Measurement of catalase in cells undergoing apoptosis

Catalase has a dual function: (a) dismutation of hydrogen peroxide (H202) to oxygen and water (catalytic activity) (see eqn 3) and (b) oxidation of hydrogen donors such as methanol, ethanol, formic acid, and phenols, with the decomposition of 1 mol of peroxide (peroxidic activity) (see eqn 4) (39).

Because the kinetics of catalase do not obey a normal pattern, measurements of the enzyme activity at substrate saturation, or determination of the Ks is virtually impossible (40). It is not possible to saturate the enzyme with substrate within the physiological range; however, at H202 concentrations above 0.1 M there is a rapid inactivation of catalase. Hence, to avoid a rapid decrease in the initial rate of the reaction, the spectrophotometric assay described below [which is fundamentally that of Aebi (40)] must be carried out at low H202 concentration. The decomposition of H202 can be followed directly by the decrease in the absorbance measured at 240 nm in a spectrophotometer. It follows initially (0-30 sec) a first-order reaction, with H202

concentration between 0.01-0.05 M. The decrease in absorbance per unit time is a measure of catalase activity. Owing to the abnormal kinetics it is not possible to define international catalase units. Thus, one unit of catalase is defined as the amount of enzyme that catalyses the decomposition of 1 (xmol H202/min. The calculation of activity (U/ml) can be determined using eqn 5:

activity = (zL4240/i)/e X assay volume X 1000/sample volume (5)

where AA240/i is the decrease in the absorbance at 240 nm measured spectro-photometrically over a period of time and e is the extinction coefficient of catalase (40 mM_1cm_1). The specific activity of the enzyme can be obtained by dividing the activity by the protein concentration in the sample (U/mg protein).

Protocol 9. Measurement of catalase in cells Reagents

• Assay buffer: 50 mM phosphate buffer • H202 (BDH) is diluted prior to the assay with pH 7.0 assay buffer to 30 mM

Method

1. Harvest by centrifugation (200 g, 5 min) 6-10x10® cells.

2. Resuspend the cells in ~600 fd assay buffer and freeze the cells at -20°C, until the experiment is carried out.

3. Thaw the cells at room temperature and refreeze at -70°C.

4. Freeze-thaw the cells twice more, as described in step 3.a

5. Centrifuge the cells at 200 g for 5 min.

6. In a 1 cm light path cuvette, mix 1 ml assay buffer and 500 |d supernatant.

7. To initiate the reaction add 1 ml H202 to the cuvette and monitor the absorbance at 240 nm over 30 sec.b

"Once diluted, catalase is riot a very stable enzyme, therefore the assay should be carried out within 10 min of the final freeze-thaw procedure.

'The blank should contain 2 ml assay buffer and 500 (d supernatant.

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