Biology
Mr. Kreuzberg
Kastan Day
1/22/13
Enzyme Lab Report
Question:
This investigation is designed to answer the question of: What factors affect enzyme
activity?
Research:
The two major materials used in this lab, other than pH buffer, are hydrogen peroxide
(H2O2) and catalase. Hydrogen peroxide is a strong oxidizer and oftentimes used as a cleaning
agent. Hydrogen peroxide is a colorless, and odorless solution when diluted. Catalase as used in
the lab is extracted from potatoes, in a concentration of 60%. Catalase is a catalyst, specifically
and enzyme and speeds up reactions in organic organisms. It is important to note that catalysts
cannot start reactions that would otherwise not be happening, they only speed up reactions (up to
1000 times faster or more) that would already be taking place. However the effectiveness of
catalase is strongly affected by three major factors.
Temperature, the pH of the solution and the concentration of the catalase all play a key
role in the functionality of the catalase. When dealing with catalase, there is an optimum
temperature range in which catalase functions the best. If catalase gets too cold it is not able to
“meet” the substrates because intermolecular movement is low. If it gets too hot then its form
will be altered and resulting its function will change also. Additionally, if the solution becomes
too acidic or too basic then this will also prevent the catalase from functioning properly. Lastly,
if the concentration of the catalase is higher there is a greater chance of catalase molecules
meeting substrates that they can bind with.
Hypothesis:
If the catalase splits hydrogen and oxygen fastest around 36˚C, at a pH of around 7 and in
greater concentrations then catalase is in an environment where it functions best because it splits
hydrogen and oxygen fastest.
Materials:
Variable: pH - Kastan Day, Chi Kyu Lee, Morgan Himmer.
1. Graduated cylinders (25mL)
2. Test tubes (3)
3. Pressure sensor(s)
4. Stopper(s)
5. Hydrogen peroxide (3% solution)
6. Buffer solutions: pH 4.00, pH 7.00, pH 10.00
7. Catalase (extracted from potatoes, 60% solution)
8. Pipets (4 or more, scaled 3mL)
9. Logger pro program (or equivalent)
10. Eye protection
Variable: Temperature – Peter Cleveland, Brian Yu
1. Graduated cylinders (25mL)
2. Test tubes (4)
3. Hydrogen peroxide (3% solution)
4. Buffer solutions: pH 7.00
5. Filter paper
6. Stop watch
7. Heating device (hot plate)
8. Cooling device (Snow)
9. Thermometer
Variable: Concentration - Paul Kim, Leo Tejavibulya, Liam Hassett
Materials:
1. Graduated cylinders (25mL)
2. Test tubes (4)
3. Hydrogen peroxide (3% solution)
4. Buffer solutions: pH 4.00, pH 7.00, pH 10.00
5. Catalase (extracted from potatoes, 60% solution)
6. Pipets (4 or more, scaled 3mL)
7. Ruler (mm)
Procedure:
Variable: pH
Experiment facilitated by: Kastan Day, Chi Kyu Lee, Morgan Himmer.
This picture shows a test tube that is connected to a Logger Pro pressure sensor and collecting
data. The computer in the background shows the change in pressure (measured in kPa) over time
(sec). Keep in mind that in the picture everything is “flipped” and this graph is truly going “up
and to the right”.
First (1st) pH Trial:
1. Add 20 mL of pH 4 buffer in test tube A.
2. Add 15 mL of H2O2 in test tube A.
3. Add 10 drops of catalase in test tube A.
4. Quickly(!) attach pressure gauge on test tube A.
5. Record results for at least 5 minuets.
6. Add 20 mL of pH 7 in test tube B.
7. Add 15 mL of H2O2 in test tube B.
8. Add 10 drops of catalase in test tube B.
9. Quickly(!) attach pressure gauge on test tube B.
10. Record results for at least 5 minuets.
11. Add 20 mL of pH 4 in test tube C.
12. Add 15 mL of H2O2 in test tube C.
13. Add 10 drops of catalase in test tube C.
14. Quickly(!) attach pressure gauge on test tube C.
15. Record results for at least 5 minuets.
Second (2nd) pH Trial:
1. Add 5 mL of pH 4 in test tube A.
2. Add 10 mL of H2O2 in test tube A.
3. Add 10 mL of catalase in test tube A.
4. Quickly(!) attach the pressure gauge on test tube A.
5. Record results for at least 5 minuets.
6. Repeat steps 1-5 using pH 7 buffer in test tube B and pH 10 buffer in test tube C.
Variable: Temperature – Peter Cleveland, Brian Yu
1.
Put 10 mL of hydrogen peroxide in 3 test tubes
2.
Put up test tubes with different temperatures - One test tube has ice water, on with room
temperature and one with hot water
3.
Soak a piece of filter paper in catalase using tweezers
4.
Put the soaked filter paper at the bottom of the test tube.
5.
Time how long it takes for the filter to reach the top
6.
Repeat this procedure for different temperatures
Variable: Concentration - Paul Kim, Leo Tejavibulya, Liam Hassett
1. Label the three test tubes corresponding to its amount of catalase solution.
2. Pour 10 mL of Hydrogen Peroxide into 3 different test tubes.
3. Add 10 drops of catalase solution to the test tube that is labeled as 10 drops.
4. Add 20 drops of catalase solution to the test tube that is labeled as 20 drops.
5. Add 30 drops of catalase solution to the test tube that is labeled as 30 drops.
6. Observe any reactions that take place.
7. Measure the height of the bubbles for each mixture and record the data.
Data:
Variable: pH
Experiment facilitated by: Kastan Day, Chi Kyu Lee, Morgan Himmer.
Trial 1:
Catalase: 10 drops
Buffer: 20ml
H2O2: 15ml
1st Trial pH 4
pH 7
pH 10
Initial
99.02 kPa 97.38 kPa 98.32 kPa
Final
99.56 kPa 98.02 kPa 98.57 kPa
100
99.5
99
98.5
pH 4
98
pH 7
97.5
pH 10
97
96.5
96
Initial
Final
Above is pH4 test 1.
Above is pH7 test 1. There was measureable increase in this graph.
Above is pH10 test 1.
Trial 2:
Catalase: 10 ml
Buffer: 5ml
H2O2: 10ml
1st Trial pH 4
pH 7
pH 10
Initial
10.51 kPa 100.37 kPa 99.72 kPa
Final
102.1 kPa 105.07 kPa 100.70 kPa
106
104
102
pH 4
100
pH 7
pH 10
98
96
Initial
Final
Above is pH4 Test 2.
This is a chart form the 2nd trial of pH 7.
Variable: Temperature - Peter Cleveland, Brian Yu, Will Appleton
Temperature (oC)
Amount of Peroxide (mL)
2
10
30
10
30
7
36
10
80
7
Rising time (seconds)
Did not rise
pH
7
42
10
40
7
90
80
70
60
50
temp ˚c
40
rising time (sec)
30
20
10
0
1
2
3
4
The rising time at 2˚C is infinite (because it did not rise), however for the sake of this graph I
inserted the integer of 0 in place of infinity.
Variable: Concentration - Paul Kim, Leo Tejavibulya, Liam Hassett
Amount of catalase
solution (mL)
Height of bubbles
(mm)
10
1
20
3
30
4
35
30
amount of
catalase
solution
(mL)
25
20
15
hight of
bubbles
(mm)
10
5
0
1
2
3
Analysis:
Variable: pH - Kastan Day, Chi Kyu Lee, Morgan Himmer.
When testing if pH had an effect of the effectiveness of catalase, this lab ran two different
trials. The first trial had much more buffer and not enough catalase or peroxide. In the second
trial this lab increased the peroxide and catalase while lowering the buffer. It was hypothesized
that this would yield results that were more drastic and therefore more measureable. The
hypothesis proved correct when the proportion between the different pH levels remained
relatively constant however the results increased dramatically.
Focusing on the second trial, because the results are more easily measureable, pH7
experienced the greatest change in pressure over 400 seconds. The slope of the line representing
pH7 is 0.008955 kPa/s. This is comparable to pH 4’s slope that is 0.001418 kPa/s and pH10’s
slope that is 0.00245 kPa/s. The change in pressure over 400 seconds also shows the major
difference in pressure that pH7 experienced. The difference in these slopes as well as the change
over 400 sec shows that pH7 offers a much more hospitable environment to catalase then either
pH4 or pH10.
Trial 1
pH4
pH7
pH10
Change over +0.54 +0.64 +0.25
400 sec
Trial 2
pH4
pH7
pH10
Change over +0.59 +4.70 +0.98
400 sec
Variable: Temperature - Peter Cleveland, Brian Yu, Will Appleton
When dealing with temperature, there are many different factors. If the substance is too
cold, then the molecules will not move around enough to come in contact with each other. Also
because less molecules are moving less will bind with catalase, and the reactions would be very
slow. However if the substance becomes too hot, the extreme heat can alter the form of catalase
and therefore alter its function making it an ineffective enzyme. The data that this lab acquired
shows that catalase functions best at 30˚C. If the temperature was much colder or hotter the
enzyme would either not interact enough to be effective, of so hot that its for is altered.
However a typical human’s regular body temperature is 36.5˚C, and catalase is used in
the human body as an enzyme. The results for the temperature portion of this lab are skewed
because not all of the trials were done at the same time. The 2˚C, 30˚C and 42˚C trials were all
run consecutively however the 36˚C trial was conducted much later. The H2O2 was left out to
react with the air, thus causing it to split into hydrogen and oxygen and compromising the
consistency of the lab. The H2O2 is no longer purely H2O2, now it is partly H2O2 with some
hydrogen atoms and some oxygen atoms thus causing the catalase to become less affective
because there is a lower concentration of H2O2 that can be split. The trial with a temperature of
36˚C should have had the shortest rising time because that is the optimal temperature for
catalase. The temperature results are skewed because of inconsistencies in the testing
environment.
Variable: Concentration - Paul Kim, Leo Tejavibulya, Liam Hassett
The amount of catalase solution is in direct correspondence to the height of the bubbles.
This relationship can be show with the averaged ratio of 0.127778 mm of bubbles per mL of
catalase solution. This is a logical statement because the more catalase there is the higher the
probability that it will meet substrates that it can bind to. Below is a graph showing the
relationship between substrate concentration and the rate of the reaction. This graph similarly
demonstrates the idea that the higher the likelihood that substrates and catalysts “meet” the
higher the reaction rate will be. However this only holds true to the point where there is enough
substrate, or enough of the catalyst that they are constantly able to meet. This is why the graph
sharply increases at first, but slowly peters off as the substrates (or catalyst) increases.
(Graph used from source #1)
Conclusion:
The initially hypothesis was had three major parts and they will be analyzed individually.
It was hypothesized that catalase would function best at a pH of 7 and this was confirmed by
experimentation. It was also hypothesized that the reaction rate would be the highest when the
concentration of the catalase is highest. The portion of experimentation regarding concentration
confirmed this hypothesis. It was hypothesized that the reaction rate would be fastest around
36˚C. This was not confirmed nor denied by experimentation because the temperature portion of
experimentation, specifically the 36˚C test, was skewed by poor lab practices.
Expansion:
Catalase, as is used by humans, is optimized for conditions that are in the human body,
thriving at a pH of 7 and at a temperature of 36˚C, however there are many different enzymes
that are used by living things. Enzymes that are used by evergreen trees must be able to survive
drastic changes in temperature. Evergreen trees survive in both sweltering summers and frigid
winters. Do these evergreen trees manage to keep a consistent internal temperature or do they
have highly “flexible” enzymes? As a third option, do evergreen trees have multiple enzymes
that are used during different temperatures? Evergreen could have enzymes that thrive in hot
climates, temperate ones and in very cold climates. Evergreen trees have to survive through
many different temperatures and climates, how do their enzymes manage the constant shift?
Bibliography:
Source #1
"Saturation Curve of Enzyme Reactions." Wikipedia. Wikimedia Foundation, 11 Sept. 2011.
Web. 23 Jan. 2013.