> Glutathione reductase activity kit

Catalog # ADI-900-159 Sufficient Reagents for 480 tests with 5 x 96-well plates For use with cell and tissue extracts

All reagents, except Standard and NADPH, should be stored at 4C. Store Standard and NADPH at -20C.

Check our website for additional protocols, technical notes and FAQs.

For proper performance, use the insert provided with each individual kit received.

Table of Contents 2 Introduction 2 Principle 3 Materials Supplied 3 Storage 3 Materials Needed but Not Supplied 4 Reagent Preparation 5 Sample Handling 7 Assay Procedure 8 Calculation of Results 11 Trouble Shooting 11 References 12 Limited Warranty

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

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Introduction
The Glutathione reductase activity kit is a complete kit for the measurement of glutathione reductase activity in cell and tissue extracts. Glutathione reductase (GR), a homodimeric flavoprotein disulfide oxidoreductase, plays an indirect but essential role in the prevention of oxidative damage within the cell by helping to maintain appropriate levels of intracellular reduced glutathione (GSH). Reduced glutathione is essential for maintaining the normal structure of red blood cells and for keeping hemoglobin in the ferrous state1. Glutathione reductase together with its co-factor, NADPH, catalyzes the reduction of oxidized glutathione (glutathione disulfide, GSSG) to glutathione (Figure 1). GSH is also a reactant for glutathione peroxidase, which converts hydrogen peroxide (H2O2) into water. In this assay the oxidation of NADPH to NADP+ (Figure 1) is monitored by the decrease in absorbance at 340 nm. This rate of decrease in absorbance at 340 nm is directly proportional to the glutathione reductase activity in the sample because the enzyme is present at rate limiting concentrations2-4. The unit definition for glutathione reductase activity may be expressed in terms of the oxidation of NADPH or the reduction of GSSG since their molar ratio is 1:1. One unit of glutathione reductase oxidizes 1 mol of NADPH per minute at 25C, pH 7.5.

Principle
1. 2. 3. Samples and standards are added to wells of a 96-well plate. GR Master mix and buffers are added. NADPH solution is added to the wells to initiate the reaction. The plate is transferred to a plate reader and absorbance readings are taken at 340 nm every minute for 10 minutes.

GSSG + NADPH + H
(Absorbs at 340 nm)

GLUTATHIONE REDUCTASE

2GSH + NADP+

Figure 1. Reduction of glutathione disulfide (GSSG) by glutathione reductase and NADPH.

Materials Supplied
1. 2. 3. 4. 5. 6. 7. Clear Microtiter Plate Five Plates of 96 Wells, Catalog No. 80-1639 Clear uncoated solid plates. GR Standard 1 mL, Catalogue No. 80-1640 Glutathione reductase standard with an activity of 1 unit/mL. One unit of GR oxidizes 1 mole of NADPH per minute at 25C, pH 7.5. 10X GR Buffer 20 mL, Catalog No. 80-1637 NADPH 5 lyophilized vials, Catalog No. 80-1638 One vial is sufficient for one 96-well plate. GSSG 3 mL, Catalog No. 80-1636 20% Triton X-100 1 mL, Catalog No. 80-1641 Glutathione Reductase Assay Layout Sheet 1 each, Catalog No. 30-0239

Do not mix components from different kit lots or use reagents beyond the expiration date of the kit. The physical, chemical, and toxicological properties of the chemicals and reagents contained in this kit may not yet have been fully investigated. Therefore, we recommend the use of gloves, lab coats, and eye protection while using any of these chemical reagents.

Storage
The GR Standard and NADPH should be stored at -20C. All other components of this kit are stable at 4C. All kit components are stable at their recommended storage temperatures until the kit expiration date.
Reagents require separate storage conditions.

Materials Needed but Not Supplied

1. 2. 3. PBS Distilled water Protease inhibitors (optional) such as phenylmethylsulfonyl fluoride (PMSF), Sigma P7626 or equivalent 4. Reagents to determine protein concentration 5. Ficoll-Hypaque (erythrocyte, lymphocyte and monocyte preparations) 6. Microtubes, 0.5 and 1.5 mL 7. 15 mL conical tubes (adherent and suspension cell preparation) 8. 50 mL conical tubes (tissue preparation) 9. Precision pipettes for volumes between 5-1000 L 10. Multichannel pipettor for volumes between 1 - 50 L and 50 L 200 L 11. Microplate reader capable of reading at 340 nm and taking readings every minute for ten minutes and exporting data to an Excel spreadsheet 12. Centrifuge and microfuge for processing samples

Reagent Preparation
All solutions must be prepared just before use.

1. 1X GR Buffer Dilute the 10X GR Buffer to 1X (1:10) with distilled water. The 1X GR Buffer is used to prepare dilutions of GR Standard Curve and to prepare the NADPH reagent. The 10X GR Buffer is used directly to prepare 1X Cell Extraction Buffer and Master Mix. 2. GR Standard Curve
160 L 200 L 200 L 200 L 160 L 200 L

Pre-rinse each pipet tip with reagent. Use fresh pipet tips for each sample, standard, and reagent.
S

240 450 L L

200 L

200 L

200 L

240 450 L L

200 L

Enzyme should be diluted just before use. Store enzyme on ice. Discard unused enzyme dilutions.

stock

0.020 units/50 L 0.005 units/50 L 0.0010 units/50 L 0.010 units/50 L 0.0025 units/50 L 0.0005 units/50 L

Thaw the 1 unit/mL GR Standard on ice. Label six 0.5 mL or 1.5 mL microtubes #1 through #6. Pipet 240 L of 1X GR Buffer into tube #1. Pipet 200 L of 1X GR Buffer into tubes #2, #3, #4, and #6. Pipet 240 L of 1X GR Buffer into tube #5. Add 160 L of GR Standard to tube #1 and vortex thoroughly. Add 200 L of tube #1 to tube #2 and vortex thoroughly. Continue this for tubes #3 and #4. Add 160 L of tube #4 to tube #5 and vortex thoroughly. Add 200 L of tube #5 to tube #6 and vortex thoroughly. Diluted standards may be kept at room temperature but must be used immediately. The concentrations of GR in the tubes are labeled above.

3. 1X Cell Extraction Buffer Prepare sufficient amount of Cell Extraction Buffer. Preparation for 10 mL is as follows: 10X GR Buffer 1.0 mL 20% (v/v) Triton X-100 0.2 mL Distilled water 8.8 mL 200 mM PMSF (optional) (10 L)
Store the reconstituted NADPH and NADPH solution on ice and use within 4 hours of preparation.

4.

NADPH Solution The kit contains 5 vials of lyophilized NADPH, each sufficient for 100 tests. Add 0.5 mL of 1X GR Buffer to dissolve the contents of the vial. Transfer the solution to a 15 mL conical tube. Wash the vial two more times with 0.5 mL of 1X GR Buffer and transfer to the 15 mL conical tube. Add 1X GR Buffer to the 15 mL conical tube to a final volume of 6.5 mL.

5. Master Mix Count the total number of wells needed for the samples and add 21 (for the complete standard curve and Blank wells in triplicate). Use the following formula to calculate the volume of Master Mix required. A. Total volume required [Total number of wells needed + 21] x 100 L B. Volume of 10X GR Buffer required [Total Volume Required (from A. above)] x 0.1 C. Volume of GSSG Reagent required [Total Volume required (from A. above)] x 0.04 D. Volume of distilled water required [Total Volume required (from A. above)] x 0.86 = __________ L = __________ L = __________ L = __________ L

Thaw the GSSG stock on ice. Keep the stock on ice while using.

Prepare Master Mix by combining the appropriate reagent volumes calculated in B, C, and D above. For example, to prepare 100 L of Master Mix the following volumes are combined: 10 L 10X GR Buffer, 4 L GSSG reagent, and 86 L distilled water. Biological Extracts After preparing the samples as outlined in the Sample Handling section that follows, make serial dilutions of cell or tissue extracts with 1X GR Buffer. Initial concentrations between 0.5 g/50 L to 50 g/50 L are recommended.

6.

Sample Handling
Choose the appropriate protocol in Section A to process sample before proceeding to Section B. Keep samples on ice to maintain enzyme activity. Section A. Processing Samples Suspension cells: 1. Centrifuge 2 to 6 x 106 suspension cells at 250 x g for 10 minutes at 4C. Discard the supernatant. 2. Suspend the cell pellet in 1 mL of ice-cold 1X PBS and transfer to a 1.5 mL microtube on ice. Centrifuge, discard supernatant, and place on ice. 3. Proceed to Section B. Preparation of Cytosolic Extracts Adherent cells: 1. Wash 2 to 6 x 106 adherent cells with 1X PBS. Adherent cells may be harvested by gentle trypsinization. 2. Transfer to a 15 mL tube on ice. Centrifuge at 250 x g for 10 minutes at 4C and discard the supernatant. 3. Suspend the cell pellet in 1 mL of ice-cold 1X PBS and transfer to 1.5 mL microtube on ice. Centrifuge, discard supernatant, and place on ice.. 4. Proceed to Section B. Preparation of Cytosolic Extracts.
Samples must be kept on ice to maintain enzyme activity.

Samples must be kept on ice to maintain enzyme activity.

Erythrocytes: 1. Dilute anticoagulated blood with an equal volume of PBS. Layer over Ficoll-Hypaque or similar reagent and centrifuge at 800 x g for 25 min at 12C with the BRAKE OFF in a swinging bucket rotor. 2. Collect the mononuclear cells (lymphocytes and monocytes) at the interphase and transfer to another tube. 3. Remove the remaining liquid from above the red blood cell pellet. Wash the pellet with 10 cell volumes of PBS. 4. Determine the packed cell volume and add 10 cell volumes of cold distilled water. Mix well and incubate on ice for 10-15 minutes to lyse the red blood cells. Lysis occurs when the opaque solution changes to a brilliant clear red solution, indicating the release of hemoglobin. 5. Centrifuge at 10,000 x g for 10 minutes at 4C to remove cell debris. Transfer the supernatant to a fresh tube and store on ice. Lymphocytes and Monocytes: 1. Dilute anticoagulated blood with an equal volume of PBS. Layer over Ficoll-Hypaque or similar reagent and centrifuge at 800 x g for 25 min at 12C with the BRAKE OFF in a swinging bucket rotor. 2. Collect the mononuclear cells (lymphocytes and monocytes) at the interphase and transfer to another tube. 3. Dilute the blood mononuclear cells with 5 volumes of PBS and centrifuge at 400 x g for 10 minutes at 4C. Discard the supernatant 4. Suspend the cell pellet in 1 mL of ice-cold 1X PBS and transfer to a pre-chilled 1.5 mL microtube. Centrifuge, discard supernatant, and place on ice. 5. Proceed to Section B. Preparation of Cytosolic Extracts. Tissue 1. Remove tissue and place in cold PBS in a 50 mL conical tube. Repeatedly wash the tissue with PBS to remove blood clots and other debris. 2. Transfer the tissue to a Petri dish on ice and mince the tissue to small pieces with surgical scissors. 3. Transfer the tissue pieces to a clean stainless steel sieve. Place the sieve with the tissue pieces in a Petri dish which contains about 20 mL of cold 1X PBS. 4. Create a single cell suspension of the tissue as follows: Using a pestle or a round bottom tube, grind the tissue pieces thoroughly until the bulk of the tissue passes through the sieve. 5. Transfer the PBS containing the single cell suspension to a 50 mL conical tube. Fill with cold PBS and mix by inverting the tube several times. Let the tube stand on ice for 1 minute to allow large aggregates of tissue to settle out of solution. 6. Carefully transfer the supernatant containing the single cell suspension to a clean 50 mL conical centrifuge tube. Centrifuge at 400 x g for 10 minutes at 4C. Discard the supernatant. Suspend the cell pellet in 1 mL of ice-cold 1X PBS and transfer to a prechilled 1.5 mL microtube on ice. Centrifuge, discard the supernatant, and place on ice. 7. Proceed to Section B. Preparation of Cytosolic Extracts.

Section B. Preparation of Cytosolic Extracts from Cells and Tissue 1. Measure the approximate volume of the cell pellets prepared above (except for erythrocytes) and suspend the cells in 5-10 volumes of cold 1X Cell Extraction Buffer. Incubate the cell suspensions on ice, with periodic vortexing, for 30 minutes. Microcentrifuge the disrupted cell suspension at 10,000 x g for 10 minutes at 4C to remove insoluble material. Recover the supernatant to a fresh tube pre-chilled on ice. Occasionally, the pellet may float and can easily be removed with a pipet tip. Determine the protein concentration of the cleared cell lysate. Note that the 1X GR Buffer contains BSA at a concentration of 0.1 mg/mL and this should be subtracted from your observed protein concentration. If not assaying for GR immediately, snap-freeze the cleared cell extract in 100 L aliquots by immersing the aliquots in liquid nitrogen and store at -80C. Avoid repeated freezing and thawing of the extract.

2.

3.

4.

Assay Procedure
Refer to the Assay layout Sheet to determine the number of wells to be used. 1. Pipet 50 L of the 1X GR Buffer to the bottom of the Blank wells. 2. Pipet 50 L of the prepared GR Standards #1 through #6 to the bottom of the appropriate wells. 3. Pipet 50 L of the diluted sample to the bottom of the appropriate wells. 4. Pipet 100 L of Master Mix into each well. 5. Initiate the reaction by adding 50L of NADPH Solution to all the wells using a multichannel pipet. 6. Immediately transfer the plate to a microtiter plate reader and take absorbance readings at 340 nm every minute for 10 minutes at room temperature. If possible, include a 10 second orbital shake prior to the first read.
All standards, controls, and samples should be run in triplicate. Pre-rinse each pipet tip with reagent. Use fresh pipet tips for each sample, standard, and reagent. Pipet the reagents to the sides of the wells to avoid possible contamination.

Calculation of Results
A. 1. Time Course of the Change in Absorbance at 340 nm of the GR Standard Plot the mean of the triplicate absorbance values at 340 nm versus time of the GR standard (Figure 2):

B.

Determination of GR Activity in Experimental Samples from the GR Standard Curve Determine the rate of decrease in absorbance at 340 nm per minute ( A340 nm/ min) for each of the standards and the Blank wells (Buffer control):

1.

(A340 nm @ 1 min.) - (A340 nm @ 10 min.) = A340 nm / min 9 min Note: Our plate reader takes absorbance readings beginning at the 1 minute timepoint and ends at 10 minutes. 2. Calculate the net rate for each standard by subtracting the rate obtained for the Blank (the Blank measures the spontaneous oxidation of NADPH. This value is usually small and may be negligible compared to sample values). 3. Plot the number of units of GR/well versus these new A340 nm/ min values (Figure 3):

4. Plot the absorbance values of samples versus time as shown in Figure 2. Take the slope of the linear portion of the curves. 5. Calculate the net rate for each level of the samples by subtracting the rate obtained for the blank (the blank measures the spontaneous oxidation of NADPH. (This value is usually small and may be negligible compared to sample values). 6. Determine the number of units/well of GR in your samples from the linear portion of the GR standard curve of Figure 3. C. Determination of GR Activity in Experimental Samples from the Molar Extinction Coefficient of NADPH The GR activity in the sample(s) may be calculated by using the extinction coefficient of NADPH:

1. Determine the rate of decrease in absorbance per minute for both samples and the Blank as described above. 2. Calculate the net rate of the samples by subtracting the Blank rate from the sample rate. 3. Using the Beer-Lambert Law one can determine the concentration of NADPH n solution. Convert the net rate ( A340 nm/ min) to concentration of NADPH consumed, which is equal to the activity of GR in mU/ mL. The molar extinction coefficient (EM) for NADPH is 6220 M-1cm-1 and EM = 6.22 x 10-3 nmol/mL if the pathlength is 1 cm.

One unit of glutathione reductase is defined as the amount of enzyme required to catalyze the reduction of one mole of GSSG per minute at pH 7.5 and 25C. One molecule of NADPH is oxidized per molecule of GSSG reduced. Therefore, the oxidation of NADPH (measured by loss of A340 nm) directly correlates with GSSG reduction. 1 U of GR = 1 mol GSSG reduced/min = 1 mol NADPH oxidized/min 1 mU of GR = 1 x 10-3 mol NADPH oxidized/min 1 mU of GR = 1 nmol NADPH oxidized/min If EM is the molar extinction coefficient for NADPH at 340 nm, EM = 6220 M-1cm-1 = 6220 x 10-6 M-1cm-1 = 6.22 x 10-3 M-1cm-1 = 6.22 x 10-3 L/mol/cm = 6.22 x 10-3 mL/nmol/cm Note: The path length for 200 L of reaction volume in the 96 well plate is 0.6 cm. Therefore, EM = 6.22 x 10-3 mL/nmol /cm x 0.6 cm = 3.732 x 10-3 mL/nmole A340 nm/ min = A340 nm/ min = 3.732 x 10-3 mL/nmole NADPH = Y nmole NADPH/min/mL = Y mU/mL GR 4. Correct for the sample dilution in the assay and for the sample dilution per formed prior to the assay. For example: If the sample volume was 50 L and was diluted 1/50 prior to the assay: Mean A340 nm/min (Sample) Mean A340 nm/min (Blank) Net Rate, A340 nm/min Glutathione Reductase Activity Assay Dilution Correction Sample Dilution Correction = 0.0325/min = 0.0005/ min = 0.0320/ min = 0.0320/min/3.732x10-3mL/nmolNADPH/min = 8.57 mU/mL = 200 L/50 L x 8.57 mU/mL = 34.30 mU/mL = 50 x 34.30 mU/mL = 1715 mU/mL

5. Divide the GR activity (mU/mL) by the protein concentration to determine the specific activity of GR in your sample (mU GR/mg protein).

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Trouble Shooting
PROBLEM CAUSE SOLUTION Add the GSSG reagent to the Master Mix Follow protocol for making the NADPH reagent and add 50 L to each well Contact Customer Service Extend reaction to 20 minutes Reduce the amount of distilled water in the Master Mix. Add 50 L of this modified Master Mix and 100 L of your extract to each well GR activity in cells and tissues very high Increase the dilution of the sample in order to reduce the amount of sample added to the wells No change in absorbance Failure to add GSSG to the at 340 nm with time in all Master Mix the wells Absorbance at 340 nm in wells with 1X GR Buffer alone is less than 0.5 Failure to add NADPH to the wells NADPH has degraded Change in absorbance with the GR standard is satisfactory, but no apparent change in absorbance observed in your sample GR activity in cells and tissues very low

Change in absorbance with the GR standard is satisfactory, but absorbance with sample drops to baseline within one or two minutes

References
1. Berg JM, Tymoczko JL, Styer L. 2002. Biochemistry, 5th Edition, WH Freeman and Company, New York. 2. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem., 27, 502-522 (1969). 3. Smith IK, Vierheller TL, Thorne CA. Assay of glutathione reductase in crude tissue homogenates using 5,5-dithiobis(2-nitrobenzoic acid). Anal. Biochem., 175, 408-413 (1988). 4. Dolphin, D, Poulson, R, Avramovic, O. (Eds.), Glutathione: Chemical, Biochemical, and Metabolic Aspects, Volumes A and B, Wiley and Sons (1989). 5. Dringen R, Gutterer JM. Glutathione reductase from bovine brain. Methods Enzymol. 348, 281-288 (2002).

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Use of Product
MSDS (Material Safety Data Sheet) available online This product contains research chemicals. As such, they should be used and handled only by or under the supervision of technically qualified individuals. This product is not intended for diagnostic or human use.

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Warranty
Enzo Life Sciences International, Inc. makes no warranty of any kind, expressed or implied, which extends beyond the description of the product in this brochure, except that the material will meet our specifications at the time of delivery. Enzo Life Sciences International, Inc. makes no guarantee of results and assumes no liability for injuries, damages or penalties resulting from product use, since the conditions of handling and use are beyond our control.
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Catalog No. 25-0599 August 3, 2010 2007

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