AP Biology
ASFA
Fall 2008
AP Lab 2 with Pressure Probes: Enzyme Catalysis
Background:
According to the AP Lab Manual catalase is ubiquitous in aerobic organisms. One function of catalase within cells is to
prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes.
The primary reaction catalyzed by catalase is the decomposition of H 2O2 to form water and oxygen.
2 H2O2
2 H2O + O2 (gas)
In general, enzyme reactions can be thought of as:
E+S
ES
E+P
What is conserved in this reaction?
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Why is this important?
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Much can be learned about enzymes by studying the kinetics (particularly the changes in rate) of enzyme-catalyzed
reactions. For example, it is possible to measure the amount of product formed, or the amount of substrate used, from
the moment the reactants are brought together until the reaction is stopped.
In previous years we measured the amount of substrate used by titrating the remaining product with KMnO 4, but this
year we are going to use available technology to measure the amount of product formed in real time.
For the reaction outlines above, what is the…Quick, what is the
…substrate? ___________________________________________
…product? ____________________________________________
… enzyme? ____________________________________________
Objective 1: Measure the amount of O2 produced by a reaction of 1.5% H2O2 and beef liver catalase using Probeware
(PASCO SPARK or Verneir LabQuets and a gas pressure sensor). Graph catalase activity versus time.
Objective 2: Learn to use probeware, utilize some of the data collection, graphical representation, and data analysis
functions of the probeware, and learn how to manage data generated during the experiments.
Objective 3: Design experiments to learn how environmental conditions (pH, temperature, salinity, enzyme and
substrate concentration) affect enzyme activity (the rate of the an enzyme-substrate reaction).
Ideally we can learn something about the initial rate of reaction (Ki) for catalase. The initial rate of any enzyme-catalyzed
reaction is determined by the characteristics of the enzyme molecule. It is always the same for any enzyme and its
substrate at a given temperature and pH. This also assumes the substrate is present in excess.
Experimental Set Up and Data Collection.
1. Obtain a 250 ml Nalgene Bottle, a gas pressure sensor and a data collection device. (This equipment set up is
roughly $500. We purchased it for you to use, however, it needs to last for years. Be extremely careful with this
equipment. If you’re unsure of how something works, ask. Today there are no stupid questions.)
Make sure you rinse your flask 2 times with tap water and once with Distilled water.
2. Obtain a small white cup, label it catalase and fill it with beef liver catalase extract.
3. Obtain a syringe marked catalase.
4. Obtain a small clear cup, label it and fill with 1.5% H2O2.
5. Obtain a plastic syringe marked H2O2.
6. Use syringe to place 10 ml of H2O2 in the nalgene bottle (make sure not to get any H2O2 on the sides of the bottle.
7. Practice placing the rubber stopper/pressure sensor in the top of the experimental chamber. It will be very important
to get good at capping the bottle right after the catalase has been added (see below).
8. When you and your lab partners are ready, press play, add 1 ml of catalase to the experimental chamber, quickly
place the stopper on top of the experimental chambers and continue recording data.
9. Collect data for 300 seconds (5 minutes).
10. Repeat the same experiment two more times. (We will get an average rate of enzyme activity across all three trials.)
Data Analysis.
1a. Record the pressure within the chamber for each time point denoted on the Catalase Experiment Data Table.
1b. Graph the average change in pressure versus time in graph 2.1 (see Graph Template).
1c. A graph is a way to represent data from an experiment. In other words, it should tell a story. What does every good
graph need in order for the reader to understand your story? I think there are five necessary items. Write them below,
and never forget them.
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2. Compare the average reaction rates for different intervals during this experiment.
Time Interval (seconds) Each group can conduct the experiment and then groups can compare trials for an average
rate of H202 decomposition
0 to 30
30 to 60
60 to 90
90 to 120
120 to 180
180 to 300
Rate* (Trial 1)
X
Rate (Trial 2)
Rate (Trial 3)
Avg Rate
X
X
X
* How do you calculate rate for each interval? __________________________________________________________
3. When was the reaction rate the highest. Why? ________________________________________________________
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4. Think about what environmental conditions you want to change, tomorrow we’ll design controlled experiments and
see if we get different results.
DAY 2
Before we begin we need to look at some to review some key concepts in experimental design.
I. Big, basic ideas:
A. If we change something in an experiment, we call the thing we change
the _____________________.
B. If we observe or measure a result based on the change we make, then
the observed result is known as the ________________________.
C. A condition (or experiment) that is unchanged, but still monitored is
known as a/an ______________________ experiment.
D. An environmental condition we maintain or don’t manipulate, but may
affect our experiments is a/an____________________.
II. What can we change about this experiment? What environmental factors affect enzymes?
--
--
--
--
These are all examples of…
III. Once you have been assigned an experimental condition to change ( ), then look at the experimental design
diagram sheet. (See file named 05a_Experimental Diagram Diagram.doc)
IV. How will we modify this experiment?
B. Other Experiments:
Repeat the experiment from Day 1, but for pH, we need to…
Amount of H202 (1.5%)
Amount of Acid/Base
Run 1: low pH
5 ml
3 ml 5 ml 1M HCl pH:_____
2: neutral
5 ml
3 ml 5 ml H2O pH: _____
Run 3: high pH
5 ml
3 ml 5 ml 1M NaOH pH:____
Constants
Temp (room)
Catalase ( ml)
Run time (300 sec)
Repeat the experiment from Day 1, but for change in substrate concentration you need to…
Conc. of H2O2
Amount of H202
Constants
Run 1 (low)
0.75 %
10 ml
Temp (room)
Run 2 (
)
1.5%
10 ml
Catalase
Run 3 (high)
3%
10 ml
Run time
Repeat the experiment from Day 1, but for change in temperature you need to…
Constants
Ambient Temp (C)
4
Enzyme volume
Run 1 (cold) (
C)
21
Substrate volume
Run 2 (control) (Room) (
C)
37
Substrate conc.
Run 3 (warm) (
C)
Repeat the experiment from Day 1, but for salinity, we need to…
Amount of H202 (3%)
Amount of salt solution
Run 1: normal salinity
5 ml
3 ml 5 ml DH20:_____
2: high salt
5 ml
3 ml 5 ml 10% salt solution
____
Run 3: really high salt
5 ml
3 ml 5 ml 20% salt solution
NaOH
Constants
Run Time
Constants
Temp (room)
Catalase ( ml)
Run time (300 sec)
Repeat the experiment from Day 1, but for source of catalase, we need to…
Amount of H202 (1.5%)
Mass of tissue/Volume
of water
Run 1: source 1
10 ml
2: source 2: beef
10 ml
Run 3: Source 3:
10 ml
Constants
Temp (room)
Catalase ( ml)
Run time (300 sec)
Is there another variable you would like to change? If so, let me know. Run it by me, and create a table similar to the
ones above.
C. Data analysis for independent experiment.
1a. Record the pressure within the chamber for each time point denoted on the Catalase Experiment Data Table.
1b. Graph the data from your three trials on Graph 2.2. (see Graph Template). Also, use a data table provided
2. Calculate and compare the average reaction rates for different intervals during your independent experiment
in table 2.2
Table 2.2 ____________________________________________________________________________________
Time Interval (seconds) (Here each group should conduct three trials of their experiment)
0 to 30
30 to 60
60 to 90
90 to 120
120 to 180
180 to 300
Rate* (Trial 1)
Rate (Trial 2)
Rate (Trial 3)
Avg Rate
3. When was the reaction rate the highest. Why? ________________________________________________________
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IMPORTANT! For the second experiment you and your lab partner(s) need to fill out an experimental design diagram.
The Independent Variable table will be important for your guiding your set up and execution of your experiments.
See next page for lab write up guidelines.
AP Biology.
ASFA.
R. Reardon, Instructor.
Enzyme Catalysis Lab. Fall 2009.
What’s Due?
When is this due?________
 Complete the “fill in the blank” questions In the original lab packet.

Complete data tables in original packet.

Make sure all graphs (2.1, 2.2, 2.3) are completed accurately, curves are differentiated in a readable way,
graphs contain a title, axes are labeled and scaled appropriately, units are included, and a legend, if
necessary, is included. YOU MUST HAND DRAW YOUR GRAPHS ON THE TEMPLATES PROVIDED.

Make sure both your Experimental Design Diagrams are filled out completely and accurately.

Make sure all your data tables are filled out completely and accurately.

Answer all Supplemental Questions. See below.
Supplemental Questions.
These can be typed up or hand written on a separate sheet of paper. Please limit your responses to one
paragraph. There is no need to re-write the questions, just indicate the number of the question.
Questions a-c deal with our first experiment (20Aug09): measuring the effect of catalase on the decomposition of
H2O2.
a) When was the rate of reaction the highest? Explain why.
b) When was the rate the lowest? Explain why.
c) Why do cells have catalase? What is its function? What would happen to cells if they did not produce catalase?
If you varied the catalase concentration in your experiment, answer questions 1-4.
1a) How did you vary the amounf of catalase concentration in each trial?
1b) Did varying the catalase concentration change the initial rate of reaction (Ki) for the catalase + H2O2 reaction?
Use evidence to support your claim, and provide reasoning (the biological reason) for your results.
2) What catalase concentration had the highest initial rate (Ki) of reaction?
3) Did varying the catalase concentration affect the amount of substrate remaining at the end of the run (5
minutes)? What evidence do you have to support your claim? What is the reason for the result?
4) Everyone observed the boiled beef liver extract. When I placed 0.5 ml of boiled beef catalase in the presence
of 10 ml 1.5% H2O2 , rate of O2 production over the course of the entire run was measured as _______________.
Why?
If you varied the substrate concentration in your experiment, answer questions 5-7.
5) Did varying the substrate concentration change the initial rate of reaction (K i) for catalase + H2O2 reaction? Use
evidence to support your claim, and provide reasoning (the biological reason) for your results.
6) List 4 constants in this experiment.
7) List 2 possible sources of error for this experiment.
If you varied pH in your experiment, answer questions 8-10.
8) Did varying the pH of the reaction change the initial rate of reaction (Ki) for catalase + H2O2 reaction? Use
evidence to support your claim, and provide reasoning (the biological reason) for your results.
9) List 4 constants in this experiment.
10) List 2 possible sources of error for this experiment.
If you varied the temperature in your experiment, answer questions 11-14.
11) Did varying the temperature change the initial rate of reaction (Ki) for catalase + H2O2 reaction? Use evidence
to support your claim, and provide reasoning (the biological reason) for your results. Make sure you indicate what
was happening at low temperatures and at high temperatures.
12) Was all the H2O2 consumed during each run? What evidence do you have to support your claim?
13) List 4 constants in this experiment.
14) List 2 possible sources of error for this experiment.
If you varied the source of catalase in your experiment, answer questions 15-18.
15) Did varying the source of catalase change the initial rate of reaction (K i) for catalase + H2O2 reaction? Use
evidence to support your claim, and provide reasoning (the biological reason) for your results. Do you think there
was more (or less catalase) within the cells? Or does catalase from different organisms have different activity
levels?
16) Was all the H2O2 consumed during each run? What evidence do you have to support your claim?
17) List 4 constants in this experiment.
18) List 2 possible sources of error for this experiment.
If you varied the salinity of the H202 solution, answer questions 19-22…
19) Did varying the salinity change the initial rate of reaction (K i) for catalase + H2O2 reaction? Use evidence to
support your claim, and provide reasoning (the biological reason) for your results. Make sure you indicate what
was happening as salinity increases. Don’t just say “denaturing the enzyme.’ Tell me how the enzyme is
denatured.
20) Was all the H2O2 consumed during each run? What evidence do you have to support your claim?
21) List 4 constants in this experiment.
22) List 2 possible sources of error for this experiment.
Meet CEER. This is a method of science writing developed by the University of Michigan in order to meet the
Science Education Standards generated by the National Science Resource Center I think CEER is an extremely
useful tool for organizing your thoughts and your writing. We will be using this method often in this class (AP
Biology), and in all my other classes. I think this method of writing short answers, and even longer papers will
serve you well in college.
C
E
R
R
The claim is a testable statement or conclusion that answers the original question.
The evidence is the scientific evidence that supports the claim. Evidence must be
both appropriate and sufficient to support the claim. (Does the evidence – the
data—you present support the claim you are making?)
Reasons are explanations. They are a justification that shows why the data
counts as evidence to support the claim and includes appropriate scientific
principles.
When explaining phenomena, there can be more than one possible claim or
explanation. Often the same data can be used to support an alternate claim. You
should learn to recognize alternative explanations. A good answer takes an
alternate explanation into consideration and offers a rebuttal to why an alternative
explanation is not appropriate. In other words, the rebuttal articulates why an
alternative claim is not appropriate. It also gives you another chance to articulate
your reasoning and lets the reader know you know what you’re talking about.
As you develop as a scientist, your ability to make claims, cite evidence, use
reasoning and offer rebuttals will become more sophisticated. I think this is a very
useful tool for science writing.