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(Donor:hydrogen peroxide oxidoreductase; EC

Peroxidase is an enzyme catalyzing the oxidation, by hydrogen peroxide, of a number of substrates such as ascorbate, cytochrome C and the leuco form of many dyes. A representative reaction is shown below:

H2O2 + DH2 ------> 2 H2O + D
(DH2 = leuco dye; D = dye)
Horseradish peroxidase (HRP) exists in the form of several isozymes, all containing heme as the prosthetic group. The enzyme has a molecular weight of approximately 40,000.

The ability of peroxidase to catalyze the oxidation of a number of organic compounds by hydrogen peroxide, resulting in formation of colored end-products, is utilized in several methods of determination of glucose and galactose in biological fluids. Also, recently a method has been described for the determination of plasma uric acid utilizing uricase and horseradish peroxidase (Sander, G.T.B.; Pasman, A.J. and Hoek, F.J., Clin. Chim. Acta, 101, 299, 1980).

1.Source: Horseradish
   Form: Freeze-dried powder 
   Solubility: Distilled water 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): None 
   Protein (approx.): 95% (biuret) 
   RZ Value (approx.): <0.1 
   Catalog No.: 239A0000 

The amount of enzyme causing the production of one milligram of purpurgallin in 20 seconds at 25C under the given assay conditions.

The rate of decomposition of hydrogen peroxide catalyzed by peroxidase, with 4-aminoantipyrine as hydrogen donor, is determined by measuring the increase in absorbance at 510 nm.

  1. 0.2 M Potassium phosphate, pH 7.0.
  2. 0.0017 M Hydrogen peroxide. Prepare by diluting 1 ml of 30% H2O2 to 100 ml with distilled water. Further dilute 1 ml of this solution to 50 ml using 0.2 M phosphate buffer, pH 7.0. Prepare fresh daily.
  3. 0.0025 M 4-Aminoantipyrine with 0.17 M phenol. Dissolve 810 mg phenol in 40 ml distilled water. Add 25 mg 4-aminoantipyrine and made the volume up to 50 ml with distilled water.
  4. Enzyme (peroxidase) solution. Dissolve 1 mg/ml in distilled water. Just prior to use, dilute further with distilled water to yield a concentration of 0.05-0.25 U/ml.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 510 nm and 25°C.
  2. Into the cuvette, pipette the following:
    Phenol/4-aminoantipyrine solution 1.4 ml
    Hydrogen peroxide solution 1.5 ml
  3. Mix the cuvette contents and incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibrium.
  4. Establish blank rate, if any, at 510 nm.
  5. Into the cuvette, pipette 0.1 ml enzyme solution (0.05-0.25 U/ml). Mix and record the increase in absorbance at 510 nm for 4-5 minutes.
  6. Calculate µE510 nm/min

Activity (U/mg) = (ΔE510nm/min)(Total Vol.)(Enz. Diln.)
(6.58)(Enz. Vol.)(mg Enz./ml)

Note: 1 Guiacol unit = 2 Calzyme units

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