Enzymatic Assay of Catalase (EC 1.11.1.6)

Description

This procedure may be used for all Catalase products.

The continuous spectrophotometric rate reduction determination (A240, Light path = 1 cm) is based on the following reaction:

Unit definition: One unit of catalase will decompose 1.0 µmole of H2O2 per minute at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM. The rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm.

Precautions

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

Reagents and Equipment Required

Potassium phosphate, dibasic, trihydrate (Catalog Number P5504)

Hydrogen peroxide (30% (w/w), Catalog Number H1009)

Cuvettes and thermostatted spectrophotometer

Preparation Instructions

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

1. Phosphate Buffer (50 mM Potassium Phosphate Buffer, pH 7.0 at 25 °C) – Prepare a 11.4 mg/ml solution in ultrapure water using potassium phosphate, dibasic, trihydrate (Catalog Number P5504). Adjust to pH 7.0 at 25 °C using 1 M HCl.

2. Hydrogen Peroxide Solution [0.036% (w/w)] – Prepare in Phosphate Buffer using hydrogen peroxide (30% (w/w), Catalog Number H1009). Determine the A240 of this solution using Phosphate Buffer as a blank. The A240 must be between 0.550 and 0.520 absorbance units. If necessary, add hydrogen peroxide to increase the absorbance and Phosphate Buffer to decrease the absorbance.

3. Catalase Solution –

  • Catalase products supplied as a lyophilized powder.
        - a. Prepare an initial solution of 10 mg/ml in cold Phosphate Buffer (this solution will be slightly hazy).
        - b. Immediately before use dilute to ~100 units/ml in cold Phosphate Buffer.

  • Catalase products supplied as a crystalline suspension.
        - a. Incubate product at 37 °C for one hour to obtain complete dissolution.
        - b. Then prepare, using either a cut pipette tip or a wide mouth pipette tip, an initial dilution of ~1,000 units/ml in 37 °C
               Phosphate Buffer.
        - c. Ensure “swirling” of the initial dilution is not present. If, after mixing, the initial dilution does not appear homogeneous,
               incubate initial dilution for one hour at 37 °C to obtain complete dissolution.
        - d. Immediately before use, perform a secondary dilution to ~100 units/ml in 37 °C Phosphate Buffer.

Procedure

Final Assay Concentrations – In a 3.00 mL reaction mix, the final concentrations are 50 mM potassium phosphate, 0.036% (w/w) hydrogen peroxide, and ~10 units of catalase.

1. Using a suitable thermostatted spectrophotometer, blank against a cuvette containing Phosphate Buffer.

2. Pipette 2.90 ml of Hydrogen Peroxide Solution into a suitable cuvette.

Note: Each test cuvette will need to be run one at a time, so do not prepare the next test cuvette until the run with the preceding cuvette is complete.

3. For each cuvette monitor the A240 until constant and then add 0.10 ml of Catalase Solution.

4. Immediately mix well by inversion. Record the time required for the A240 to decrease from 0.45 to 0.40 absorbance units. Take one reading per second for ~180 seconds.

Results

Calculations

1.

Units/ml enzyme = (3.45) (df)
(time) (0.1)

Where:

3.45 = decomposition of 3.45 µmoles of hydrogen peroxide in a 3.0 ml reaction mixture producing a decrease in the A240 from 0.45 to 0.40

df = dilution factor

time = minutes required for the A240 to decrease from 0.45 to 0.40

0.1 = milliliter of enzyme solution added to the cuvette

2.

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

Materials

     

References

  1. Beers, R.F. Jr., and Sizer, I.W., Journal of Biological Chemistry, 195, 133-140 (1952).
  2. Stern, K.G., Journal of Biological Chemistry, 121, 561-572 (1937).

 

03/14-1

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