Horseradish Peroxidase Enzyme Activity Assay Procedure

Chemical Principle:

Peroxidase + H2O2 → Complex

Complex + AH2 (donor) → Peroxidase + H2O + A (colored)

Assay Reagents:

Buffer: 0.01M Sodium Phosphate, pH 6.0

Enzyme: Dilute with the Buffer. Acceptable dilution: 1-2 µg/ml.

Dye: Freshly prepare 1% o-dianisidine in methanol. Note: the solid will not dissolve well. Vortex briefly and let settle before use. Store in amber bottle or wrapped in foil.

Substrate: Prepare 0.3% H20solution in deionized or distilled water. Prior to use dilute with Buffer to a final concentration of 0.003%.


  1. Add 0.05 mls of Dye to 6.0 ml of substrate and vortex. Add 2.9 mls of this mixture to a Reaction tube and 2.9 ml to a Control tube.
  2. At time=0, add 100 µl of diluted enzyme to the Reaction tube and 100 µl PBS to the Control tube. Mix thoroughly.
  3. Measure and record the optical density at 460nm (OD460) every 15 seconds for 3 minutes, or take the end point reading after 3 minutes by stopping the reaction with 100 µl of concentrated NaN3.
  4. Use this value to determine the rate of change in absorbance per minute.

Enzyme Activity Calculations:

One unit of activity is the amount of enzyme to decompose 1 µmole of peroxide/minute at 25 degrees Celsius. 11.3 x 10 3 cm -1 is the molar absorbance of H2O2.

OD460/min = (OD460/3min – OD Control/ 3 minutes) / 3 minutes

mg enzyme / ml reaction mixture = [enzyme dilution] / 30

units / mg = ( OD460 /min ) / ( 11.3 x mg enzyme) / ml reaction mixture