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 purpurogallin (20 second) unit is equivalent to ~18 µM units per minute at 25 °C.


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

Reagents and Equipment Required

Potassium Phosphate, Monobasic, Anhydrous (Catalog Number P5379)

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) – Prepare a 13.6 mg/ml solution of Potassium Phosphate, Monobasic, Anhydrous (Catalog Number P5379) using ultrapure water. Adjust pH to 6.0 at 20 °C with 1.0 M KOH. After adjusting the pH, put on ice.

Peroxide Solution (0.50% [w/w] Hydrogen Peroxide [H2O2] Solution) – Prepare 5 ml of a 1:60 solution in ultrapure water using Hydrogen Peroxide, 30% (w/w) Solution (Catalog Number H1009). PREPARE FRESH 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 in ultrapure water using Pyrogallol (Catalog Number 254002). PREPARE FRESH AND KEEP FROM LIGHT. Keep this solution on ice.

Peroxidase Solution – Prepare a 10 mg/ml solution of peroxidase in COLD Phosphate Buffer. At this concentration, the solution is stable for 15 minutes. Immediately before use, prepare a working solution containing 0.4–0.7 unit/ml of Peroxidase in COLD Phosphate Buffer.


Final Assay Concentrations – In a 3.00 ml reaction mix, the final concentrations are 14 mM potassium phosphate, 0.027% (w/w) hydrogen peroxide, 0.5% (w/v) pyrogallol, and 0.04–0.07 unit peroxidase.

1. Pipette the following reagents into suitable cuvettes:

Test Sample
Ultrapure Water
Phosphate Buffer 0.32
Peroxide Solution 0.16
Pyrogallol Solution 0.32

2. Mix by inversion and equilibrate for 10 minutes to 20 °C in the spectrophotometer. Monitor the A420 until constant, using a suitably thermostatted spectrophotometer. Then add:

Test Sample
Phosphate Buffer 0.10
Peroxidase Working Solution 0.10

3. Immediately mix by inversion and record the increase in A420 at a rate of 1 reading/second for ~3 minutes. Use the maximum linear rate to obtain the DA420/20 seconds for both the Test Sample and Blank.

4. The Test Sample enzyme concentration may have to be modified in order for the rate (DA420/20 seconds) to be within the specified absorbance change rate of 0.16–0.28.




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



Units/mg solid = units/ml enzyme
mg solid/ml enzyme



Units/mg protein = units/ml enzyme
mg protein/ml enzyme




  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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