ACTIVITY IX-ORGANIC CHEMISTRY
ENZYMES ARE AFFECTED BY CHANGES IN TEMPERATURE, pH, SALT
CONCENTRATION, CONCENTATION OF ENZYME OR SUBSTRATE
LAB-FACTORS INFLUENCING ENZYME ACTIVITY
EXPERIMENT 1: HOW DOES ENZYME CONCENTRATION AFFECT THE RATE OF
REACTION?
MATERIALS: Chilled distilled water, mortar & pestle, potato, scale, 100 ml 2
graduated cylinders, cheesecloth or coffee filter, two 50 ml beakers or small cups,
ice bath, 3% hydrogen peroxide, room temperature distilled water, 5 test tubes, test
tube rack, dropper pipet, forceps, & filter disks
PREDICT: What will increasing or decreasing the amounts of enzyme do to the
rate of reaction? ________________________________________________________________________
PROCEDURE:
1. Grind up 20 grams of peeled potato cubes with 20 ml of chilled distilled
water using a mortar and pestle.
2. Filter the solution into a 100 ml graduated cylinder using cheesecloth or
coffee filter.
3. Add chilled distilled water to graduated cylinder to bring volume up to 2o ml.
4. Pour into 50 ml beaker or small cup that is chilled on ice and label 100%
catalase.
5. Using graduated cylinder measure 15 ml of 3% hydrogen peroxide and then
add 15 ml of room temperature distilled water (you now have 30 ml of a
1.5% hydrogen peroxide).
6. Pour the 1.5 % hydrogen peroxide into a 50 ml beaker or small cup and label.
This will be your reaction vessel.
7. During the experiment, when you drop a disk soaked in catalase into the
reaction vessel, the hydrogen peroxide will decompose, forming oxygen gas
bubbles on the disk that will eventually lift it to the surface.
8. Label five test tubes 0%, 25%, 50%, 75%, and 100% respectively. These
represent the concentrations of the chilled catalase.
9. Using standard drops from a dropper pipet (20 drops =1 ml), add the
following drops of catalase to each test tube, followed with drops of cold
distilled water to obtain a final volume of 1 ml per test tube. Use chart below
as a guide:
1
CREATING CATALASE CONCENTRATIONS
0% CATALASE
25% CATALASE
50% CATALASE
75% CATALASE
100% CATALASE
0 Drops of
catalase
5 Drops of
catalase
10 Drops of
catalase
15 Drops of
catalase
20 Drops of
catalase
20 Drops of
water
15 Drops of
water
10 Drops of
water
5 Drops of water
0 Drops of water
10. Keep the five test tubes in an ice bath to preserve catalase solution.
11. With a pair of forceps, take a dry filter disk out of the petri dish lid and dip it
into 0% test tube solution for five seconds.
12. Remove the disk and blot its edge on the top of the test tube.
13. Start your timer as you place the disk on the bottom of the reaction vessel
containing 30 ml of 1.5% hydrogen peroxide.
14. Time in seconds how long it takes the disk to rise to the surface of the
peroxide. Stop timing after three minutes if there is no activity.
15. Remove the use disk. Perform another trial with a new catalase-soaked disk
for 0% catalase.
16. Conduct two trials for each of the remaining catalase solutions (25%, 50%,
75%, & 100%). For each percent solution, average the time for floating the
disks. Always remember to remove the used disk before starting the next
trial.
17. The rate of each reaction can be calculated by the formula R=d/t, where
d=the distance from the bottom of the container to the surface in cm, and
t=seconds. The unit for rate is cm/sec. If the disk remains on the bottom of
the container after 3 minutes, the distance (d) will be zero.
18. Plot as graph for each data point and draw a best-fit line.
ENZYME REACTION TIME FOR DIFFERENT CONCENTRATIONS OF CATALASE IN
SECONDS
0%
25%
50%
75%
100%
catalase
catalase
catalase
catalase
catalase
TRIAL 1
TIME
TRIAL 2
TIME
AVG. TIME
RATE (d/t)
2
19. After experimenting and analyzing your data, do you agree or disagree with your
prediction? Why?
EXPERIMENT 2:WHAT IS THE AFFECT OF SUBSTRATE CONCENTRATION ON
ENZYME ACTIVITY?
PREDICT: What will increasing or decreasing the amounts of substrate do to
the rate of reaction?
DESIGN YOUR OWN EXPERIMENT. BELOW IS HOW TO MAKE THE DIFFERENT
CONCENTRATIONS OF SUBSTRATE (HYDROGEN PEROXIDE)
CREATING HYDROGEN PEROXIDE CONCENTRATIONS
O%
hydrogen 0.3%
peroxide
peroxide
hydrogen 1.5%
peroxide
hydrogen 3.0%
hydrogen
peroxide
0
Drops
of 6 Drops of hydrogen 10
Drops
of 20
Drops
of
hydrogen peroxide peroxide
hydrogen peroxide
hydrogen peroxide
20 Drops of water
14 Drops of water
10 Drops of water
0 Drops of water
MATERIALS: Chilled distilled water, mortar & pestle, potato, scale, 100 ml 2
graduated cylinders, cheesecloth or coffee filter, two 50 ml beakers or small cups,
ice bath, 3% hydrogen peroxide, room temperature distilled water, 5 test tubes, test
tube rack, dropper pipet, forceps, & filter disks
1.
2.
3.
4.
5.
Form a prediction
Write out a detailed procedure
Have procedure checked by teacher
Perform experiment
After experimenting and analyzing your data, do you agree or disagree with
your prediction? Why?
EXPERIMENT 3: WHAT IS THE AFFECT OF TEMPERATURE ON ENZYME
ACTIVITY?
PREDICT: What will be the effect of low temperature and high temperature on
enzyme activity? _______________________________________________________________________
3
PROCEDURE:
1. Grind up 20 grams of peeled potato cubes with 20 ml of chilled distilled
water using a mortar and pestle.
2. Filter the solution into a 100 ml graduated cylinder using cheesecloth or
coffee filter.
3. Add chilled distilled water to graduated cylinder to bring volume up to 2o ml.
4. Pour into 50 ml beaker or small cup that is chilled on ice and label 100%
catalase.
5. Using graduated cylinder measure 15 ml of 3% hydrogen peroxide and then
add 15 ml of room temperature distilled water (you now have 30 ml of a
1.5% hydrogen peroxide).
6. Pour the 1.5 % hydrogen peroxide into a 50 ml beaker or small cup and
label. This will be your reaction vessel.
7. During the experiment, when you drop a disk soaked in catalase into the
reaction vessel, the hydrogen peroxide will decompose, forming oxygen gas
bubbles on the disk that will eventually lift it to the surface.
8. Label three test tubes 5 C, 37 C, and 100 C respectively.
9. Add 5ml of 100% catalase to each tube
10. Place test tube labeled 5 C in ice bath for five minutes, test tube labeled 100 C
in boiling water for five minutes, and test tube labeled 37 C in warm water
bath for five minutes.
11. Measure temperature of each test tube to ensure correct temperature.
12. With a pair of forceps, take a dry filter disk out of the petri dish lid and dip it
into 5 C test tube solution for five seconds.
13. Remove the disk and blot its edge on the top of the test tube.
14. Start your timer as you place the disk on the bottom of the reaction vessel
containing 30 ml of 1.5% hydrogen peroxide.
15. Time in seconds how long it takes the disk to rise to the surface of the
peroxide. Stop timing after three minutes if there is no activity.
16. Remove the use disk. Perform another trial with a new catalase-soaked disk
for 5 C catalase.
17. Conduct two trials for each of the remaining catalase solutions (37 C & 100
C). For each Celsius solution, average the time for floating the disks. Always
remember to remove the used disk before starting the next trial.
18. The rate of each reaction can be calculated by the formula R=d/t, where
d=the distance from the bottom of the container to the surface in cm, and
t=seconds. The unit for rate is cm/sec. If the disk remains on the bottom of
the container after 3 minutes, the distance (d) will be zero.
19. Plot as graph for each data point and draw a best-fit line.
20. After experimenting and analyzing your data, do you agree or disagree with
your prediction? Why?
4
ENZYME REACTION TIME AT DIFFERENT TEMPERATURES OF CATALASE
5 C Catalase
37 C
Catalase
100 C
Catalase
TRIAL 1 TIME
TRIAL 2 TIME
AVG. TIME
RATE (d/t)
EXPERIMENT 4: WHAT IS THE AFFECT OF pH ON ENZYME ACTIVITY?
PREDICT: What will be the effect of acid or basic pH on enzyme activity?
______________________________________________________________________________________________
PROCEDURE:
1. Grind up 20 grams of peeled potato cubes with 20 ml of chilled distilled
water using a mortar and pestle.
2. Filter the solution into a 100 ml graduated cylinder using cheesecloth or
coffee filter.
3. Add chilled distilled water to graduated cylinder to bring volume up to 2o ml.
4. Pour into 50 ml beaker or small cup that is chilled on ice and label 100%
catalase.
5. Using graduated cylinder measure 15 ml of 3% hydrogen peroxide and then
add 15 ml of room temperature distilled water (you now have 30 ml of a
1.5% hydrogen peroxide).
5
6. Pour the 1.5 % hydrogen peroxide into a 50 ml beaker or small cup and
label. This will be your reaction vessel.
7. During the experiment, when you drop a disk soaked in catalase into the
reaction vessel, the hydrogen peroxide will decompose, forming oxygen gas
bubbles on the disk that will eventually lift it to the surface.
8. Label three test tubes pH 4, pH 7, and pH 10 respectively.
9. Add 5ml of 100% catalase to each tube.
10. Add drops of HCL to pH 4 test tube until you have a pH reading of 4
11. Add drops of NaOH to pH 10 test tube until you have a reading of 10
12. Add distilled water to pH 7 test tube until you have a pH reading of 7
13. With a pair of forceps, take a dry filter disk out of the petri dish lid and dip it
into pH 4 test tube solution for five seconds.
14. Remove the disk and blot its edge on the top of the test tube.
15. Start your timer as you place the disk on the bottom of the reaction vessel
containing 30 ml of 1.5% hydrogen peroxide.
16. Time in seconds how long it takes the disk to rise to the surface of the
peroxide. Stop timing after three minutes if there is no activity.
17. Remove the use disk. Perform another trial with a new catalase-soaked disk
for pH 4 catalase.
18. Conduct two trials for each of the remaining catalase solutions (pH 7 & pH
10). For each pH solution, average the time for floating the disks. Always
remember to remove the used disk before starting the next trial.
19. The rate of each reaction can be calculated by the formula R=d/t, where
d=the distance from the bottom of the container to the surface in cm, and
t=seconds. The unit for rate is cm/sec. If the disk remains on the bottom of
the container after 3 minutes, the distance (d) will be zero.
20. Plot as graph for each data point and draw a best-fit line.
ENZYME REACTION TIME AT DIFFERENT pH’s OF CATALASE
pH 4
pH 7
pH 10
TRIAL 1 TIME
TRIAL 2 TIME
AVG. TIME
RATE (d/t)
21. After experimenting and analyzing your data, do you agree or disagree with your
prediction? Why?
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