How to Measure Catalase Concentrations

Catalase is a biological (organic) catalyst. This enzyme is one of the most potent catalysts known and has beneficial effects on the human body and is crucial to life. Catalase acts as a catalyst in speeding up the breakdown of hydrogen peroxide. Hydrogen peroxide is a metabolic byproduct that is poisonous to the cells if not removed. Catalase assists in breaking down hydrogen peroxide into water and oxygen. Catalase also aids in oxidizing other toxins such as phenols, formic acid, formaldehyde and alcohols.

Things You'll Need

• Two 110 mL of chromogen in phosphate buffer
• 300 μL substrate - 30% hydrogen peroxide
• 400 μL.HRP - horseradish peroxidase in phosphate buffer
• 60 mL phosphate buffer
• 250 mL sample diluent surfactant in phosphate buffer
• Two 30 mL substrate diluent phosphate buffer
• Two 30 mL stop reagent - sodium azide
• Two vials 160 U/vial standard catalase
• Spectrophotometer
• 10 μL adjustable pipettes
• 1 cm path-length spectrophotometric cuvettes
• 1.5 mL polypropylene microtubes with cap
• Test tubes - 12x75 millimeter (2.5 mL each)
• Timer
• Deionized water

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Instructions

• 1

  Prepare the stock of catalase that you will be measuring by adding 200 microlitre (μL) of deionized water to the catalase concentrations in 50 milliliter (ml) vials. The level of the catalese concentration should be 160 units per milliliter (U/ml).

• 2

  Add 30 μL of diluted catalase into tubes. Add 500 μL of the substrate (hydrogen peroxide - 10mm H2O2) into each tube. Be careful not to splash the hydrogen peroxide when working with it.

• 3

  Incubate each individual tube for one minute at room temperature.

• 4

  Add 500 μL of reagent to each vial. Cap each vial and mix by inverting the vial up and down. Do not swirl the mixture, shake the vial or stir it with an object.

• 5

  Add 20 μL of each reaction mixture into the test tubes. Add 2 milliliters of the HRP reagent into each test tube. Again, mix the solution by inverting the test tubes several times.

• 6

  Incubate the solution again, this time for 10 minutes at room temperature.

• 7

  Read the absorbance level of the solution with a spectrophotometer. The spectrophotometer measures light wavelengths to determine exactly what substances are present and and what concentrations. This method will provide an accurate reading of the catalase concentrations. The information is useful in the context of the interaction of catalase with hydrogen peroxide, as in this experiment, and as takes place naturally in the human body.