Enzymatic Assay of Peroxidase (EC 1.11.1.7)

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.

Precautions

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 (Storage/Stability)

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.

Procedure

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:

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:

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.

Results

Calculations

1.

Where:
3 = Volume (in milliliters) of assay
df = Dilution factor
12.0 = Extinction coefficient of 1 mg/mL of Purpurogallin at 420 nm (determined internally)
0.1 = Volume (in milliliters) of enzyme used

2.

3.