Glutathione Reductases (GR) Activity Assay Kit (UV Micro Method)

Cat. No.: G1506769

GRADE & PURITY BioReagent

Overview

  Glutathione reductase (GR) (EC 1.6.4.2) is a flavoprotein oxidoreductase widely found in eukaryotes and prokaryotes. GR catalyzes the reduction of glutathione disulfide (GSSG) to regenerate glutathione (GSH), making it a key enzyme in the glutathione redox cycle (typically, GR in insects is replaced by thioredoxin reductase, TrxR). GR catalyzes the NADPH-dependent reduction of GSSG to GSH, helping to maintain the balance of the GSH/GSSG ratio in vivo. GR plays a critical role in scavenging reactive oxygen species under oxidative stress. Additionally, GR is involved in the ascorbate-glutathione cycle pathway.

Detection Principle

  GR catalyzes the NADPH-dependent reduction of GSSG to regenerate GSH, simultaneously dehydrogenating NADPH to generate NADP⁺. NADPH has a characteristic absorption peak at 340 nm, whereas NADP⁺ does not absorb at this wavelength. The GR activity is determined by measuring the rate of decrease in absorbance at 340 nm, which corresponds to the rate of NADPH dehydrogenation.

Applicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma).

G1506769	Component	96 T	Storage
G1506769A	Assay Buffer	70 mL×2	2-8℃
G1506769B	Substrate	2 mL	2-8℃. Store in the dark.
G1506769C	GR Cofactor	1 EA	-20℃. Store in the dark.

Please verify the quantity of all components before commencing the experiment.

An additional 10% of each component is supplied beyond the specified volume/amount to facilitate standard curve preparation or preliminary testing.

Materials Required but Not Provided

Category	Resource Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 340 nm
Consumables	96-well Microplate	UV-transparent plate
Reagents	PBS / Deionized Water	For sample washing / reagent preparation
Others	Homogenizer (for tissue samples), Incubator, Ice Bucket, Refrigerated Centrifuge, Adjustable Pipettes and Tips	For high-throughput detection, using a multichannel pipette can improve efficiency.

Experimental Procedure

1. Reagent Preparation

Reagent	Preparation	Notes
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Substrate	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light.
GR Cofactor	Prepare immediately before use. Add 2.4 mL deionized water to each vial and mix thoroughly.	Keep protected from light on ice during the experiment. Unused reagent can be aliquoted and stored at -20°C protected from light for 1 month; avoid repeated freeze-thaw cycles.

2. Sample Preparation

Notes:

① Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.

② All sample processing steps should be performed on ice. Enzyme activity should be measured on the same day. Avoid repeated freeze-thaw cycles of the homogenate.

③ For GR activity measurement in cells, the cell number should be between 3-5×10⁶. Use Assay Buffer for homogenization or ultrasonic disruption during GR extraction from cells; do not use cell lysis buffer.

④ Assay Buffer contains a certain concentration of protein (approx. 1 mg/mL). If determining protein concentration, subtract the protein content of the Assay Buffer itself.

2.1 Animal/Plant Tissues

Weigh approximately 0.1 g of sample, add 1 mL of pre-cooled Assay Buffer, and homogenize in an ice bath. Centrifuge at 10,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.

2.2 Cells or Bacteria

Collect 5×10⁶ cells or bacteria. Wash with pre-cooled PBS, centrifuge at 800 × g for 2 minutes, and discard the supernatant. Add 1 mL of pre-cooled Assay Buffer and disrupt using an ultrasonic cell disruptor on ice. Ultrasonicate for 5 minutes (20% power or 200 W, pulse for 3 s, pause for 7 s, repeat 30 times). Centrifuge at 10,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.

2.3 Serum (Plasma)

Assay directly.

3.. Assay Procedure

3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 340 nm.

3.2 Reagent Pre-incubation: Pre-warm the Assay Buffer at 25°C (for common species) or 37°C (for mammals) for at least 30 minutes before the assay.

3.3 Assay Setup: One blank well is sufficient. Ensure the absorbance change is linear within 180s. Mammalian tissues generally need to be diluted 2-5 times with Assay Buffer. As enzyme activity is calculated from the reaction rate, control the number of samples measured at once based on operation speed when using a 96-well UV plate. Add reagents to the 96-well UV plate as follows:

Reagent (μL)	Blank Well	Test Well
Sample	0	20
Assay Buffer	170	150
Substrate	10	10
GR Cofactor	20	20

3.4 Absorbance Measurement:

Mix rapidly immediately after adding the GR Cofactor. Measure the absorbance at 340 nm. Record the absorbance at 10 seconds as A1 and at 190 seconds as A2. For the Test well, record as A1_test and A2_test; for the Blank well, record as A1_blank and A2_blank.

4. Calculation of Results

The following provides both the detailed derivation formula and the simplified formula. They are exactly equivalent.

4.1 Data Processing

Calculate ΔA = A1 - A2 for each well.

ΔA_test = A1_test - A2_test ΔA_blank = A1_blank - A2_blank ΔΔA = ΔA_test - ΔA_blank 

4.2 Sample GR Activity Calculation 

(1) Calculation based on protein concentration 

(1) Calculation based on protein concentration Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute per mg of protein at 25°C or 37°C, pH 8.0. GR Activity (U/mg prot) = [ΔΔA ÷ (ε × d) × V_{reaction total} × 10⁶] ÷ (Cpr × V_sample) ÷ T = 1.072 × ΔΔA ÷ Cpr 

(2) Calculation based on sample fresh weight 

(2) Calculation based on sample fresh weight Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute per gram of sample at 25°C or 37°C, pH 8.0. GR Activity (U/g) = [ΔΔA ÷ (ε × d) × V_{reaction total} × 10⁶] ÷ (V_sample ÷ V_{sample total} × W) ÷ T = 1.072 × ΔΔA ÷ W 

(3) Calculation based on cell or bacterial count 

(3) Calculation based on cell or bacterial count Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute per 10⁴ cells or bacteria at 25°C or 37°C, pH 8.0. GR Activity (U/10⁴) = [ΔΔA ÷ (ε × d) × V_{reaction total} × 10⁶] ÷ (N × V_sample ÷ V_{sample total}) ÷ T = 1.072 × ΔΔA ÷ N 

(4) Calculation based on liquid volume 

Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute per mL of liquid at 25°C or 37°C, pH 8.0. GR Activity (U/mL) = [ΔΔA ÷ (ε × d) × V_{reaction total} × 10⁶] ÷ V_sample ÷ T = 1.072 × ΔΔA 

Parameter Description: 

ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm 

d: Light path of the 96-well UV plate, 0.5 cm 

V_{reaction total}: Total reaction volume, 200 μL = 2 × 10⁻⁴ L 

10⁶: Unit conversion factor, 1 mol = 1 × 10⁶ μmol 

Cpr: Protein concentration of the supernatant, mg/mL 

W: Sample weight, g 

V_sample: Volume of supernatant added to the reaction system, 20 μL = 2 × 10⁻² mL 

V_{sample total}: Volume of extraction buffer, 1 mL 

T: Reaction time, 3 min 

N: Cell or bacterial count, in units of 10⁴ (e.g., for a cell count of 5×10⁶, N = 500). 

5. Result Demonstration 

Using 0.1 g of mouse liver and following the assay procedure with a 96-well UV plate: 

Measured A1_test = 0.664, A2_test = 0.487, thus ΔA_test = 0.664 - 0.487 = 0.177. 

Measured A1_blank = 0.288, A2_blank = 0.286, thus ΔA_blank = 0.288 - 0.286 = 0.002. 

ΔΔA = ΔA_test - ΔA_blank = 0.177 - 0.002 = 0.175. Calculated GR Activity (U/g) based on sample weight = 1.072 × 0.175 ÷ 0.1 = 1.876 U/g.

Frequently Asked Questions

Q: What should I do if the measured ΔΔA for the sample is too high or too low?

A: If ΔΔA is less than 0.02, consider appropriately increasing the sample amount. If ΔΔA is greater than 1.0, the sample can be further diluted with Assay Buffer, or the sample amount used for extraction can be reduced, before re-assaying.

Precautions

1. Before formal testing, it is recommended to perform preliminary assays using 2-3 samples expected to have significant differences.

2. For tissue and cell samples, results can be normalized by measuring protein concentration. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, etc., and perform experiments in a fume hood or biosafety cabinet.

5. This product is for research use only. Not for use in diagnostic procedures.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.