2. To be able to know the properties of an enzyme
To be able to test for the presence of an enzyme
To be able to test the specificity of an enzyme
Objectives
3. Procedures
Preparation for Potato Extract:
1. Peel and grate a potato
into 100 mL distilled water.
2. Let it stand for 10 minutes.
Stir occasionally.
3. After 10 minutes, strain
through cheese cloth.
4. Filter the extract using
filter paper.
Use the extract in the
following tests.
4. Procedures
Biuret Test
1. To 1 mL of extract, add 2
mL of 10% NaOH
Solution.
2. Mix thoroughly and add
2-3 drops of 1% Copper
Sulfate solution.
Observe the
appearance for violet color.
6. Procedures
Test for Catalase Activity
1. To 5mL of extract, mix it with
1 mL of 3% H2O2.
2. Observe whether the gas
evolve supports
combustion, by way of
holding a splinter over the
mouth of the test tube.
3. Add 1 mL of 0.5% Benzidine.
Note the formation of
the blue to green coloration.
8. Procedures
Preparation of a dilute
solution of Salivary Amylase
1. Rinse mouth several times
with water.
2. Collect 1 mL of saliva.
3. Prepare a 1:8 solution of
saliva by adding 0.25 mL of
saliva to 20 mL of distilled
water.
Use prepared test in
the following tests.
9. Procedures
Test for Specificity of Enzyme Action
1. Prepare two test tubes, each
containing 2 mL of 0.02M Phosphate
buffer pH 6.7 and 1 mL of 0.9% Sodium
Chloride solution.
2. Test tube no. 1: Add 1 mL cooked starch
and 1 mL of salivary amylase solution.
3. Test tube no. 2: Add 1 mL 1% glycogen
solution and 1 mL of salivary amylase
solution.
4. Stand for 15 minutes at room
temperature.
5. Stir the mixture in both test tubes.
Place one drop of each solution in
separate evaporating dish or spot
plate.
6. Add a drop of Iodine solution. Observe
the color produced.
7. Repeat the test at 5 minutes intervals
for an hour. Tabulate results.
10. Result:
Results and
Observations
Time
(minutes)
Test Tube
No. 1
Changes Test Tube
No. 2
Changes
5 positive As time
passes,
the color
turns
lighter.
Negative No
change.
10 Positive Negative
15 Positive Negative
20 Positive Negative
25 Positive Negative
30 Positive Negative
35 Positive Negative
40 Positive Negative
45 Positive Negative
50 Positive Negative
55 Positive Negative
60 Positive Negative
11. 1. Principle involved in the use of Biuret Test.
Biuret Test is based on the ability Cu (II) ions to form a violet-colored chelate
complex with peptide bonds (-CONH-groups) in alkaline conditions.
2. How will you prove the presence of catalase? Show chemical equation.
A rapid appearance of sustained gas bubbles is an indication of the presence of a
catalase.
2 H2O2 2 H2O + O2
3. How will you prove the specific action of salivary amylase?
Enzymes are normally very specific , catalyzing at most only a few types of
chemical reactions. The enzyme amylase acts on the substrate starch catalyzing its break
down into simple sugars. Amylase will not catalyze the break down of the complex sugar
sucrose into simple sugars. Indicators can be used to test for the presence of these
reactions like iodine solution in the iodine test, amylase in the starch (straight chain)
forms helices where iodine molecules assemble and forming blue-black color. While
amylopectin (branched portion) in glycogen forms much shorter helices and iodine are
unable to assemble and only forming orange/yellow hue.
Analysis and Conclusion
13. Describe the effect of temperature and pH in enzyme
action.
Explain how changes in pH and temperature affect
enzyme action.
Objectives
14. Procedures
Effect of Temperature
1. Prepare three test tubes and label
them respectively.
2. Test tube no. 1: 5 mL 1% cooked
starch solution + 1mL saliva. Keep in
water bath at 40ºC.
3. Test tube no. 2: 5 mL 1% cooked
starch solution + 1 mL saliva. Keep
in water bath at 60ºC.
4. Test tube no. 3: 5 mL 1% cooked
starch solution + 1 mL saliva. Keep
in water bath at 10ºC.
5. At 15 minutes interval, take a drop
of the reaction mixture from each
test tube, stir the mixture first
before taking a drop, and test with
iodine solution. Perform the iodine
test for a period of an hour.
At 60ºC
At 10ºC
At 40ºC
15. Result
Results and
Observations
Time
(minutes)
Test tube
no.1 @ 40ºC
Test tube
no. 2 @
60ºC
Test tube no. 3
@ 10ºC
15 White
surrounded
by black
ring
Blue black
or
something
dark in
color
Light yellow
with visible
black ring
formation in
center
Orange or
something
light in
color
Yellowish
black
*after 6
minutes –
violet
Blue black
or
something
dark in color
30 Light yellow
surrounded
by black
ring
Blue black
or
something
dark in
color
White with
visible black
ring
formation in
the middle
Orange or
something
light in
color
Yellowish
black
*after 40
minutes –
black
Blue black
or
something
dark in color
45 yellow
surrounded
by black
ring
Blue black
or
something
dark in
color
Light yellow
with visible
scattered
black
particles
Orange or
something
light in
color
(darker)
yellowish
black
*after 10 mins.
– Light Violet
Blue black
or
something
dark in color
60 Dark yellow Blue black
or
something
dark in
color
violet Orange or
something
light in
color
Dark yellow Blue black
or
something
dark in color
At
40ºC
At
60ºC
At
10ºC
16. Procedures
Effect of pH
1. Prepare four test tubes containing equal
amounts of egg white. Place the test tubes
in hot water bath. When the egg white is
coagulated, cool n=and mark the height of
the solidified egg white.
2. Test tube no. 1: 5 mL 2% pepsin + 10 drops
0.4% HCl
3. Test tube no. 2: 5 mL 2% pepsin + 10 drops
of 0.4% Na2CO3
4. Test tube no. 3: 5 mL 2% pancreatin + 10
drops of 0.4% HCl
5. Test tube no. 4: 5 mL pancreatin + 10 drops
0.4% Na2CO3
6. Keep the test tubes in a beaker of water
controlled at 40ºC for one hour.
7. Use a pH paper to determine specific pH of
each test tube.
8. Determine the extent of digestion in each
test tube by noting the amount of protein
dissolved or disintegrated.
9. Perform Biuret Test of 1 mL of supernatant
liquid from each test tube.
10. Compare the colors obtained with 0.5%
peptone standard.
17. Result
Results and
Observations
Test tube pH Color Expect
ed
color
Result
No 1.
pepsin +
HCl
Acidic Purple Purple +
No. 2
pepsin +
Na2CO3
Basis Purple Purple +
No. 3
pancreatin
+ HCl
Acidic Purple Purple +
No. 4
pancreatin
+ Na2CO3
Basic Purple Purple +
18. 1. Explain the mechanism of enzyme action at varying temperature. Give
the principle involved.
Being a protein, an enzyme is denatured at high temperature
and becomes inactive. Initially as the temperature increases, the rate
of enzymatic reaction also increases but it has a limitation. When the
temperature affects the molecular structure of the enzyme it gets
inactivated.
2. How does different pH affect enzyme action? Explain your results using
this principle. Discuss Isoelectric pH enzyme action.
Enzymes are picky with pH levels, as they are with everything
else. They have an optimal level at which they work the best, and
anything above or below that level, their activity begins to slow down
until they shut down all together.
Analysis and Conclusion
