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.

Reagents and Equipment Required

1.0 M Potassium phosphate dibasic solution (Catalog Number P8584)

1.0 M Potassium phosphate monobasic solution (Catalog Number P8709)

Potassium hydroxide (Catalog Number P1767)

Hydrochloric acid (Catalog Number H1758)

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

Cuvettes and thermostatted spectrophotometer

Precautions

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

Preparation Instructions (Storage/Stability)

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)
   To prepare 200 mL:
   • Add 6.15 mL of 1.0 M Potassium phosphate dibasic solution (Catalog Number P8584) into a beaker.
   • Add 3.85 mL of 1.0 M Potassium phosphate monobasic solution (Catalog Number P8709).
   • Make up the final volume to 200 mL using ultrapure water.
   • Adjust the pH to 7.0 at 25 °C using 1 M KOH or 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 or Phosphate Buffer to decrease the absorbance. Keep solution chilled on ice during assay.
   
   
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. The secondary dilution
   must be prepared fresh each time.
   • Catalase products supplied as a crystalline suspension, such as Catalog Number C30.
   a. Incubate product at 37 °C for ~1 hour to obtain complete dissolution.
   b. Using a positive displacement pipette, prepare an initial dilution of ~1,000 units/mL in 37 °C
   Phosphate Buffer.
   c. Incubate initial dilution for ~1 hour at 37 °C until dissolution is achieved (no swirls).
   d. Immediately before use, perform a secondary dilution to ~100 units/mL in 37 °C Phosphate Buffer.
   The secondary dilution must be prepared fresh each time.

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 set at A₂₄₀ and 25 °C, blank the instrument versus a quartz cuvette containing Phosphate Buffer.
2. Pipette 2.90 mL of Hydrogen Peroxide Solution into a quartz cuvette.
   Note: Run only one Test at a time.
3. Place the cuvette in the spectrophotometer and allow the substrate to equilibrate to 25 °C.
4. Then add 0.10 mL of Catalase Solution to the cuvette. Immediately mix by inversion and monitor the decrease in absorbance by taking one reading per second for ~180 seconds.
5. Record the time required for the A₂₄₀ to decrease from 0.45 to 0.40 absorbance units.
6. Perform steps 1–5 in triplicate.

Results

Calculations

1.	Units/mL enzyme =		(3.45) (df)
			(time) (0.1)

Where:

3.45 = corresponds to the 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 absorbance units
0.1 = milliliter of enzyme added to the cuvette

2.	Units/mg solid =		units/mL enzyme
			mg solid/mL enzyme

1. Beers Jr. R, Sizer I. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem.. 195(1):133-40.

2. Stern K. 1937. On the absorption spectrum of catalase. J. Biol. Chem.. 121561-72.