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Glutathione Peroxidase

(Glutathione: hydrogen peroxide oxidoreductase; EC 2GSH + H2O2 ------> GSSG + 2H2O Glutathione peroxidase catalyzes the reduction of various organic hydroperoxides, as well as hydrogen peroxide, with glutathione as hydrogen donor. It has been suggested that this enzyme functions in more times as a mechanism of protecting the cellular membrane system against peroxidative damage. And the importance of selenium as an essential trace element is further concerned with this suggested function of the enzyme. It has a molecular weight of 84,000 and 4 subunits per mol of enzyme. The enzyme is useful for enzymatic determination of lipid hydroperoxide.

The amount of enzyme which causes the oxidation of one micromole of GSH (half a micromole of NADPH) per minute at 25C and pH 7.

The assay is based on the coupled reaction with glutathione reductase. The decrease of NADPH is measured at 340 nm by spectrophotometer

  1. Reduced glutathione (GSH) - 10 mM (3.07 mg GSH/ml of H2O) (make fresh)
  2. Hydrogen peroxide (H2O2) - 4.0 mM [Dilute 0.0455 ml of 30% (W/V) H2O2 to 100 ml with H2O (prepare fresh)]
  3. Buffer - 0.1 M phosphate buffer, pH7 containing 5.0 mM EDTA
  4. NADPH - 1.6 mM [5 mg/ml]
  5. Glutathione reductase - 2.5 U/ml
  6. Sodium azide (NaN3) - 10 mM (6.5 mg/ml of distilled water)
  7. Glutathione peroxidase. Using phosphate buffer, prepare a solution to yield 2-5 U/ml. Prepare fresh prior to assay.

  1. Set spectrophotometer at 340 mm and 25C.
  2. Into a cuvette pipette the following reagants in the amounts indicated:

    Phosphate buffer 1.0 ml
    GSH 0.2 ml
    NADPH 0.2 ml
    GSSG-reductase 0.2 ml
    NaN3 0.2 ml

  3. Record the absorbance at 340 nm (blank) if any
  4. Initiate the reaction by adding 10 l of enzyme glutathione peroxidase. Record degrees in absorption at 340 nm for 5 minutes. Calculate (delta)E340 nm/min

Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)(2)
(6.22)(Enz. Vol.)(mg Enz./ml)

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