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354104 Glutathione Peroxidase, Cellular Assay Kit

Glutathione Peroxidase, Cellular Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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Overview

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Description
Overview

This product has been discontinued.

We are offering Glutathione Peroxidase Assay Kit (Cat. No. 353919) as a possible alternative. Please read the alternative product documentation carefully and contact technical service if you need additional information.





A spectrophotometric assay kit based on the method described by Paglia and Valentine. Glutathione peroxidase (GPx) catalyzes the reduction of an organic peroxide while forming oxidized glutathione (GSSG). GSSG is reduced to GSH by the enzyme glutathione reductase, and NADPH is oxidized to NADP in the catalytic cycle. GPx activity is quantitated by measuring the change in absorbance at 340 nm caused by the oxidation of NADPH.
Each kit can be used to perform up to 100 assays.
Catalogue Number354104
Brand Family Calbiochem®
Application Data
Cellular Glutathione Peroxidase at a 288 mU/ml assayed at 23°C.
Materials Required but Not Delivered• Spectrophotometer, preferably equipped with a temperature-controlled cuvette chamber capable of measuring the absorbance at 340 nm. • Cuvettes with 1-cm path length
References
ReferencesThompson, C.D., et al. 1977. Br. J. Nutr. 37, 457. Paglia, D.E., and Valentine, W.N. 1967. J. Lab. Clin. Med. 70, 158.
Product Information
Unit of DefinitionOne unit is defined as the amount of enzyme that will cause the oxidation of 1.0 μM of NADPH to NADP per minute at 37°C measured at 340 nm.
Detection methodColorimetric
Form100 Tests
FormatCuvette
Kit containsGlutathione, glutathione reductase, NADPH, t-butylhydroperoxide, buffer, and a user protocol.
Applications
Biological Information
Assay range5.6-24 mU/ml
Assay time2 h
Sample TypeCell lysates, tissue extracts, erythrocyte lysates
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 8-20/21/22-34-40-44

Contact with combustible material may cause fire.
Harmful by inhalation, in contact with skin and if swallowed.
Causes burns.
Limited evidence of a carcinogenic effect.
Risk of explosion if heated under confinement.
S PhraseS: 23-36/37/39-16-17

Do not breathe fumes.
Wear suitable protective clothing, gloves and eye/face protection.
Keep away from sources of ignition - No Smoking.
Keep away from combustible material.
Product Usage Statements
Storage and Shipping Information
Ship Code Multiple Storage Conditions
Toxicity Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage Multiple Storage Requirements
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsGlutathione, glutathione reductase, NADPH, t-butylhydroperoxide, buffer, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
354104 0

Documentation

Glutathione Peroxidase, Cellular Assay Kit Certificates of Analysis

TitleLot Number
354104

References

Reference overview
Thompson, C.D., et al. 1977. Br. J. Nutr. 37, 457. Paglia, D.E., and Valentine, W.N. 1967. J. Lab. Clin. Med. 70, 158.
User Protocol

Form100 Tests
FormatCuvette
Detection methodColorimetric
BackgroundCellular glutathione peroxidase (cytoplasmic GPx, c-GPx, EC 1.11.1.9) is a member of a family of GPx enzymes whose function is to detoxify peroxides in the cell. Because peroxides can decompose to form highly reactive radicals, the GPx enzymes play a critical role in protecting the cell from free radical damage, particularly lipid peroxidation. The GPx enzymes catalyze the reduction of H2O2 to the water and organic peroxidase (R-O-O-H) to the corresponding stable alcohols (R-O-H) using glutathione (GSH) as a source of reducing equivalents.

Figure 1: Characteristics

The GPx enzymes catalyze the reduction of H2O2 to water and organic peroxides (R-O-O-H) to the corresponding stable alcohols (R-O-H) using glutathione (GSH) as a source of reducing equivalents:

With the exception of phospholipid-hydroperoxide GPx, a monomer, all of the GPx enzymes are tetramers of four identical subunits (monomer Mr 22-23 kDa). Each subunit contains a selenocysteine in the active site which participates directly in electron donation to the peroxide substrate and becomes oxidized in the process. The enzyme then uses GSH as a donor to regenerate the reduced form of the selenocysteine. The GPx enzymes accept a wide variety of organic peroxides as substrates. However, with the exception of phospholipid-hydroperoxide GPx and perhaps plasma GPx, the enzymes exhibit a strong preference for glutathione as a source of reducing equivalents. Cellular GPx is present in all tissues, while many of the other GPx enzymes show tissue-specific expression patterns.
Principles of the assayCalbiochem®'s Cellular Glutathione Peroxidase (c-GPx) Assay Kit measures c-GPx activity indirectly. Oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by c-GPc, is recycled to its reduced state by glutathione reductase (GR). The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm that provides a spectrophotometric means of monitoring GPx activity. To assay c-GPx, the sample containing c-GPx is added to a solution containing GSH, glutathione reductase, and NADPH. The reaction is initiated by the addition of an organic peroxide (tert-butyl hydroperoxide) and the A₃₄₀ is recorded. The rate of decrease in the A₃₄₀ is directly proportional to the GPx activity in the sample.

Figure 2: Principle of the Assay
Materials provided• Assay Buffer: 1 x 120 ml, 50 mM Tris-HCl, pH 7.6, 5 mM EDTA • tert-Butyl Hydroperoxide: 1 x 2.0 ml, (70% aqueous solution) • NADPH Reagent: 5 x 1 vial, each vial contains: 24 µmol GSH, 4.8 µmol NADPH, and ≥12 U Glutathione Reductase. • Control Sample Diluent: 1 x 20 ml, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1 mM Ergothioneine, 1 mg/ml Bovine IgG • Cellular Glutathione Peroxidase (c-GPx) Control* 2 x 0.5 ml, in 50 mM Tris-HCl, pH 7.3, 5 mM EDTA, 1 mM Ergothioneine, 1 mM DTT, 1 mg/ml Bovine IgG, derived from bovine erythrocytes *The activity of this component varies from lot to lot; please refer to the vial label for activity.
Materials Required but not provided• Spectrophotometer, preferably equipped with a temperature-controlled cuvette chamber capable of measuring the absorbance at 340 nm. • Cuvettes with 1-cm path length
Precautions and recommendations• Tissue extracts containing enzymes that consume NADPH can cause the GPx levels to be overestimated. A blank without tert-butyl hydroperoxide included should be performed to assess nonspecific oxidation of NADPH. • High concentrations of reducing agents (≥ 0.1 mM final concentration) such as dithiothreitol or β-mercaptoethanol. The use of polyethoxy-nonionic detergents (e.g. TWEEN® 20 or TRITON® X-100) is not recommended, as they often contain peroxides. If these detergents are absolutely needed, we recommend the use of Calbiochem® Protein Grade® detergents. Note: It has been reported that heme peroxidase activity of hemoglobin can lead to falsely elevated values for GPx activity in erythrocyte lysates. Direct testing revealed no significant effect in the cellular GPx assay using tert-butyl hydroperoxide as a substrate. Therefore, it is not necessary to use Drabkin's reagent (potassium ferricyanide/potassium cyanide) to convert hemoglobin to cyanmethemoglobin in the sample as this can inactivate GPx.
Preparation• Erythrocyte lysate 1. Collect blood using an anticoagulant such as heparin, citrate, or EDTA. 2. Centrifuge blood at 1500 x g for 10 min at 4°C and discard the supernatant. 3. Wash the red blood cells with 10 volumes of cold saline. 4. Lyse the erythrocyte pellet in 4 times its volume of ice-cold deionized water. 5. Remove the red cell stroma by centrifugation (e.g. 8500 x g for 10 min at 4°C). 6. Collect the resulting clarified supernatant for use in the assay. Store on ice. If not assaying the lysate the same day, the sample should be frozen at -70°C where it will maintain activity for at least 6 months. 7. It is recommended that 0.2-0.5 mg of protein from the clarified lysate be used per assay (e.g. 70 µl of 7 mg/ml sample) in initial experiments. • Tissue Homogenates 1. Prior to dissection, perfuse tissue with 0.9% NaCl containing 0.16 mg/ml heparin to remove any red blood cells and clots from the tissues of interest. 2. Homogenize the tissue in 4-8 ml of cold buffer (e.g. 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM DTT) per gram tissue. 3. Centrifuge at 5000-10,000 x g for 10-20 min at 4°C. 4. Remove the supernant for assay. Store on ice. If it is not assayed on the same day, freeze it at -70°C here it will maintain activity for at least 6 months. 5. It is recommended that 0.1-1.0 mg of protein be used per assay (e.g. 70 µl of a 1.5-15 mg/ml sample) in initial experiments. • Cell Lysates 1. Collect cells by centrifugation. For adherent cells, do not harvest using proteolytic enzymes; rather use a rubber policeman. 2. Homogenize pellet in cold buffer (e.g. 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM DTT). 3. Centrifuge at 10,000 x g for 15-20 min at 4°C. 4. Remove the supernatant for assay. Store on ice. If not assaying the lysate the same day, the sample should be frozen at -70°C where it will maintain activity for at least 6 months. 5. It is recommended that 0.1-1.0 mg of protein be used per assay (e.g. 70 µl of a 1.5-15 mg/ml sample) in initial experiments.
Reagent preparation1. Assay Buffer: Estimate the amount of assay buffer required to run the intended number of samples for the day. Transfer that amount of Assay Buffer into a separate container, preferably with a cap, and bring to assay temperature (23-25°C). Discard unused buffer at the end of the day. DO NOT RETURN UNUSED ASSAY BUFFER TO THE ORIGINAL KIT BOTTLE. 2. NADPH Reagent: Add 7.5 ml of Assay Buffer to each of the NADPH Reagent bottles you intend to use (20 tests per vial). Recap the vial, invert gently to ensure uniform dissolution of the contents, and bring the reagent to assay temperature (23-25°C). If capped and protected from light, each vial can be kept at room temperature up to 9 h without significantly affecting assay performance. If kept on ice or in the refrigerator, this reagent may be used the next day before being discarded. DO NOT REFREEZE THE RECONSTITUTED NADPH REAGENT. 3. tert-Butyl Hydroperoxide: Dilute the tert-butyl Hydroperoxide substrate 1:10,000 in deionized water. Place in a closed container and bring to assay temperature (23-25°C). The working solution (0.007%) may be kept at assay temperature for up to 8 h. It should be made fresh each day. 4. Thaw Glutathione Peroxidase samples and/or GPx control, mix thoroughly, and place on ice.
Detailed protocolFinal Assay Conditions: 1 mM GSH, 0.2 mM NADPH, 0.22 mM tert-Butyl Hydroperoxide, >0.4 U/ml glutathione reductase, pH 7.6 1. Equilibrate the assay buffer, NADPH Reagent, and tert-Butyl Hydroperoxide to the assay temperature. 2. Zero the spectrophotometer at 340 nm using a cuvette filled with water. 3. Immediately prior to assay, dilute the sample into Assay Buffer as needed (typically, a 1:10 dilution is required to obtain absorbance readings between 0.035 to 0.15 A340 /min). NOTE: Purified GPx, such as the control sample provided should be diluted in the Control Sample Diluent rather than Assay Buffer. 4. Add the following to designated cuvettes:

Table 1: Cuvette Guidelines

5. Place cuvette in a spectrophotometer which has been set to the assay temperature. 6. Add 350 µl of diluted tert-Butyl Hydroperoxide to the cuvette. Mix by pipetting up and down twice (avoid making bubbles). NOTE: It is advisable to prepare an additional control which includes water instead of tert-Butyl Hydroperoxide (see interferences). 7. Record the absorbance at 340 nm for 3 min by either manually recording the absorbance at least every 30 s or automatically recording the absorbance over a 3 min interval using a recording spectrophotometer. NOTE: If the A340 at t = 0 is < 0.8, the NADPH reagent vial in use should be discarded and another vial reconstituted.
CalculationsNOTE: The first 15 s of the reaction (after adding the substrate) should be excluded from data analysis as the rates may not be representative of the GPx activity due to sample mixing. 1. Determine the rate of decrease in A340 per min for both samples and controls by either calculating the difference in A340 between 60 and 120 s or by averaging the rate of change for several timed intervals (e.g. 15 or 30 s intervals) or performing regression analysis on A340 as a function of time to obtain the slope (rate). 2. Calculate the net rate for the sample by subtracting the rate observed for a water blank (water instead of sample) from the sample rate. 3. To ensure accurate measurement of GPx, it is desirable to obtain absorbance values from 0.035-0.15 A340/minute. If your samples are not in this range, repeat the assay using a higher or lower sample dilution in step 2 of the assay procedure. 4. Convert the net A340/min for the sample to NADPH consumed in mU/ml using the following relationship:

Table 2: Calculations

5. Correct for the dilution of sample. There is a 16-fold dilution in the assay (70 µl diluted to 1120 µl), plus the initial sample dilution you performed in step 2 of the assay procedure. Total dilution = 16 X (initial sample dilution).
Example data

Figure 3: Sample Data and Calculations

Cellular Glutathione Peroxidase at a 288 mU/ml assayed at 23°C.
Sensitivity NotesSamples that produce a change in absorbance of less than 0.035 A₃₄₀/min can be accurately assayed if the protocol is modified (longer reaction times); however, we recommend that you obtain values from 0.035 to 0.15 A₃₄₀ /min using the protocol provided. This corresponds to approximately 5.6 to 24 mU/ml.
Assay Range5.6-24 mU/ml
PrecisionWhen a series of 20 measurements are performed on 8 different days under the same experimental conditions at three different GPx activity levels (6.2, 18.7 and 31.25 mU/ml), the standard deviations were 0.25, 0.78, and 1.52, respectively, and the coefficients of variation were 4.0, 4.1, and 4.9%, respectively.
Technical AppendixThose researchers who prefer to use a microplate reader capable of measuring absorbance at 340 nm may adapt the Calbiochem® GPx Assay Kit for use in this format. The calculations given in this procedure assume a path length of 1 cm. For plate readers the path length may not be 1 cm. Hence, it is necessary to determine a correction factor for the path length in the plate reader using a specific plate. A comparison of the rates obtained in a 1 cm path spectrophotometer with those obtained in a plates using the same samples may be used to determine a correction factor for the path length. The following reagent volumes can be used for assay in a plate:

Table 3: Adaption of Assay to a Plate Reader
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicalss, Inc. Triton® is a registered trademark of Dow Chemical Company Tween® is a registered trademark of ICI Americas, Inc. Interactive Pathways™ is a trademark of EMD Chemicalss, Inc.