In this article we will discuss about the tests for estimation of peroxidase in plants.
Principle:
The enzyme peroxidase catalyses the oxidation of substrate by oxygen generated from the decomposition of hydrogen peroxide:
2H2O2 → 2H2O + O2
Substrate + O2 → Oxidized substrate.
Requirements:
(a) Reagents:
1. Hydrogen peroxide (H2O2) — 0.2%
2. Catechol soln. — 0.5%
(b) Glass-wares:
1. Test tubes, Pipette, Mortar and Pestle, Measuring Cylinder, Linen cloth, Balance with weight box etc.
(c) Preparation of enzyme extract:
Fresh seedlings of rice are taken and the endosperms are removed from the seedlings. Then 5 gm. of plant material is weighed and crushed in a mortar with the help of a pestle. Then an extract of about 50 ml is prepared and strained through a linen cloth and final volume is made up to 50 ml my addition of dist. water.
Test Procedure:
1. The reaction mixture is made in the following manner. In each tube 1 ml of catechol and 1 ml of H2O2 soln. are taken. Then 1 ml of enzyme extract is added to it.
2. Finally, the mixture is shaken gently and allowed to stand for 1-5 minutes to develop colour.
3. The intensity of colour formed in each reaction mixture at varied duration is recorded optically by denoting ‘+’ sign.
4. Each set is replicated at least thrice for getting better results.