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Enzymes are thermo-labile organic catalysts which are very much sensitive to heat and pH. The activity of a number of respiratory enzymes could be detected within plant tissue by suitable tests.
(a) Detection of Dehydrogenase:
This enzyme accomplishes intracellular oxidation and reduction by the transfer of hydrogen from one type of molecule to another. The importance of such enzyme is that of activating the hydrogen of metabolites. This enzyme is widely noted in plant systems.
Material:
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Potato tuber.
Chemicals:
1. Triphenyl tetraxolium chloride (0.2%).
2. Methylene blue (0.1%).
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Experiment:
Test 1:
A potato tuber is taken, washed and peeled. With the help of cork borer, a number of cylinders are cut and then sliced. The slices are kept in water, in two separate test tubes containing cold and hot water, slices are placed for a few minutes. Then a few drops of triphenyl-tetrazolium chloride solution is added to each tube.
Test 2:
Slices of potato tuber are made as before — then some of the slices are boiled in water in a tube while others are placed in cold water. Then a few drops of methylene blue solution is added to both the tubes and corked carefully. The tubes are kept overnight in an incubator at 30-35°C.
Observations:
The test tube containing living slices (i.e. slices in cold water containing tube) show red coloration. But boiled slices do not produce any colour.
The tube containing living tissue (i.e. slices of the tube containing cold water) shows dehydrogenase activity, as such, methylene blue colour is discharged. The colour of the slices of the other tube remain unchanged.
Inference:
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The slices showing red coloration possess dehydrogenase activity.
Presence of dehydrogenase activity in living slices.
(b) Detection of Oxidase:
This enzyme activates the reaction leading to the formation of oxidized substrate and water under aerobic condition.
Material:
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Potato tuber (sliced).
Chemicals:
Catechal solution (2%).
Experiment:
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The slices of potato tuber are placed in a tube containing catechal solution.
Some slices are boiled in water and then placed in a tube containing catechal solution.
Observations:
The slices turn yellowish brown due to oxidase activity.
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No change of colour is observed.
Inference:
Presence of Oxidase activity.
Absence of Oxidase activity.
(c) Detection of Peroxidase:
The enzyme peroxidase oxidizes a substrate by O, obtained from decomposition of H2O2.
Material: Potato tuber (sliced)
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Chemicals: Catechal solution (2%), H2O2 (2 volumes)
Experiment:
Potato slices are placed in a tube containing catechal for 2 mins. Then catechal is drained off and H2O2 is added.
The same test is performed with boiled slices.
Observations:
The slices turn pink in colour.
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No change of colour of the slices.
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Inference:
Presence of Peroxidase enzyme activity.
Absence of enzyme activity as the tissue is dead.
(d) Detection of Catalase:
This enzyme catalyses the decomposition of H2O2 into H2 and
Material: Potato tuber (sliced)
Chemicals: H2O2 (2 volumes)
Experiment:
To potato slices, H2O2 is added. To boiled slices, H2O2 is added.
Observations:
Evolution of O2 is noted.
No evolution of O2.
Inference:
Presence of Catalase activity.
Absence of enzyme activity as the tissue is dead.
