CATALASE LAB PROCEDURE
Part A Purpose: What is the effect of substrate concentration on enzyme activity?
Hypothesis:
Lab Safety:
Wear appropriate lab attire (i.e., safety goggles, closed toe shoes) before commencing the
lab. Long hair should be tied back, and loose clothing and contact lens should not be
worn in the lab. During the lab, keep the container of H2O2 closed at all times. Not only
does this prevent the decomposition of the substrate, but it also prevents hazardous spills.
Procedure:
1.
Dissolve approximately 30 grains of yeast in 50 mL of distilled water. Stir until you
have a unified suspension. This is your 100% by volume catalase solution. This will
be called Suspension C.
2.
Obtain 4 separate beakers. For each beaker, label and prepare the dilution of
substrate (hydrogen peroxide) as described below.
Beaker Label
Dilution Instructions
4
3.0% H2O2
40 mL of 3% H2O2
3
1.5% H2O2
Add 20 mL of distilled water to 20 mL of 3% H2O2
2
0.75% H2O2
Add 30 mL of distilled water to 10 mL of 3% H2O2
1
0.0% H2O2
40 mL of distilled water
3.
Using forceps, drop a filter paper disc into the 100% suspension of catalase. Make
sure it is saturated with the suspension C before removing it with the forceps.
4.
Reset your stopwatch. Start the stopwatch at the moment you drop the filter paper
disc that has been coated with suspension C into beaker 1.
5.
Use the stopwatch to time how long it takes the filter paper disc to rise to the top of
the beaker 1 solution. Record all results in the data table below.
6.
Repeat Steps 4 – 5 for the remaining 3 beakers.
Results:
Beaker
H2O2
Time A (s): for filter
Rate of Enzyme
Class Average
Concentration
paper disc to sink and then
Activity (floats/s) =
(floats/s)
(X)
float to surface.
1 divided by Time A.
(3 dec. places)
(3 dec. places)
(Y)
1
0.0%
Inf.
2
0.75%
31.75, 31, 33.5
0.030, 0.032, 0.029
0.030
3
1.5%
19.97, 5,19.28
0.050, 0.200, 0.051
0.100
4
3.0%
10.19, 12, 7.44
0.098, 0.083, 0.134
0.105
Part B Purpose: What is the effect of enzyme concentration on enzyme activity?
Hypothesis:
Lab Safety:
Wear appropriate lab attire (i.e., safety goggles, closed toe shoes) before commencing the
lab. Long hair should be tied back, and loose clothing and contact lens should not be
worn in the lab. During the lab, keep the container of H2O2 closed at all times. Not only
does this prevent the decomposition of the substrate, but it also prevents hazardous spills.
Procedure:
1.
Prepare 5 separate beakers with 40 mL of 3% hydrogen peroxide each and label them 1
through 5.
2.
Dissolve approximately 60 grains of yeast in 100 mL of distilled water. Stir until you
have a unified suspension. This is your 100% by volume catalase solution. This will be
called Suspension C and will be used to make enzyme solutions 2 to 4 in the chart
below.
3.
Prepare each of the following enzyme dilutions in separate beakers.
Enzyme Solution
Desired Enzyme Concentration
Dilution Instructions
5
100% catalase by volume
20 mL of the 100% suspension C
4
75% catalase by volume
Add 5 mL of distilled water to 15 mL of suspension C
3
50% catalase by volume
Add 10 mL of distilled water to 10 mL of suspension C
2
25% catalase by volume
Add 15 mL of distilled water to 5 mL of suspension C
1
0% catalase by volume
20 mL of distilled water.
4.
Using forceps, drop a filter paper disc into Solution 1. Make sure it is saturated before
removing it with the forceps.
5.
With forceps take the saturated disc and drop it into beaker 1 with the 3% hydrogen
peroxide solution. Use the stopwatch to time how long it takes the filter disc to sink and
then to rise to the top of the 40 mL solution. Record all results in the data table below.
6.
Repeat Steps 4 – 5 for the remaining 4 beakers, being sure to saturate a new filter disc in
the correct enzyme solution each time.
Results:
Beaker
Enzyme
Time A (s): for filter paper disc to
Rate of Enzyme
Class Average
Concentration
sink and then float to surface.
Activity (floats/s) =
(float/s)
1 divided by Time A.
(3 dec. places)
(3 dec. places)
(Y)
68.09, 82.03
0.014, 0.012
0.013
24.15,18.81
0.04, 0.053
0.046
15.53, 8.25
0.064, 0.121
0.092
4.57, 6.56
0.219, 0.152
0.186
5.72, 6.03
0.175, 0.166
0.171
(X)
1
0% catalase
by volume
2
25% catalase
by volume
3
50% catalase
by volume
4
75% catalase
by volume
5
100% catalase
by volume
Part C Purpose: What is the effect of temperature on enzyme activity?
Hypothesis:
Lab Safety:
Wear appropriate lab attire (i.e., safety goggles, closed toe shoes) before commencing the
lab. Long hair should be tied back, and loose clothing and contact lens should not be
worn in the lab. During the lab, keep the container of H2O2 closed at all times. Not only
does this prevent the decomposition of the substrate, but it also prevents hazardous spills.
Procedure:
1.
Prepare 4 separate beakers with 40 mL of 3% hydrogen peroxide each and label them 1
through 4.
2.
Dissolve 30 grains of yeast in 50 mL of distilled water. Stir until you have a unified
suspension. This is your 100% by volume catalase solution. This will be called
Suspension C.
3.
Fill four test tubes with suspension C. Fill them each about ¾ full. Label the test tubes
as 0°C, Room Temp, 37°C, and 100°C.
4.
Take Beaker 1 with 40mL of 3% hydrogen peroxide and place in ice bath. Leave
beakers 2 – 4 at room temperature. This is because heat will destroy hydrogen peroxide.
DO NOT BOIL HYDROGEN PEROXIDE!
5.
Take each test tube with Suspension C and incubate in the water bathes at each
temperature for 5 minutes.
6.
Using test tube clamps hold the test tube, take forceps and drop a filter paper disc into
test tube 1. Make sure it is saturated with suspension C before removing it with the
forceps.
7.
With forceps take the saturated disc and drop it into beaker 1 with the 3% hydrogen
peroxide solution. Use the stopwatch to time how long it takes the filter disc to sink and
then to rise to the top of the 40 mL solution. Record all results in the data table below.
8.
Repeat Steps 6-7 for the remaining 3 beakers, being sure to saturate a new filter disc in
the correct enzyme solution each time.
Results:
Beaker
Enzyme
Time A (s): for filter paper disc to
Rate of Enzyme
Class Average
Temperature
sink and then float to surface.
Activity (floats/s) =
(float/s)
1 divided by Time A.
(3 dec. places)
(3 dec. places)
(Y)
(X)
1
0°C
135.6, 312
0.007, 0.0032
0.0051
2
Room Temp
144, 120
0.007, 0.0083
0.0076
3
37°C
N.V., 480
0.0020
0.0020
4
100°C
N.V., 300
0.0033
0.0033
Lab Report Marks Checklist:
1.
Appropriate title for this lab (1 mark)
2.
Introduction (4 marks): Write a brief paragraph describing the background and what
the purpose of this lab was. Include any equations for the reactions and graphs that
that give important information about the concepts in this lab.
3.
What was your hypothesis for what will happen during each part of the lab (3
marks)?
4.
Data Charts and Analysis (12 marks): You should have 3 data tables (including raw
data from your group and class averages for all three parts of the lab. 3 graphs should
be prepared, one for each variable that was manipulated in this lab (see first Lab
package). 1 Additional graph for the results specific to your group.
5.
Conclusion (14 marks): Succinctly answer the questions in the first lab package
6.
Neatness/3rd person (6 marks): The lab and charts must be typed. Graphs may be
hand drawn but must be neat and legible. The lab should be written in 3rd person.
Do not use the pronouns I, we, us etc.