Hello,

You can find you answer by searching the forum, because this question has been arose and answered several times. Overall, the widely-used method is the rate of H2O2 degradation. Follow Sigma protocol, and you will succeed.

C cell-free extracts are prepared after 72 h of cultivation and harvest by centrifugation at 800 x g for 10 minutes and wash it twice with distilled H2O. Cell wall disruption is carried out by zymolyase according to the procedure of Defontaine et al. 1991. The cell debris is removed by centrifugation at 1000 x g for 10 min. Then the cell free extracts are frozen and thawed 3 times in order to disrupt mitochondria and centrifuged at 15 000 x g for 10 min. Obtained supernatants are used for enzymatic analyses. In mitochondria these enzymes are dare isolated from spheroplasts after osmotic shock in distilled water applying the procedure of Holtta, 1977. The cell debris is harvested at 1000 x g for 10 min and the heavy mitochondrial fraction is isolated by centrifugation at 3500 x g for 20 min. Then the crude mitochondrial pellet is carefully washed with buffer, containing 0.5 M sorbitol, 50 mM Tris, 10 mM EDTA, pH 7.5. This procedure is repeated three times and for collection of  mitochondria. These are resuspended in desionized water and lyse by freezing and thawing. The obtained sample of disrupted mitochondria are subjected to further biochemical analyses.

Catalase [EC: 1.11.1.6] activity was determined spectrophotometrically according to Aebi, 1970. One unit of enzyme activity is expressed as D E/min/mg protein.

Please click the following link to download the protocol for the determination of catalase activity.

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/cat100bul.pdf