Glutathione Peroxidase Cellular Activity Assay Kit

Catalog Number CGP1

Storage Temperature –20 °C

TECHNICAL BULLETIN

Product Description This kit uses an indirect determination method. It is

Glutathione peroxidase (GPx, EC 1.11.1.9) provides a based on the oxidation of glutathione (GSH) to oxidized
mechanism for detoxification of peroxides in living glutathione (GSSG) catalyzed by GPx, which is then
1,2
cells. This reaction plays a crucial role in protecting coupled to the recycling of GSSG back to GSH utilizing
cells from damage by free radicals, which are formed glutathione reductase (GR) and NADPH
by peroxide decomposition. Lipid components of the (β-Nicotinamide Adenine Dinucleotide Phosphate,
cell are especially susceptible to reactions with free Reduced).
radicals, resulting in lipid peroxidation. GPx enzymes
reduce peroxides to alcohols using glutathione, thus The decrease in NADPH absorbance measured at
preventing the formation of free radicals. 340 nm during the oxidation of NADPH to NADP+ is
indicative of GPx activity, since GPx is the rate limiting
GPx enzymes will catalyze the reduction of hydrogen factor of the coupled reactions.1,6
peroxide (H2O2) and a wide variety of organic peroxides
(R-OOH) to the corresponding stable alcohols (R-OH) GPx
and water using cellular glutathione as the reducing R-OOH + 2 GSH → R-OH + GSSG + H2O
reagent.
GR
Most cellular glutathione peroxidases are tetrameric GSSG + NADPH + H+ → 2 GSH + NADP+
enzymes consisting of four 22 kDa monomers, each of
which contains a selenocysteine moiety in the active GPx is glutathione peroxidase, GR is glutathione
site.2 The selenocysteine participates directly in reductase, and R-OOH is an organic peroxide.
electron donation to the peroxide substrate and
becomes oxidized in the process. The enzyme then The reaction is performed at 25 °C and pH 8.0,10 and is
uses reduced glutathione as a hydrogen donor to started by adding an organic peroxide, tert-butyl
regenerate the selenocysteine. GPx enzymes also exist hydroperoxide (t-Bu-OOH).11 This substrate is suitable
as non-selenium (non-Se) containing enzymes.3 for the assay since its spontaneous reaction with GSH
is low and it is not metabolized by catalase. The
Cellular GPx is present in all tissues; however, various reaction with tert-butyl hydroperoxide measures the
diseases may influence its level. An increase in the amount of selenium-containing glutathione peroxidase
level of glutathione peroxidase has been observed in activity present.
reticulocytes of diabetic rats. The level returned to
normal after administration of insulin.6 A decrease in If the presence of non-Se enzymes is suspected,
the level of the enzyme has been observed in patients cumene hydroperoxide can be used as the substrate at
7
suffering from diseases such as Favism (a disease 4,5
a concentration of 0.25–1.0 mM. This will measure
associated with extreme hemolytic crisis) or hairy cell the total GPx (Se and non-Se enzymes) activity. The
leukemia.8 difference between the activity observed with cumene
hydroperoxide and the tert-butyl hydroperoxide activity
is the non-Se glutathione peroxidase activity.
2

Reagents provided Preparation Instructions

Sufficient for 100 tests Glutathione Peroxidase Assay Buffer – Bring a suitable
• Glutathione Peroxidase Assay Buffer, 120 ml aliquot to room temperature. For long term stability of
Catalog Number G8664 the solution after opening, handle the solution in an
50 mM Tris HCl, pH 8.0, aseptic manner.
containing 0.5 mM EDTA
• NADPH Assay Reagent, 5 vials NADPH Assay Reagent – Reconstitute 1 vial in 1.25 ml
Catalog Number N5283 of water. Store the solution at 2–8 °C. This
When reconstituted with 1.25 ml of water, reconstituted solution should be used within 3 hours.
each vial will prepare a solution containing Each vial is sufficient for at least 20 tests. Do not freeze
5 mM NADPH, 42 mM reduced glutathione, the solution for reuse.
and 10 units/ml of glutathione reductase
• Luperox TBH70X 1 ml 30 mM tert-Butyl Hydroperoxide Solution – Dilute
Catalog Number 458139 21.5 µl of Luperox TBH70X, Catalog Number 458139,
70% aqueous solution of tert-Butyl Hydroperoxide to a total volume of 5 ml with water to prepare a 30 mM
tert-Butyl Hydroperoxide solution.
Reagents and equipment required but not provided
• UV/Vis spectrophotometer with thermostated Glutathione Peroxidase Standard (not supplied) –
cuvette holder and a kinetic program Dissolve a 100 unit vial of glutathione peroxidase,
• Quartz cuvette Catalog Number G6137, in 1 ml of Glutathione
• Cumene hydroperoxide, Catalog Number 513296, if Peroxidase Assay Buffer. Dilute to 0.25 unit/ml with
total GPx level is to be measured. Glutathione Peroxidase Assay Buffer supplemented
• Glutathione peroxidase, Catalog Number G6137, with 1 mg/ml IgG, Catalog Number I4506, and 1 mM
for use as control enzyme DTT, Catalog Number D0632.
• Human immunoglobulin G, Catalog Number I4506, Notes:
and dithiothreitol, Catalog Number D0632, for use • The Glutathione Peroxidase Standard is used as a
with the control enzyme glutathione peroxidase qualitative positive control to assure the reaction is
working. The 100 units/ml solution is diluted
Precautions and Disclaimer 400-fold to a Working Stock Solution of
This product is for R&D use only, not for drug, 0.25 units/ml. Use 20–50 µl of the Working Stock
household, or other uses. Please consult the Material Solution in the reaction to obtain values within the
Safety Data Sheet for information regarding hazards linear range of 0.005–0.02 units per reaction
and safe handling practices. volume of 1 ml. This is equivalent to an absorbance
decrease of 0.032–0.13 per minute.
Storage/Stability • The IgG is required for stabilization of the enzyme.
Store the kit at –20 °C. If daily use of the kit is needed, It is only required in cases of very pure and dilute
the assay buffer and tert-butyl hydroperoxide may be enzyme solutions.
stored for convenience at 2–8 °C. The components in
this kit are stable for 24 months when unopened. Sample preparation – Dilute crude biological samples in
Glutathione Peroxidase Assay Buffer.
Notes:
• Perform several dilutions of the sample for use in
the reaction in order to obtain results within the
linear range.
• The final concentration of the reagents in the assay
mixture is 0.25 mM NADPH, 2.1 mM reduced
glutathione, 0.5 unit/ml glutathione reductase, and
300 µM t-Bu-OOH.
 3

Procedure
Tissue extracts may contain enzymes that utilize 2. Add 50 µl of the NADPH Assay Reagent and
NADPH and skew the results. A blank without 10–50 µl of sample or 20–50 µl of enzyme to the
t-Bu-OOH can be used as a control for these cuvette and mix by inversion. The total volume in
endogenous activities. When liver samples are the cuvette should be 1.00 ml.
analyzed, H2O2 should be used to start the reaction 3. Start the reaction by addition of 10 µl of the 30 mM
(0.15–0.30 mM solution) since t-Bu-OOH is also a tert-Butyl Hydroperoxide Solution. Mix by inversion.
substrate for glutathione S-transferase. In this case, 4. Follow the decrease in absorbance at 340 nm using
catalase must be blocked by addition of 1 mM NaN3 in a kinetic program. The following program is
12
the reaction mixture. In addition, when using H2O2 recommended:
instead of t-Bu-OOH, the pH of the Glutathione Wavelength: 340 nm
Peroxidase Assay Buffer should be adjusted to pH 7.0 Initial delay: 15 seconds
with HCl. At a pH greater than 7.0 there will be a Interval: 10 seconds
spontaneous reaction of hydrogen peroxide with Number of readings: 6
reduced glutathione. 5. Calculate the amount of enzyme in the sample.

High concentrations of reducing agents such as DTT or Calculation

2-mercaptoethanol (>0.1 mM final concentration in the The spectrophotometer should give the A340/min from
assay system) will depress the measured activity by the reaction automatically. If the absorbance is
∼40% at 0.15 mM and up to 70% at 1 mM concentration measured manually, calculate this value for the blank,
in the assay. EDTA at 5 mM in the assay will depress positive controls, and all samples.
the activity by ∼50%.
The activity of Glutathione Peroxidase in the sample
Nonionic detergents such as TWEEN 20 and can be calculated using the formula:
Triton X-100 that contain high levels of endogenous
peroxides will raise the apparent activity. If these Activity per extract (mmol/min/ml = Units/ml)
detergents are vital to the extraction of the proteins of
interest, a low peroxide detergent should be used, such ∆A340 × DF
as Catalog Numbers X100PC (Triton X-100), P6585 6.22 × V
(TWEEN 20), or P8192 (TWEEN 80).
∆A340 = A340/min(blank) – A340/min(sample)
1. Pipette the volume of Glutathione Peroxidase 6.22 = εmM for NADPH
Assay Buffer indicated in Table 1 into a 1 ml quartz DF = dilution factor of sample before adding to
cuvette. Keep the temperature of the assay buffer reaction
in the spectrophotometer at 25 °C by using a V = sample volume in ml
thermostated cell holder.
Unit definition: 1 unit of glutathione peroxidase will
Table 1. cause the formation of 1.0 µmol of NADP+ from NADPH
Glutathione Peroxidase Reaction Scheme per minute at pH 8.0 at 25 °C in a coupled reaction in
the presence of reduced glutathione, glutathione
GPx NADPH Enzyme reductase, and tert-butyl hydroperoxide.
30 mM
Assay Assay (0.25 Sample
t-Bu-OOH
Buffer Reagent units/ml) (µl)
(µl)
(µl) (µl) (µl)
Blank 940 50 – – 10
Positive
890–920 50 20–50 – 10
control
Sample 890–930 50 – 10–50 10
4

References
1. Mannervik, B., Methods in Enzymol., 113, 490-495 8. Arruda, V.R. et al., Neoplasma, 43, 99–102 (1996).
(1985). 9. Paglia, D.E., and Valentine, W.N., J. Lab. And Clin.
2. Ursini, F. et al., Methods in Enzymol., 252, 38-52 Med., 70, 158-169 (1967).
(1995). 10. Beutler, E. et al., Br. J. Haematol., 35, 331-340
3. Wendel, A., in “ Enzymatic Basis of Detoxification” (1977).
Vol. 1, Academic Press (New York, NY: 1980) 11. Thomson, C.D. et al., Br. J. Nutr., 37, 457-460
pp. 333-353. (1977).
4. Thomson, C.D., Biochem. Int., 10, 673-679 (1985). 12. Flohe, L., and Gunzler, W.W., Methods in
5. Carmagnol, F. et al., Biochim. Biophys. Acta, 759, Enzymol., 105, 114 -121 (1984).
49-57 (1983).
6. Gupta, B.L., and Baquer, N.Z., Biochem. Mol. Biol. Triton is a trademark of the Dow Chemical Co.
Int., 46, 1145-1152 (1998). TWEEN is a registered trademark of the ICI Group
7. Mavelli, I. et al., Eur. J. Biochem., 139, 13-18 Luperox is a registered trademark of Arkema Inc.
(1984).
EM,ESS,NDH,PHC,MAM 02/12-1

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