Peroxidase + H2O2 → Complex
Complex + AH2 (donor) → Peroxidase + H2O + A (colored)
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% H202 solution in deionized or distilled water. Prior to use dilute with Buffer to a final concentration of 0.003%.
- 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.
- At time=0, add 100 µl of diluted enzyme to the Reaction tube and 100 µl PBS to the Control tube. Mix thoroughly.
- 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.
- 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