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Final Enzyme LAb

The document outlines an experiment to investigate the effect of pH levels (6.0 to 8.0) on the activity of the enzyme catalase in yeast, specifically measuring the production of oxygen gas from the breakdown of hydrogen peroxide. The experiment controls various factors such as temperature and concentration of reactants, and utilizes a gas pressure sensor to measure oxygen output. The hypothesis suggests that catalase activity will peak at a neutral pH of 7.0, with deviations resulting in reduced activity.

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0% found this document useful (0 votes)
10 views14 pages

Final Enzyme LAb

The document outlines an experiment to investigate the effect of pH levels (6.0 to 8.0) on the activity of the enzyme catalase in yeast, specifically measuring the production of oxygen gas from the breakdown of hydrogen peroxide. The experiment controls various factors such as temperature and concentration of reactants, and utilizes a gas pressure sensor to measure oxygen output. The hypothesis suggests that catalase activity will peak at a neutral pH of 7.0, with deviations resulting in reduced activity.

Uploaded by

dkmkdvjbjf
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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BACKGROUND

Enzymes are proteins that speed up chemical reactions by lowering the activation
energy, which is the energy required for a reaction to take place. There are several
different types of enzymes, and they are found in all living organisms. Our bodies
naturally produce enzymes, but they can also be produced and modified within a lab
setting. Enzymes are highly specific to a specific molecule, the substrate, and are
importantly not used up in a reaction, and can be used more than once. The enzyme
catalyzed reaction follows a pattern of:

Enzyme + Substrate ⇆ Enzyme-Substrate Complex ⇆ Enzyme + Product


(Enzyme comes in contact with its specific substrate and an enzyme substrate complex
is formed, after which a desired product is made, leaving the enzyme unchanged, and
ready to accept another substrate molecule to perform the same reaction again)

Enzyme activity is affected by various factors, such as temperature, pH levels, and


concentrations. Catalase is a specific enzyme found in many organisms, including
yeast. Catalase catalyzes the breakdown of hydrogen peroxide (H2O2) into water
(H2O) and oxygen gas (O2). In this experiment, we will use the catalase present in
yeast to test the effects of different pH levels (6.0, 6.5, 7.0, 7.5, 8.0) on the production of
oxygen gas, which will be measured via a pressure sensor, with a higher pressure
reading indicating a higher catalase activity. pH levels will act as our manipulated
variable, with oxygen gas produced being our responding variable. We expect the
catalase activity to be highest at the pH of 7.0 due to a pH of 7.0 being neutral. We
would suspect that at other pH levels (more acidic or basic) catalase activity will be
lower. By measuring the oxygen produced at various pH levels, we aim to determine the
optimal pH level for catalase effectiveness, which, according to literature, should fall
within a pH range of 6.0-8.0 (Ota, A).

FOCUS QUESTION
How does changing the pH (6.0, 6.5, 7.0, 7.5, 8.0) of the hydrogen peroxide solution
impact the amount of oxygen gas produced when the enzyme catalase from a yeast
solution reacts with the substrate, hydrogen peroxide.
EXPERIMENTAL VARIABLES/DESIGN
Manipulated Variable: pH of the hydrogen peroxide solution. Our group decided to
choose pH levels of 6.0, 6.5, 7.0, 7.5, 8.0, because it tests equal magnitudes of basic
and acidic solutions. Because pH of 7.0 is neutral, we are able to capture both sides of
the pH scale. We chose stepwise increments of 0.5 because it allows us to see a clear
trend spanning different pH levels.
Responding Variable: Oxygen gas produced, tested by monitoring changes in
pressure in kPa.
Controlled Variable: Time of reaction in each trial, Temperature of the solution,
Concentration of Yeast (Catalase) Solution, Amount of Buffer Solution Added, Initial
Concentration of Hydrogen Peroxide (originally 3%)
1.​ Time of reaction in each trial: It is important that we keep the time of each trial
the same, as longer or shorter times of reaction per trial could potentially change
the outcome and our data. We will keep each reaction the same length of time by
using a stopwatch, timing each reaction to (30) seconds.
2.​ Temperature of the solution: Temperature has a known impact on reaction rates,
therefore a difference in temperatures would significantly impact the quality of our
data. We won't be able to directly control the temperature, but we will be able to
monitor it and ensure that it remains similar across the different trials and pH
levels. We will record the temperature for all trials and pH levels separately in a
data table. (below) will control the temperature of the solutions by keeping them
all in the same area of the same room, making sure that they are all room
temperature.
3.​ Concentration of Yeast (Catalase): The concentration of yeast is an important
reactant in this experiment, and ensuring that the concentration of yeast is the
same from trial to trial is key in reaching accurate data. We will control this
variable by measuring out the amount of yeast by weight (measured via
electronic balance), and mix this yeast with a consistent amount of water
(measured via graduated cylinder) to ensure that the yeast concentration
remains consistent for all trials.
4.​ Volume of Buffer Solution: The volume of the pH buffer solution must remain
consistent across all trials to ensure that the only variable affecting the catalase
activity is the pH level itself. In this experiment, 2 mL of the specific pH buffer
solution (6.0, 6.5, 7.0, 7.5, or 8.0) will be added to each of the five test tubes.
This will help ensure that differences in oxygen production are due to the pH
level and not variations in the volume of the buffer.
5.​ Concentration of Hydrogen Peroxide (diluted from 3% to 1%): Keeping the
concentration of Hydrogen Peroxide consistent is important because if our trials
had different concentrations of hydrogen peroxide, our data would likely be
different, peroxide is a known participant in the reaction. We will keep this
consistent by using the same 1% hydrogen peroxide solution that was prepared
at the start of the experiment.

MATERIALS & METHODS/PROCEDURE


1.​ Dry Active Yeast
2.​ Granulated White Sugar
3.​ Distilled Water Jug
4.​ Hydrogen Peroxide (3%)
5.​ 10 mL Graduated Cylinder
6.​ 100 mL Graduated Cylinder
7.​ 400 mL Beaker (2
8.​ Stirring Rod
9.​ Pipettes (7)
10.​Electronic Balance
11.​Buffer Solutions (pH 6.0, 6.5, 7.0, 7.5, 8.0)
12.​Vernier Gas Pressure Sensor
13.​LabQuest Interface
14.​Stopwatch
15.​Rubber Corks (attached to gas pressure sensor)
16.​10 mL Test Tubes (5)
17.​Test Tube Rack
18.​Thermometer
19.​Clean Paper Towels
20.​Spreadsheet to Record Data
21.​Graphing Software

(1) Preparation of Yeast Catalase Solution

1)​ Weigh yeast and sugar: Using an electronic balance, measure out 11.0 g of dry
yeast and 5.0 g of white sugar and place both in a 400 mL beaker.
2)​ Measure water: Using a 100 mL graduated cylinder, measure out 60 mL of room
temperature distilled water (20-22°C). Check the water temperature with a
thermometer to confirm the temperature.
3)​ Mix Ingredients: Pour the 60mL of distilled water into the 400 mL beaker with the
sugar and yeast. Stir the mixture with a stirring rod until everything appears to
have dissolved. Continue stirring every 10 minutes to keep the solution
well-mixed.
(2) Preparation of 1% Hydrogen Peroxide Solution

1)​ Prepare the hydrogen peroxide solution: Using a clean 100 mL graduated
cylinder, measure 50 mL of 3% hydrogen peroxide and pour into a clean 400 mL
beaker.
2)​ Dilute with Water: Using a 100 mL graduated cylinder, measure 100 mL of
distilled water and pour it into the 400 mL beaker with the 1% hydrogen peroxide
solution. Stir the solution using a stir rod to ensure thorough mixing.

(3) Preparation of Five 1% Hydrogen Peroxide Solutions with Varying pH Levels

1)​ Prepare test tubes: Place 5 clean test tubes onto a test tube rack.
2)​ Prepare Hydrogen Peroxide Solutions: Use a pipette to transfer 4 mL of your 1%
hydrogen peroxide solution into a 10 mL graduated cylinder. Pour the 4 mL of 1%
hydrogen peroxide solution into the first test tube. Repeat this step to add 4 mL
of 1% hydrogen peroxide solution to each of the 5 test tubes on the test tube
rack.
3)​ Add pH Buffers: Using a different pipette for each pH buffer, transfer 2 mL of the
corresponding buffer solution (first pH solution will be 6.0. The order of trials will
be: pH 6.0, 6.5, 7.0, 7.5, 8.0) to a clean 10 mL graduated cylinder. Pour the 2 mL
of buffer solution into the first test tube. Repeat this step to add 2 mL of the
specific buffer solution being tested to each of the 5 test tubes on the test tube
rack. You should now have 5 test tubes, each with 4 mL of hydrogen peroxide
solution and 2 mL of buffer mixed.

(4) Reaction with Catalase and Substrate

1)​ Set Up Vernier Gas Pressure Sensor: Plug in the Vernier Gas Pressure Sensor
and ensure it is operating correctly. If the screen does not turn on, it may not be
charged. If this is the case, either grab a different device, or plug in the one you
already have to charge while you use it.
2)​ Measure Temperature: Before beginning the reaction, measure and record the
temperature of the test tube being tested. You will measure the temperature right
before adding the yeast solution to the test tube for each given trial. This is to
monitor any changes from trial to trial.
3)​ Using a new pipette, pour 1 mL of the yeast solution into the first test tube to
begin the reaction with the 1% hydrogen peroxide and the buffer solution.
Immediately after the solution has been added to the test tube, seal the tube with
the rubber cork attached to the vernier pressure sensor. Once sealed, start a
timer for 30 seconds.
4)​ At the 30-second mark, record the observed gas pressure reading from the
Vernier gas pressure sensor.

(5) Clean and Repeat

1)​ Using a dry paper towel, wipe the Vernier gas pressure sensor’s rubber end to
remove any existing residue from the previous trial.
2)​ Wash the test tubes, pipette, and graduated cylinder with water from the sink,
drying with a paper towel afterwards.
3)​ Set up the five test tubes back on the test tube rack
4)​ Repeat steps 3 and 4 with pH solutions of 6.5, 7.0, 7.5, and 8.0, with step 5 in
between each to ensure clean equipment.
5)​ After completing, record pressure levels for each of the pH solutions (6.5, 7.0, 7.5
8.0). You should have 25 data points in total, five solutions and five trials per
solution.

(6) Process the Data:

1)​ Record Pressure Data: Use a spreadsheet to log all pressure data for each pH
level.
2)​ Calculate Averages: Using the recorded data, calculate the average pressures
for each of the five varying pH levels. Do this by adding the 5 pressure
measurements that were recorded for each pH level and dividing that total by 5.
The average will fall somewhere between your highest and lowest measurement.
3)​ Plot the Data: Plot the average pressure for each pH level on a graph.​
Use this graph to identify any trends in the data. Select a line of best fit to
represent the trend.
Data Table: Pressure (kPa) Measurements of Oxygen Gas Produced at varying pH
Levels
Ph of 1% Gas Pressure of Oxygen Gas (kPa) Observations
Hydrogen
Peroxide
Solution Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Average
(+/-0.1) Pressure Pressur Pressu Pressure Pressure Pressure (kPa)
(kPa +/-4) e (kPA re (kPa (kPa +/-4) (kPa +/-4)
+/-4) +/-4)

6.0 138.74 135.77 139.00 143.35 144.45 140.262 (+/- 4.34) Audible bubble
popping sounds
as the stopper
was released.

6.5 142.31 140.23 141.99 142.3 140.70 141.506 (+/-1.04) The cap popped
off in two of our
trials when we
released the
pressure on the
stopper. Test
tube also
fogged up.

7.0 148.76 141.36 151.98 146.17 154.27 148.508(+/- 6.46) Test tubes
fogged up more
than others,
also was more
bubbling than
others.

7.5 128.25 138.70 127.22 139.67 147.32 136.23 (+/- 10.05) Audible bubble
popping sounds
as we released
the stopper.

8.0 120.3 121.63 125.65 128.29 123.5 123.87 (+/- 3.99) There was
minimal
bubbling and no
sound when we
released the
stopper.
Data Table: Temperature of Hydrogen Peroxide Solutions at Varying pH Levels
Ph of 1% Initial Temperature Before Reaction (°C)
Hydrogen
Peroxide
Solution Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
(+/-0.1) Temperature °C Temperature °C Temperature °C Temperature °C Temperature°C
(+/- 0.5) (+/- 0.5) (+/- 0.5) (+/- 0.5) (+/- 0.5)

6.0 21 21 21 21 21

6.5 21 21 21 21 21

7.0 21 21 21 21 21

7.5 21 21 21 21 21

8.0 21 21 21 21 21

Data Table Annotations:


Uncertainty for the pH measurements were calculated by our teacher, and the
uncertainty for the pH levels was (+/-0.1). The uncertainty for pressure measurements
kPa was found by referencing the uncertainty values for the Vernier gas pressure
sensor that are posted online. We found that the uncertainty of this device was (+/- 4
kPa). We calculated the uncertainty for our average kPa by using the 4 step method
(shown below in example calculations). For our second data table, in which we recorded
the temperatures, we found our uncertainty for temperature to be (+/- 0.5 °C).
Example Calculation: Average Pressure (kPa) for pH of 6.0. Trials 1-5
1.​ Given: 2.​ Equation:
Trial 1: 138.74 (Trial 1 results + Trial 2 results + Trial 3
Trial 2: 135.77 results + Trial 4 results+ Trial 5 results) /
Trial 3: 139.00 Total # of trials
Trial 4: 143.35
Trial 5: 144.45

3.​ Set up equation: 4.​ Answer:


(138.74 + 135.77 + 139.00 + 143.35 + Average of 140.262 kPa
144.45) / 5

Example Calculation: Uncertainty of the calculated average pressure (kPa) for pH


of 6.0. (Error Bar Calculation for pH of 6.0)

Given: Equation:
Trial 1: 138.74 range / 2
Trial 2: 135.77
Trial 3: 139.00 (largest value - smallest value) / 2
Trial 4: 143.35
Trial 5: 144.45

Step 3: Set up the equation Step 4: Answer

(144.45 - 135.77) / 2 4.34 kPa


Graph Annotation: The graph shows that as the pH level increases in 0.5 increments
from 6.0-7.0, the pressure increases, and subsequently decreases at pH levels higher
than 7. The line that best fits the graph is a quadratic equation because it increases,
reaches a peak and then falls back down again. Our error bar for pH was (+/-0.1),
shown on the graph as the error bars for the X values. For our Y values (Pressure), we
found the uncertainty of each average using the 4 step method. For the graph, we used
the highest uncertainty out of all the averages, which was (+/- 10.05 kPa). We used the
highest uncertainty among the averages to ensure the error bars cover the maximum
possible variation in the data. This ensures the most reliable and conservative estimates
on our potential error.
Conclusion:
Our focus question for this experiment was: How does changing the pH (6.0, 6.5, 7.0,
7.5, 8.0) of a hydrogen peroxide solution impact the amount of oxygen gas produced
when the enzyme catalase from a yeast solution reacts with the substrate, hydrogen
peroxide? Our results indicate that the pH of the hydrogen peroxide solution has a clear
impact on catalase activity. The highest level of oxygen production, determined by the
highest observed pressure, was observed in trials at pH 7.0, with an average pressure
of 148.51 kPa (+/- 6.46). Given the range of pH levels tested, and that pressures were
lower for the slightly acidic and slightly basic pH levels tested, this suggests that
catalase is most active at neutral pH and deviations from neutral alter the enzyme and
its effectiveness. The first pH level tested was at a slightly acidic level of 6.0, with
pressures averaging to 140.262 kPa (+/- 4.34). When the pH level rose to 6.5, the
average pressure rose to 141.506 kPa (+/-1.04). After hitting its peak average pressure
of 148.51 kPa (+/- 6.46) at pH of 7.0, the pressures started to decrease as the pH level
rose beyond 7.0 and into the basic zone. At pH 7.5, the pressure averaged to 136.23
kPA (+/- 10.05). At the highest pH tested, 8.0, the pressure reached its lowest average
level of 123.87 kPa (+/- 3.99). This trend supports the hypothesis that catalase activity is
optimal at a neutral pH, with less oxygen production occurring at acidic and basic pH
levels. In terms of uncertainty, the Vernier gas pressure sensor used in the experiment
has an uncertainty of +/- 4 kPa, which was factored into the pressure readings. We
calculated the average uncertainty for each of the pH trials. Our uncertainty did have a
larger amount of variability than we wanted, with our lowest level of uncertainty being +/-
1.04 kPa at pH 6.5 and our highest uncertainty being +/- 10.05 kPa at pH 7.5. This high
variability in uncertainty suggests some inconsistencies within our experiment and
potentially the design. When comparing our results to results obtained by the NIH (Ota,
A), they align well with what was observed about catalase enzyme function at varying
pH levels. Although our data does agree with accepted science, our uncertainties for
pressure were too high and inconsistent to have a high confidence level of our lab's
results. Accounting for the relatively large uncertainties, I would claim that our lab
should have a moderate confidence level.
Evaluation:
Strengths of Procedure:

Describe Strength of Procedure Impact on Procedure

Having five trials for each pH level Having five trials for each of the five pH
levels strengthens the validity of the
results. This minimizes the chance of
having an outlier have a large effect on
the entire experiment. With fewer trials
our group's data would have been much
more volatile, as an outlier or error in the
experiment would have skewed the data
significantly more.

Choosing intervals of 0.5 for the pH levels A 0.5 pH difference between trials allows
for a clear identification of trends in
catalase activity for our experiment. We
capture both slightly acidic and slightly
basic ranges of pH, by observing both
sides of the 7.0 pH mark equally. This
also helps the graph show a clear trend,
and one that agrees with modern science.
If we had picked larger or smaller
intervals of pH, there would have been a
less consistent or recognizable trend both
in our data and graph.

Effective Cleaning Protocol Our group ensured that we thoroughly


cleaned test tubes and pipettes between
trials. This helps reduce the risk of
cross-contamination between trials and
also ensures that the results are not
influenced by leftover solutions from
previous trial runs.
Weakness of Procedure:

Describe Weakness Error Impact on Data Describe specific way to


or limitation improve this if we were
to repeat the experiment

1.​ Potential difference While we controlled most We could fix this by timing
in swirling or mixing of the variables, there may exactly when we mix each
of the pH and have been slight solution and also use the
peroxide solutions inconsistencies in how same amount of swirls
with the yeast. thoroughly the yeast around the solution to mix
catalase solution was it consistently. This would
mixed with the hydrogen remove some of the
peroxide and pH buffer possible uncertainty and
solution in each trial. We errors in our data.
did not time exactly when
to mix our how many swirls
we would use to mix it.
This could have changed
our data slightly as the
uneven distribution of the
yeast enzyme may have
resulted in some trials
having higher enzyme
activity than others, even
at the same pH level.

2. Differences on the seal We had different group If we were to repeat this


of the cork on the test tube. members perform the seal experiment, next time we
(Having multiple group of the test tube using the would have the same
members seal the test voronoi sensor's top. This group member hold the
tube) could have had a large cork down on the test tube
impact on the pressure, as instead of multiple group
different group members members doing it. This
possess different strength would reduce possible
and where potentially errors within our data, as
holding down the verniers the force that the cork is
pressure sensors cork with held down by would be
varying levels of force. For much more constant
example, if group member instead of varying from
A held the cork down with person to person.
more force than group
member B, this would
cause the data for group
member A to have more
pressure than the data of
group member B.

3. Yeast solution sitting After making our yeast Next time we could use 5
and being possibly solution, we tried our best test tube holders and 25
different for trial 1 and trial to perform all 5 trials for all test tubes in order to do all
25. 5 pH levels as quickly as of our reactions right after
we could. Unfortunately, another. We could also
each trial was 30 seconds have 2 group members
long, and there was more work on one of the
time in between trials reactions, with one timing
cleaning and preparing the and the other using the
next pH level. This caused stopper, with the other 2
the effect of the yeast group members doing the
solution possibly being same on a different pH
slightly different as it sat in levels trial.
the water longer. For
example, the yeast solution
in trial 1 may be different
from the yeast solution for
the 25th trial, due to it
soaking in water for all of
the time in between trial 1
and 25.
Bibliography:

Vernier Software & Technology. Gas Pressure Sensor. Vernier Software & Technology,
www.vernier.com/product/gas-pressure-sensor/?srsltid=AfmBOoqTFqb7FBDzV7mvCJt1
ReaYcvai7VdvOScreB9DozWYSWJCCnDd

Science Buddies. Experiment with the pH Scale. Science Buddies,


www.sciencebuddies.org/science-fair-projects/science-projects/experiment-with-the-ph-
scale

Ota A. Properties of catalase activity in vegetative and sporulating cells of yeast


Saccharomyces cerevisiae. J Cell Biochem. 1986;30(4):331-9. doi:
10.1002/jcb.240300406. PMID: 3519626.

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