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14. Investigation of the effect of enzyme concentration on enzyme activities

Catalase is an enzyme present in nearly all living cells. It catalyses the decomposition of hydrogen peroxide to water and oxygen. 2H2O2 2H2O + O2

Hydrogen peroxide is produced in cells as a by-product in a number of chemical reactions involving oxidative enzymes. It is chemically very reactive, and toxic to cells. Catalase inside cell organelles is believed to protect the cells by breaking down hydrogen peroxide. The activity of catalase on the decomposition of hydrogen peroxide to water and oxygen can be determined by: (1) measurement of the heat production of the catalase reaction. The maximum rise in temperature after the start of the reaction can serve as an approximate measure of the enzyme activity; (2) direct measurement of the amount of end-product (oxygen); or (3) indirect measurement of the amount of substrate (hydrogen peroxide) not acted upon. In this investigation, the third method is used to study the effect of enzyme concentration on the action of catalase on hydrogen peroxide.

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Assay of catalase activity by iodometric method The activity of catalase can be determined by measuring the amount of hydrogen peroxide that has not undergone degradation. This is done by adding the remaining mixture to excess potassium iodide and then titrating the liberated iodine with sodium thiosulphate. Hydrogen peroxide reacts with iodide in an acid medium. Ammonium molybdate is added as a catalyst. H2O2 + 2H + 2I2H2O + I2

The iodine liberated is then titrated with standard sodium thiosulphate solution using starch as the indicator. I2 + 2S2O32Procedure 1. 2. 3. Label six 250 cm3 conical flasks from 1A to 6A and six test tubes from 1B to 6B. Use a cork borer to cut out 6 potato cylinders with a length of about 5 cm and diameter 0.8 cm. Trim one end of the cylinders with a sharp razor blade. Then slice off 15 discs, each about 2 mm thick, from each cylinder. 4. Add 4 cm3 of 2M sulphuric acid, 3 cm3 of 10% potassium iodide to each conical flask. Mix the contents well. 5. 6. Add 10 cm3 of hydrogen peroxide (1 vol or about 0.1M) to each of the 6 test tubes. Place the potato discs into the six test tubes as shown below and start timing instantaneously. S4O62- + 2I-

Tube Number of potato discs

1B 4

2B 8

3B 12

4B 16

5B 20

6B 25

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7.

After exactly 10 minutes, transfer a 5 cm3 sample from each test tube to the correspondingly labelled conical flask.

8.

Add 1 drop of 3% ammonium molybdate solution to the mixture. Shake to mix the contents well. Allow the contents to stand for 2 minutes.

9.

Titrate the iodine that has been set free with standard (0.1M) sodium thiosulphate solution until a faint straw colour develops.

10. Add 2 cm3 of 1% starch solution and continue the titration until the blue colour is just discharged. 11. The volume of sodium thiosulphate required for titration is used as an indication of the relative rate of catalase activity.