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Peroxidase

(Donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7)

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:

         PEROXIDASE
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 Roots
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20° C; -4° F 
   Activity: 200 U/mg 
   RZ Value: >= 1.00 
   Protein: 85% (biuret) 
   Catalog No.: 100A0200 
$.20/mg
2.Source: Horseradish Roots
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20° C; -4° F 
   Activity: 400 U/mg 
   RZ Value: >= 2.00 
   Protein: 95% (biuret) 
   Catalog No.: 100A0400 
$.30/mg
3.Source: Horseradish Roots
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20° C; -4° F 
   Activity: 600 U/mg 
   RZ Value: >= 3.00 
   Protein: 95% (biuret) 
   Catalog No.: 100A0600 
$.40/mg
4.Source: Horseradish Roots
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20° C; -4° F 
   Activity: 160 U/mg 
   RZ Value: >= 3.4 
   Protein: 95% (biuret) 
   Isoenzyme: Acidic 
   Catalog No.: 100A0160 
$1.50/mg
5.Source: Zucchini Squash
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20° C; -4° F 
   Activity: 10-20 U/mg 
   Protein: 95% (biuret) 
   Purity: 95% 
   Catalog No.: 244A0000 
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The amount of enzyme causing the production of one milligram of purpurgallin in 20 seconds at 25°C 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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