Glutathione Reductase (GR) Assay Kit (ab83461)

Glutathione Reductase Assay Kit (ab83461) is a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring GR activity in biological samples.

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Key facts

Detection method: Colorimetric
Sample types: Urine, Plasma, Tissue Extracts, Serum, Other biological fluids, Cell Lysate
Assay type: Enzyme activity
Range: 0.1 - 40 mU/mL
Assay time: 40m
Sensitivity: > 0.1 mU/mL

Target data

See full target information GSR

Function

Maintains high levels of reduced glutathione in the cytosol.

Alternative names

GLUR, GRD1, GSR, GR, GRase

What's included?

200 Test

Components

Assay Buffer 41

1 x 100 mL

Catalase

1 x 1 Vial

DTNB

1 x 1 Vial

GR Positive Control

1 x 1 Vial

GSSG

1 x 1 Vial

Hemin Substrate

1 x 1 mL

NADPH Generating Mix

2 x 1 Vial

TNB Standard

1 x 1 Vial

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Glutathione Reductase Assay Kit (ab83461) is a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring GR activity in biological samples.

Key facts

Detection method: Colorimetric
Sample types: Urine, Plasma, Tissue Extracts, Serum, Other biological fluids, Cell Lysate
Assay type: Enzyme activity
Range: 0.1 - 40 mU/mL
Assay time: 40m
Assay Platform: Microplate reader
Sensitivity: > 0.1 mU/mL

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: -20°C
Appropriate long-term storage conditions: -20°C
Storage information: Please refer to protocols

Notes

Glutathione Reductase Assay Kit (ab83461) is a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring GR activity in biological samples. In the assay, GR reduces GSSG to GSH, which reacts with 5, 5'-Dithiobis (2-nitrobenzoic acid) (DTNB) to generate TNB2- (yellow color, λmax = 405 nm). The assay can detect 0.1-40 mU/ml GR in various samples.

Since Glutathione Reductase has significantly higher concentrations in cells (mM range) compared to Thioredoxin Reductase (μM range), we predict that ab83461 will detect mostly GR activity in samples.

Glutathione Reductase (GR, EC 1.8.1.7) catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), which plays an important role in the GSH redox cycle that maintains adequate levels of reduced GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress. **Related products** Review the to learn about more assays for oxidative stress.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Glutathione reductase also known as GSR or GSH reductase is an enzyme critical for maintaining the cellular redox balance by reducing glutathione disulfide (GSSG) to its sulfhydryl form GSH. It has a molecular mass of approximately 100 kDa and is widely expressed in various tissues with high levels found in the liver and red blood cells. This enzyme is a flavoprotein that requires FAD as a cofactor to drive the reduction process playing an important role in the recycling of glutathione which is essential for detoxification processes in the cell.

Biological function summary

Glutathione reductase activity supports cellular defense against oxidative stress by regenerating reduced glutathione. It is not part of a larger complex but interacts with other antioxidants to protect cells from damage. The enzyme works continuously to keep the intracellular environment balanced by maintaining an adequate GSH/GSSG ratio. This balance is vital for neutralizing reactive oxygen species (ROS) which can otherwise cause cellular damage and contribute to disease development.

Pathways

Glutathione reductase function is integral to the glutathione metabolism and pentose phosphate pathways. It collaborates closely with related proteins like glutathione peroxidase which uses GSH to reduce hydrogen peroxide therefore preventing its toxic accumulation. By ensuring a steady supply of GSH glutathione reductase supports the antioxidant defenses necessary for cellular survival under stress conditions linking it to various signaling cascades that monitor and manage oxidative stress levels.

Associated diseases and disorders

Glutathione reductase has significant implications in conditions characterized by oxidative stress such as Alzheimer's disease and cardiovascular diseases. Impairments in glutathione reductase activity can lead to insufficient detoxification of ROS contributing to the pathophysiology of these conditions. In Alzheimer's the enzyme's dysfunction allows for increased oxidative damage often involving interactions with other proteins such as amyloid-beta which exacerbates neuronal damage. Similarly in cardiovascular diseases disrupted glutathione homeostasis can drive oxidative stress-related damage to vascular tissues.

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7 product images

Cao, Zeyu et al., Scientific reports: vol. 7,1 16278., Fig 5, doi:10.1038/s41598-017-16446-7
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
ICAC decreased the ROS level induced by EV71 infection via modulating the antioxidant enzymes involved in GSH metabolism. Infected (100 TCID50 EV71) Vero cells were treated with medium or 100 µM ICAC for 12 h. Uninfected cells were used as the control group. The antioxidant enzymes activities of the Vero cells were detected (n = 3). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the EV71 group at the P < 0.05 level according to one-way analysis of variance.
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
Glutathione reductase measured in mouse tissue lysates showing activity (mU) per mg of extracted protein (T₁=2 min; T₂=30 min).
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
Glutathione reductase measured in cell lysates (mU) per 10⁶ cells (T₁=2 min; T₂=30 min)
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
Glutathione reductase measured in human plasma and serum (T₁=2 min; T₂=30 min) showing activity (mU) per ml of tested sample. No activity was detected in saliva or urine.
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
TNB Standard Curve using ab83461.
Functional Studies - Glutathione Reductase (GR) Assay Kit (ab83461)
Functional Studies - Glutathione Reductase Assay Kit (ab83461) Glutathione Reductase assay time line using ab83461.
Biochemical assay - Glutathione Reductase (GR) Assay Kit (ab83461)
Pesti-Asboth et al used Lipid Peroxidation (MDA) Assay Kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Vitamin C Assay Kit Ascorbic Acid Assay Kit (Biological Samples) ab65656, Glutathione Peroxidase Assay Kit Glutathione Peroxidase Assay Kit (Colorimetric) ab102530, Glutathione Reductase Assay Kit ab83461, and Superoxide Dismutase Assay Kit Superoxide Dismutase Activity Assay Kit (Colorimetric) ab65354 to investigate changes in redox in the plasma of fast growing broiler chickens.
Changes in the redox parameters in blood plasma at Days 3, 8, 21, 32, and 42. Significant differences were determined by comparing the data from each time point with the data from the first time point. Data are expressed as the means ±8 SEMs; *P<0.05, ** p<0.005, *** p<0.001.
Lipid peroxidation was determined using a commercially available assay kit (Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Abcam, Cambridge, United Kingdom). The measurements were based on the reaction between MDA and thiobarbituric acid (TBA). All reagents and standard solutions were prepared according to the manufacturer’s instructions. Plasma samples (20 μl) were mixed with 500 μL of 42 mM H2SO4, 125 μL of phosphotungstic acid solution was added, and each sample was mixed by vortexing and incubated at room temperature for 5 minutes. After incubation, we centrifuged the samples at 13000 × g for 3 minutes. The pellet was collected and resuspended in 100 μL of distilled water on ice with 2 μL of butylated hydroxytoluene (BHT) (100-fold dilution). The final volume was adjusted to 200 μL with distilled water. After sample preparation, the assay was performed. The MDA-TBA adduct was generated in each sample and standard solution. The TBA reagents (600 μl) were added to the samples and standards for incubation at 95°C for 60 minutes. The reaction mixes were cooled in an ice bath for 10 minutes after incubation. The samples and standards were placed in duplicate in a 96-well microplate for analysis. The absorbance of the MDA-TBA adduct was measured at 532 nm, and the calculated concentrations are expressed in nmol/ml.
To determine the vitamin C concentration, a plasma-specific assay kit was used (Ascorbic Acid Assay Kit (Biological Samples) ab65656, Abcam, Cambridge, United Kingdom) [34–36]. The measurements were performed according to the protocol in the manual with some modifications. The kit’s supernatant assay buffer was set to pH 7.0 with NaOH (10 M). The kit provides a sensitive method for vitamin C measurement. Plasma vitamin C reduces Fe3+ to Fe2+, which shows strong absorbance that can be monitored between 545–600 nm. The reagent and standard were prepared as described in the protocol. Fifty microliters of each plasma sample, run in duplicate, was diluted two fold for measurement. The assay procedure was followed as described in the manual. The absorbance of each sample was measured at 593 nm, and the concentrations were calculated as recommended and are expressed in nmol/ml.
The activity of Glutathione peroxidase (GPx) was measured with a commercially available kit (Glutathione Peroxidase Assay Kit (Colorimetric) ab102530, Abcam, Cambridge, United Kingdom). In the assay, GSSG produced after GPx oxidizes GSH during H2O2 reduction. GSSG is reduced back to GSH by GR with the aid of nicotinamide adenine dinucleotide phosphate (NADPH). The reduction of NADPH is proportional to GPx activity and can be measured colorimetrically at 340 nm. The kit reagents were dissolved as described in the manual. A standard curve was prepared as described in the kit. The activity of GPx is expressed as mU/ml.
The activity of Glutathione reductase (GR) in plasma was determined using a specific assay kit (ab83461, Abcam, Cambridge, United Kingdom). In this assay, GSH is formed from GSSG by GR; then, GSH reacts with DTNB, and a 2-nitro-5-thiobenzoate anion (TNB2-) is generated. The change in absorbance was measured at 405 nm. The kit reagents were dissolved as described in the “components and storage” section. The protocol recommends pretreating the samples. First, 5 μL of 3% H2O2 was added to 100 μL of each sample. The samples were incubated at 25°C for 5 min. Then, 5 μL of catalase was added to each sample, and we incubated the samples again at 25°C for another 5 min. After the pretreatment procedure, 50 μL of each pretreated sample was added to the sample wells. The standard curve was prepared as described in the manual, and 50 μL of diluted standard solution was added to each well. GR activity is expressed in nmol/min/mL = mU/ml.
The superoxide dismutase(SOD)inhibition rate was measured with a specific assay kit (Superoxide Dismutase Activity Assay Kit (Colorimetric) ab65354, Abcam, Cambridge, United Kingdom). In this assay, xanthine oxidase produced superoxide anions, and the conversion of superoxide anions into hydrogen peroxide was catalyzed by SOD. Superoxide anions and the water-soluble tetrazolium salt WST-1 can react to produce a water-soluble formazan dye, the absorbance of which was detected at 450 nm. The reagents were prepared as described in the kit. First, 20 μL of Blank 1, Blank 2, Blank 3 or sample was added to a 96-well plate in duplicate. Then, WST-1 solution (200 μl) was added to each blank and sample. Twenty microliters of dilution buffer were added to the Blank 2 and Blank 3 solutions. Enzyme working solution (20 μl) was added to each sample and Blank. Then, the plate was incubated at 37°C for 20 minutes. The absorbance (A) was then measured at 450 nm.