Kinetic Parameters of Catalase
Aya Essam El Garhy
Ola El Rakhawy
Students in Biochemistry Lab
at German University in Cairo
The activity of catalase enzyme towards hydrogen peroxide is tested by measuring the amount of H2O2 consumed by the enzyme catalase (by determining the decrease in H2O2 concentration) within a time interval of 5 minutes. Then ending the reaction by the addition of H2SO4 (by changing the PH of the medium). The reaction is carried out with different H2O2 concentrations and a blank is done for each concentration then the amount of remaining H2O2 is determined by titration versus KMnO4. The titration is done for both reaction mixture and blank and ...view middle of the document...
This enzyme is considered among the most efficient known enzymes as it has a rate 200,000 catalytic events/second/subunit (near the diffusion-controlled limit) (1). The optimum pH for catalase is approximately neutral (pH=7), while the optimum temperature varies by species.
Since catalase plays an important role in controlling the concentration of H2O2, it protects pancreatic ÃŸ-cells from the harmful effects caused by H2O2 (4). Low catalase activities have been seen in patients suffering from schizophrenia and atherosclerosis. While the absence of catalase may lead to peroxisomal disorder acatalasia(4).
Materials and Methods:
â€¢ 1. H2O2 of different concentrations : 1.65mM, 0.8mM, 0.4mM, 0.2mM and another solution of unknown concentration (from Morgan Chemical. Ind. Company),
â€¢ M KMnO4 (from (ADWIC) Elnasr pharmaceutical chemicals company)
â€¢ 20% H2SO4 (from (ADWIC) Elnasr pharmaceutical chemicals company),
â€¢ Catalase enzyme in buffer (catalase bovine liver) from Fluka (sigma Aldrich)
â€¢ Buffer prepared by 3.522 gm KH2PO4 (from WINLAB) + 7.26 gm Na2HPO4 (from ADWIC Elnasr pharmaceutical chemicals company and complete to 1 L with H2O which adjusts the pH to 7)
In a glass stoppered flask, place the amounts of H2O2 solution that are stated in the following table. Add 1 ml catalase, mix well but slowly (by rotation) and leave for 5 minutes.
After exactly 5 minutes time, stop the reaction by addition of 2 ml of 20 % H2SO4. Titrate the mixture against KMnO4. The end point is the appearance of 1st pink
Repeat the above steps for each H2O2 concentration.
A Blank is done for each H2O2 concentration by adding 5 ml of H2O2 followed by 2 ml of H2SO4, then 1 ml of the catalase solution. The blank is also titrated against KMnO4.then the results were recorded in the following table
|Exp. No. |1 |2 |3 |4 |Unknown |
|Catalase soln (ml) |1 |1 |1 |1 |1 |
|[H2O2] (mM) |1.65 |0.8 |0.4 |0.2 |---------- |
|= [S] | | | | | |
|20 % H2SO4 (ml) |2 |2 |2 |2 |2 |
| | | | | | |
|KMnO4 soln |12.6 |5.2 |2.2 |1.1 |7.7 |
|(ml) Reaction | | | | | |
|ml KMnO4 used | | ...