Estimation of Catalase | Enzyme Activity

In this article you will discuss about the tests for estimation of catalase in plants.

Principle:

The enzyme catalase is responsible for catalysing the decomposition of H₂O into H₂O and O₂. The assay of enzyme depends on estimation of residual H₂O₂ by titration with KMnO₄.

Requirements:

(a) Reagents:

1. H₂O₂ —0.2 volume

2. H₂SO₄—10%

3. 0.05 NKMnO₄ solution.

(b) Glass-wares:

1. Conical flasks, Pipette, Burette, Measuring cylinder etc.

(c) Preparation of enzyme extract:

5 gms of germinating seeds are taken in a mortar and crushed with a pestle. An extract of about 50 ml is made and strained through a linen cloth and finally the filtrate is made up to 50 ml by addition of dist. water.

Test Procedure:

1. 5 ml of distilled water, 1 ml of H₂O, substrate and 1 ml of enzyme extract are taken in a conical flask. The mixture is incubated in an incubator at 28 ± 1°C for 15 minutes.

2. Finally, the enzyme activity is stopped by adding 5 ml of 10% H₂SO₄ to the reaction mixture.

3. Then residual H₂O₂ content of the mixture is estimated through titration against KMnO₄ soln. Each set is replicated thrice.

4. A control set titration is also made without the addition of enzyme to the reaction mixture (only it contains 6 ml dist. water and 1 ml H₂O₂ substrate). The set is also incubated in the same manner as before and the reaction whatsoever is stopped by the addition of 5 ml of 10% H₂SO₄ before titration against KMn0₄ soln. The set is replicated thrice.

5. The difference between the titration reading of the control set (without enzyme) and reaction mixture (with enzyme) provides the amount of H₂O₂ which was decomposed by the enzyme action.

6. During titration readings are taken when a faint pink colour persists for at least 10 sec.

7. Catalase activity is expressed in terms of mgm H₂O₂ decomposed/gm. of fresh wt. of tissue/1 ml of 1 N KMnO₄ = 17 mg of H₂O₂.