1. The document describes experiments to test the effects of temperature, pH, and substrate concentration on enzyme activity. 2. In the temperature experiment, salivary amylase activity was highest at 37°C, the optimum temperature. 3. The pH experiment found that pepsin only catalyzes protein hydrolysis in acidic conditions. 4. Increasing substrate (starch) concentration increased the reaction rate of salivary amylase up to 0.4% concentration, beyond which the rate plateaued.

1. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 10
Activity 4.1
Aim To differentiate between reducing and non-reducing sugars.
Materials 5% glucose solution, 5% fructose solution, 5% galactose solution, 5% maltose solution, 5% lactose solution, 5% sucrose solution, dilute
hydrochloric acid, water, sodium hydrogen carbonate powder and Benedict’s solution
Apparatus Test tubes, beaker, Bunsen burner, tripod stand, wire gauze and test tube holder
A.Testing for reducing sugars
Procedure
1. 2 ml of Benedict’s solution are added to 2 ml of glucose solution in a test tube
2. The test tube is placed in a water bath of boiling water for 5 minutes.
3. The colour of the precipitate that is formed (if any) is recorded in the table below.
4. Steps 1 to 3 are repeated for all the other sugar solutions.
Results Sugar solution Benedict’s solution Sugar solution Benedict’s solution
Glucose Brick-red precipitate Maltose Brick-red precipitate
Fructose Brick-red precipitate Lactose Brick-red precipitate
Galactose Brick-red precipitate Sucrose Blue solution
Discussion 1. Glucose, fructose, galactose, maltose and lactose are reducing sugars because they give a positive Benedict’s test.
2. Sucrose is a non-reducing sugar because it gives a negative Benedict’s test.
B.Testing for non-reducing sugar
Procedure 1. 2 ml of sucrose solution and 1 ml of dilute hydrochloric acid are added to a test tube
2. The test tube is placed in a water bath and boiled for 5 minutes.
3. The test tube is cooled under a tap and sodium hydrogen carbonate powder is added to neutralize the excess acid.
4. When it stops fizzing, 2 ml of Benedict’s solution are added to the mixture in the test tube。
5. The test tube is placed in the water in the water bath again and boiled for 5 minutes.
Observation A brick red precipitate is formed in the test tube.

2. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 11
Discussion 1. Sucrose is hydrolysed into glucose and fructose by boiling the sucrose solution with dilute hydrochloride acid.
2. When the solution containing glucose and fructose is boiled with Benedict’s solution, it produces a brick-red precipitate.
Conclusion Glucose, fructose, maltose are reducing sugars. Sucrose is non-reducing sugar.

3. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 12
Experiment 4.1
Aim To study the effects of temperature on the activity of salivary amylase.
Problem statement What are the effects of temperature on the activity of salivary amylase?
Hypothesis The optimum temperature for salivary amylase is 37oC.
Variables (a) Manipulated variable: Temperature
(b) Responding variable: Rate of reaction
(c) Fixed variable: Volume and concentration of starch suspension, volume and concentration of saliva suspension, pH of starch and
saliva suspensions.
Apparatus Beakers, test tubes, tripod stand, wire gauze, Bunsen burner, thermometer, dropper, glass rod, syringes, stopwatch, spotting tile and test
tube rack
Materials 1% starch starch suspension, saliva, iodine solution, ice cubes and distilled water
Technique Recording the time taken for the complete hydrolysis of starch with a stopwatch. The presence of starch is indicated by the iodine test.

4. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 13
Procedure
1. The mouth is rinsed with distilled water.
2. The saliva suspension is prepared by spitting into a small beaker and diluting the saliva with an amount of distilled water equal to
the amount of saliva.
3. Five water baths are prepared at the following temperatures: 0o
C, 20 o
C, 37 o
C, 50 o
C and 60 o
C.
4. Two test tubes are labelled P and Q.
5. 4 ml of starch suspension are placed in test tube P and 1 ml of saliva suspension in test tube Q.
6. Both test tubes are placed into the first water bath (at 60 o
C) for 5 minutes.
7. A drop of iodine solution is placed into each groove of a clean spotting tile.
8. A drop of suspension from test tube P is poured into the saliva suspension in test tube Q. The stopwatch is started immediately.
9. A drop of suspension is taken out from the test tube Q every minute and tested with iodine solution on the spotting tile.
10. Test tube Q is kept in the water bath thoughout the experiment.
11. The time taken for the complete hydrolysis of starch is recorded in the table, that is, when the iodine solution does not turn blue
anymore..

5. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 14
12. Steps 4 to 9 are repeated for temperatures 20 o
C, 37 o
C, 50 o
C and 60 o
C.
13. The results are recorded in the table below.
14. A graph of rate of reaction against temperature (o
C) is plotted.
Results
Temperature of water
bath (o
C)
Time taken for complete
hydrolysis of starch, t (minute)
Rate of reaction,
(minute-1
)
0 >30 <0.03
20 8 0.13
37 0.33 3
50 6 0.17
60 >30. <0.03
Discussion 1. The test tubes P and Q are kept in the water bath for 5 minutes to stabilize the temperature of the suspensions in the two test tubes.
2. When the amylase in the saliva has completely hydrolysed the starch, the dark blue colour does not form in the iodine test.
3. At low temperatures, the reaction progresses slowly. As the temperature increases, the rate of reaction increases until the optimum
temperature of 37o
C is reached. Above the optimum temperature, the rate of reaction falls rapidly.
Conclusion The rate of reaction is highest at the temperature 37o
C. the hypothesis is accepted.

6. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 15
Experiment 4.2
Aim To study the effects of pH on the activity of pepsin.
Problem statement What are the effects of pH on the activity of pepsin?
Hypothesis The enzyme pepsin works best in acidic conditions.
Variables (a) Manipulated variable: pH of medium
(b) Responding variable: Rate of reaction
(c) Fixed variable: Volume and concentration of egg albumen suspension, volume and concentration of pepsin solution, temperature of
medium
Materials Egg albumen suspension, dilute hydrochloride acid, dilute sodium hydroxide solution, 1% of pepsin solution, pH paper and distilled
water
Apparatus Beaker, tripod stand, wire gauze, Bunsen burner, thermometer, dropper, test tubes, test tube rack and syringes
Procedure
1. The egg albumen suspension is prepared by adding 1g of dried egg albumen to 100 cm3
of water. The mixture is then heated to
90o
C.
2. Three test tubes are labelled P,Q and R.
3. 5 ml of egg albumen suspension and 1 ml of pepsin solution are placed in each test tube.
4. 5 drops of distilled water are added to test tube P.
5. 5 drops of dilute hydrochloric acid are added to test tube Q.
6. 5 drops of dilute sodium hydroxide solution are added to test tube R.
7. All the three test tubes are placed into a water bath at 37o
C for 20 minutes.
8. The appearance of the contents in each test tube is recorded at the beginning and the end of the experiment.
9. The contents in each test tube are tested with pH paper.
10. The results are recorded in a table.

7. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 16
Results Test
tube
pH Contents
Appearance of contents
At the beginning of the experiment At the end of the experiment
P 7 Albumen + pepsin + distilled water White suspension White suspension
Q 2
Albumen + pepsin + dilute
hydrochloric acid
White suspension Clear solution
R 9
Albumen + pepsin + dilute sodium
hydroxide solution
White suspension White suspension
Discussion 1. The test tubes are kept in a water bath maintained at 37o
C because that is the optimum temperature for pepsin.
2. The mixture in test tube Q is clear at the end of the experiment because the albumen has been hydrolysed.
3. Pepsin does not catalyse the hydrolysis of albumen in the neutral and in alkaline conditions.
Conclusion Pepsin catalyses the hydrolysis of protein only in acidic conditions. The hypothesis is accepted.

8. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 17
Experiment 4.3
Aim To study the effects of substrate concentration on salivary amylase activity.
Problem statement What are the effects of substrate concentration on salivary amylase activity?
Hypothesis The rate of enzymatic reaction increases with the increase in substrate concentration until it reaches a maximum rate.
Variables (a) Manipulated variable: Substrate concentration
(b) Responding variable: Rate of concentration
(c) Fixed variable: Enzyme concentration, temperature, pH of starch and saliva suspension, volume of starch and saliva suspension
Materials 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6% starch suspensions, saliva, iodine solution and distilled water
Apparatus Beakers, test tubes, tripod stand, wire gauze, Bunsen burner, thermometer, dropper, glass rod, syringes, stopwatch, spotting tile and test
tube rack
Technique Recording the time taken for the complete hydrolysis of starch with a stopwatch. The presence of starch is indicated by the iodine test.
Procedure 1. The mouth is rinsed with distilled water.
2. The saliva suspension is prepared by spitting into a small beaker and diluting the saliva with an amount of distilled water is equal to
the amount of saliva.
3. Six test tubes are labelled P, Q, R, S, T and Uare filled with the following starch suspensions using different syringe.
Test tube Type of starch suspension
P 4 ml 0.1% starch suspension
Q 4 ml 0.2% starch suspension
R 4 ml 0.3% starch suspension
S 4 ml 0.4% starch suspension
T 4 ml 0.5% starch suspension
U 4 ml 0.6% starch suspension
4. 1 ml of saliva suspension is placed in a test tube.
5. A drop of iodine solution is placed into each groove of a clean spotting tile.
6. Test tube P and the test tube containing the saliva suspension are placed into a water bath at 37o
C.
7. After 5 minutes, the saliva suspension is poured into the starch suspension in test tube P. the stopwatch is started immediately.
8. A drop of suspension is taken from test tube P every minute and tested with iodine solution on the spotting tile.
9. Test tube P is kept in the water bath throughout the experiment.
10. The time taken for the complete hydrolysis of starch is recorded in the table, that is, when the iodine solution does not turn blue
anymore.
11. Steps 4 to 10 are repeated for test tubes Q, R, S, T and U.
12. The results are recorded in the table below.
13. The graph of rate of reaction (% minute-1
) against concentration of starch suspension (%) is plotted.

9. Biology |Form 4 | Chapter 4: Chemical Composition in Cell 18
Results Concentration of starch
suspension, C (%)
Time taken for the complete hydrolysis of starch,t Rate of reaction = C/t
(% minute-1
)(seconds) (minutes)
0.1 300 5.00 0.02
0.2 300 5.00 0.04
0.3 300 5.00 0.06
0.4 300 5.00 0.08
0.5 375 6.25 0.08
0.6 450 7.50 0.08
Discussion 1. The rate of reaction increases with the increase in the concentration of the substrate (starch) concentration, that is from 0.1% to
0.4% starch suspension.
2. The maximum rate is reached at 0.4% starch suspension. At this point all the active sites of the enzymes are filled at anyone one
time. This is referred to as saturated point.
3. Beyond the saturation point, the rate of reaction does not increases even through the substrate concentration is increased because the
concentration of enzyme becomes a limiting concentration.
Conclusion The rate of enzymatic reaction increases with the increase in substrate concentration until it reaches a maximum rate. The hypothesis is
accepted.