Catalase Assay

There are numerous assays for catalase. Gregory and Fridovich (1974) report on a sensitive activity stain for catalase applicable to a polyacrylamide gel electrophoretogram, Haining and Legan (1972) describe a polarographic assay utilizable in tissue homogenates, and Kroll et al. (1989) discuss a rapid method for estimating the bacterial content of foods. The subject has been reviewed by Maehly and Chance (1954) and Chance and Maehly (1955). The assay used at Worthington follows:

 

Method: Essentially that described by Beers and Sizer (1952) in which the disappearance of peroxide is followed spectrophotometrically at 240 nm. One Unit decomposes one micromole of H₂O₂ per minute at 25°C and pH 7.0 under the specified conditions.

Reagents

• 0.05 M Potassium phosphate, pH 7.0
• 0.059 M Hydrogen peroxide (30%) in 0.05 M potassium phosphate, pH 7.0

Enzyme

Immediately prior to use dilute the enzyme in 0.05 M phosphate buffer, pH 7.0 to obtain a rate of 0.03-0.07 ΔA/min.

Procedure

Adjust the spectrophotometer to 240 nm and 25°C.

Pipette into each cuvette as follows:

Reagent grade water	1.9 ml
0.059 M Hydrogen peroxide	1.0 ml

Incubate in spectrophotometer for 4-5 minutes to achieve temperature equilibration and to establish blank rate if any. Add 0.1 ml of diluted enzyme and record decrease in absorbance at 240 nm for 2-3 minutes. Calculate ΔA₂₄₀/min from the initial (45 second) linear portion of the curve.

Calculation