ACTIVITY OF ENZYMES

Enzymes are protein catalysts that can increase the rate of chemical reactions
within the cell. Thus, they play a pivotal role in the synthesis and degradation of
biological compounds and in the regulation of cellular functions. Enzyme activity
can also be used for diagnostic purposes. For example, increased levels of lactate
dehydrogenase (LDH) are found in the blood of patients after a heart attack.
Enzymes are also used for industrial purposes, where the specificity of these
catalysts is particularly useful.
Enzyme reactions can be detected by determining the amount of product
formed or the disappearance of the substrate. In those reactions that involve
coenzymes or other factors, the production or disappearance of those factors can be
used as an indicator to monitor the reaction. ln Studying enzyme kinetics, the most
important step is to design a suitable enzyme assay that includes designing an
optimal detection method and determination of optimal reaction conditions
(optimum temperature, pH, substrate, and enzyme concentration). Most often,
enzymatic reactions are monitored with the use of spectrophotometry.
Peroxidase/Catalase
During biological oxidation, enzymes known as dehydrogenases transfer
hydrogen from substrate to oxygen, via carrier substances, forming oxidized
substrate and water. However, there are few dehydrogenase enzymes, known as
aerobic dehydrogenases, which transfer hydrogen directly to oxygen, forming the
oxidized substrate and hydrogen peroxide as a byproduct. Hydrogen peroxide (H2O2)
is a toxic substance and is removed by different mechanisms in plant and animal
cells. The enzyme Peroxidase deactivates H2O2 in plant cells while the enzyme
catalase is responsible for removal of H2O2 in animal cells.
Tests for enzymes
1) Test for Amylase
1) To 1 ml of enzyme extraction add 0.5 ml of 1% starch solution and 1.0 ml of
phosphate buffer (pH 6.5). Prepare a blank with water.
2) Incubate at 37C for 30 min.
3) Add 0.5 ml of the reaction mixture into a fresh test tube and add 4 drops of
distilled water, 2 drops of Fehlings A and 2 drops of Fehling's B solution.
4) Boil this mixture.
5) Appearance of a brick-red precipitate indicates the presence of Amylase
2) Test for Trypsin
1) Take two test tubes and add 1 ml of the enzyme extraction to the one tube
2) Add 1 ml of milk solution, 1 ml of the phosphate buffer and a small amount
of Congo-Red solution to each tube.
3) Red solution to each tube.
4) Mix and place in a place in a water bath at 37C, for 15 min.
5) Note any changes in turbidity. Explain your observations.
3) Test for Lipase
1) To 4 ml of 90% alcohol add 10 drops of the given oil sample and warm
gently until it dissolves.
2) Add an equal amount of distilled water and 10 drops of phenol red.
3) Add 1% Na2CO3 dropwise until a faint color appears.
4) To 0.5 ml of the prepared oil emulsion add 1 ml of the enzyme extraction.
5) Incubate at 37C for 30 min.
6) A change in color from pink to yellow indicates the presence of lipase.
4) Peroxidase activity in Mung beans
ln plants peroxidase enzyme, in the presence of certain phenolic and a romatic
amino compounds, transfers the oxygen from hydrogen peroxide to these
compounds resulting in the formation of colored oxidation product.
1) Make an extract of the green gram seeds provided.
2) Add 2 ml of extract to a test tube.
3) Add 10 drops of 4% orthotolidine (or benzidine reagent) and 3 drops of 3%
hydrogen peroxide.
4) Note the color change.
5) Place another 2 ml of Mung bean extract, boil for minute and repeat the
experiment.
5) Catalase activity in Liver tissue:
Catalase is present in all animal tissues in varying amounts It catalyzes the
decomposition of hydrogen peroxide to water and oxygen. It occurs at even 0C
and of the fastest reaction.
1) Take about 100 mg of liver tissue and grind it well
2) Place the extract in a test tube and the tube in an ice bath
3) After few minutes add few drops of hydrogen peroxide.