The amount of enzyme causing the production of one milligram of purpurgallin in 20 seconds at 25°C under the given assay conditions.
Assay Method
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.
Applications
Peroxidase (EC 1.11.1.7) 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.
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.
Reagents
0.2 M Potassium phosphate, pH 7.0.
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.
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.
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. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 510 nm and 25°C.
Procedure
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 510 nm and 25°C.
Into the cuvette, pipette the following:
Phenol/4-aminoantipyrine solution 1.4 ml
Hydrogen peroxide solution 1.5 ml
Mix the cuvette contents and incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibrium.
Establish blank rate, if any, at 510 nm.
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.
