Enzymatic Assay of Peroxidase (EC


This procedure is for the determination of Peroxidase enzymatic activity using Pyrogallol as the substrate. Products using this method include, but are not limited to, P1709, P2088, P6140, P6782, P8125, P8170, P8250, P8375, P8415, and P8651.

The continuous spectrophotometric rate determination (A420, Light path = 1 cm) is based on the following reaction:

Unit Definition: One unit of peroxidase will form 1.0 milligram of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20 °C. This unit is equivalent to ~18 µM units per minute at 25 °C.


Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

1.0 M Potassium phosphate monobasic solution (Catalog Number P8709)

1.0 M Potassium phosphate dibasic solution (Catalog Number P8584)

Hydrogen Peroxide, 30% (w/w) Solution (Catalog Number H1009)

Pyrogallol (Catalog Number 254002)

Cuvettes and thermostatted spectrophotometer

Preparation Instructions

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Phosphate Buffer (100 mM Potassium Phosphate Buffer, pH 6.0 at 20 °C) – Add 17.36 ml of 1.0 M Potassium phosphate monobasic solution (Catalog Number P8709). Add 2.64 ml of 1.0 M Potassium phosphate dibasic solution (Catalog Number P8584) and adjust to final volume of 200 ml using ultrapure water. Adjust to pH 6.0 at 20 °C using 1 N KOH or 1 N HCl. Store the Phosphate Buffer on ice.

Peroxide Solution (0.50% [w/w] Hydrogen Peroxide [H2O2] Solution) – Prepare a 1:60 dilution using Hydrogen Peroxide, 30% (w/w) Solution (Catalog Number H1009) in ultrapure water.

Note: Prepare fresh dilutions for control and sample, and store the solution in a capped 4 dram vial on ice to reduce exposure to air.

Pyrogallol Solution (5% [w/v] Pyrogallol Solution) – Prepare a 50 mg/ml solution using Pyrogallol (Catalog Number 254002) in ultrapure water in an amber vial (or cover with foil).

Note: Prepare fresh and keep this solution on ice protected from light.

Peroxidase Solution – Prepare a 10 mg/ml solution of peroxidase in COLD Phosphate Buffer. Immediately before use, prepare a working solution containing 0.45–0.75 unit/ml of Peroxidase in COLD Phosphate Buffer.

Note: The 10 mg/ml stock solution may be used for the RZ Factor Determination.


Final Assay Concentrations – In a 3.00 ml reaction mix, the final concentrations are 14 mM potassium phosphate, 0.027% (v/v) hydrogen peroxide, 0.5% (w/v) pyrogallol, and 0.45–0.75 unit peroxidase.

Note: Run one cuvette at a time as the maximum rate is in the first minute.

1. Pipette the following reagents into suitable cuvettes:

Reagent Blank
Test 1
Test 2
Test 3
Ultrapure Water 2.1 2.1 2.1 2.1
Phosphate Buffer 0.32 0.32 0.32 0.32
Peroxide Solution 0.16 0.16 0.16 0.16
Pyrogallol Solution 0.32 0.32 0.32 0.32


2. Mix by inversion and equilibrate to 20 °C in a suitably thermostatted spectrophotometer for ~10 minutes.

Note: All the cuvettes can be incubated at the same time but read only one cuvette at a time.


3. Then add:

Reagent Blank
Test 1
Test 2
Test 3
Phosphate Buffer 0.1
Peroxidase working Solution 0.1 0.1 0.1


4. Immediately mix by inversion and record the increase in A420 for 3 minutes. Obtain the DA420/20 seconds using the maximum linear rate or 0.5 minute interval for all the Tests and Blank.

Suitability Criteria: The rate DA420/20 seconds must be within 0.18-0.34. The enzyme concentration may be modified, if necessary.




3 = Volume (in milliliters) of assay
df = Dilution factor
12.0 = Extinction coefficient of 1 mg/ml of Purpurogallin at 420 nm (determined by MilliporeSigma)
0.1 = Volume (in milliliters) of enzyme used







  1. Chance, B., and Maehly, A.C., Methods in Enzymology, 2, 773-775 (1955).
  2. Shannon, L.M. et al., Journal of Biological Chemistry, 241, 2166-2172 (1966).



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