, adjustable pipette, mortar, distilled water, 1× PBS buffer.

II. Extraction of Crude Enzyme

1.Tissues:

Homogenize on ice at a ratio of tissue mass (g) : CB0136S-A volume (mL) = 1 : 5–10

(Recommended: weigh ~0.1 g tissue and add 1 mL CB0136S-A).

Centrifuge at 8000 × g, 4 °C for 10 min. Collect the supernatant and keep on ice for analysis.

2.Bacteria / cells:

Use a ratio of cell number (×10⁴ cells) : CB0136S-A volume (mL) = 500–1000 : 1

(Recommended: add 1 mL CB0136S-A to 5 × 10⁶ cells).

Lyse cells by ultrasonication on ice (300 W; 3 s on / 7 s off; total 3 min).

Then centrifuge at 8000 × g, 4 °C for 10 min. Collect the supernatant and keep on ice for analysis.

3.Serum and other liquid samples: Measure directly.

III. Assay Procedure

1.Preheat the spectrophotometer for 30 minutes, set the wavelength to 412 nm, and zero the instrument using distilled water.

2.Preheat CB0136S-A at 25 °C (for general species) or 37 °C (for mammals) for 30 minutes. Before measurement, mix the sample supernatant with CB0136S-D at a volume ratio of 50:1 (i.e., add 2 μL of CB0136S-D to 100 μL of supernatant). Incubate in a 37 °C water bath for 30 minutes, then place on ice.

3.Add the following reagents

Note: Blank tubes only need to be measured 1–2 times.

IV. Calculation of TrxR Activity

1.Based on protein concentration

At 25 °C or 37 °C, the amount of enzyme that catalyzes the reduction of 1 nmol of DTNB per minute per milligram of protein is defined as one unit of enzyme activity.

TrxR (U/mg prot) = (ΔA sample - ΔA blank)÷ε÷d×109×V1÷(Cpr×V2)÷T= 147×(ΔA sample - ΔA blank)÷Cpr

2.Based on sample mass

At 25 °C or 37 °C, one enzyme activity unit is defined as the amount of enzyme that catalyzes the reduction of 1 nmol of DTNB per minute per gram of sample.

TrxR (U/g) = (ΔA sample - ΔA blank)÷ε÷d×109×V1÷(W×V2÷V3)÷T = 147×(ΔA sample - ΔA blank)÷W

3.Based on bacterial or cell density

At 25 °C or 37 °C, one enzyme activity unit is defined as the amount of enzyme that catalyzes the reduction of 1 nmol of DTNB per minute per 10⁴ cells.

TrxR (U/104 cell) = (ΔAsample - ΔA blank)÷ε÷d×109×V1÷(cell number×V2÷V3)÷T= 147×(ΔA sample - ΔA blank)÷cell number

4.Based on Liquid Volume

At 25 °C or 37 °C, one enzyme activity unit is defined as the amount of enzyme that catalyzes the reduction of 1 nmol of DTNB per minute per milliliter of liquid.

TrxR (U/mL) = (ΔA sample - ΔA blank)÷ε÷d×109×V1÷V2÷T= 147×(ΔA sample - ΔA blank)

Note:

ε: Molar extinction coefficient of TNB at 412 nm, 1.36 × 10⁴ L/mol/cm;

d: Optical path length of the cuvette, 1 cm;

V1: Total volume of the reaction system (L), 1000 μL = 1 × 10-3 L;

Cpr: Protein concentration of the supernatant (mg/mL), to be determined separately;

W: Sample mass;

V2: Volume of supernatant added to the reaction system (mL), 100 μL = 0.1 mL;

V3: Volume of extraction buffer, 1 mL;

T: Reaction time (min), 5 min Cell number: Expressed in units of 10⁴ cells (ten thousand cells);

10⁹: Unit conversion factor, 1 mol = 10⁹ nmol

1.Before formal measurement, perform a preliminary test using 1–2 samples to ensure that the absorbance shows a linear change within 5 minutes. For TrxR activity assays of mammalian tissues and blood products, samples generally need to be diluted approximately 5-fold with distilled water. All procedures during the assay should be carried out rapidly.

2.Since CB0136S-A contains a certain concentration of protein (approximately 0.1 mg/mL), the protein contributed by the extraction buffer itself must be subtracted when determining the protein concentration of the samples.

3.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

4.Please wear a lab coat and disposable gloves.

• CB0136S-Instruction Manual(274 KB)