Sample Liver Enzyme Lab

Sample Liver Enzyme Lab
Design Aspect 1: Research Question
This lab will be driven by the research question, Do changes in temperature have an
effect on the activity of the enzyme catalase? Pearson Baccalaureate: Standard Level Biology
Developed Specifically for the IB Diploma defines enzymes as “protein molecules which act as
catalysts for reactions. As catalysts, the real function of enzymes is to lower the activation
energy of the reactions that they catalyze” (166). Enzymes are proteins; therefore the liver has a
particularly high concentration of catalase. When hydrogen peroxide (H2O2) is added to liver,
catalase catalyzes a reaction in which the hydrogen peroxide is broken down into oxygen gas
(O2) and liquid water (H2O). Hydrogen peroxide is a toxic chemical that is produced as a
byproduct of many normal cellular reactions, so it is crucial that catalase in the liver breaks the
hydrogen peroxide down into the two harmless substances of oxygen gas and liquid water. The
hydrogen peroxide must be quickly degraded or converted, and catalase accomplishes this task
because one molecule of catalase can deal with six million molecules of hydrogen peroxide in
one minute. Enzymes and the temperature of their environment are particularly important to the
human body because “Many of the reactions which represent the digestive process would need
far higher temperatures than we are able to maintain safely if enzymes were not involved” (166).
This lab will be using beef liver, which contains the specific enzyme catalase, and by
placing the liver into different temperatures it will be assessed how catalase performs under
certain conditions. After the liver has been placed in different temperatures, hydrogen peroxide
will be added to each piece of liver, and by measuring the height of the chemical reaction it will
be determined which temperatures catalase performs the best in. Reactions with a high height
will represent catalase quickly and efficiently breaking down the hydrogen peroxide, and
reactions with a low height will represent catalase slowly and inefficiently breaking down the
hydrogen peroxide. This lab will serve as a model for the role of enzymes in the human body,
and will outline the importance of enzymes for the human body.
Hypothesis
If liver is placed in different temperatures of 0˚C, 7˚C, 19˚C, 37˚C, and 100˚C and
hydrogen peroxide is added to each piece of liver, then the liver placed in 37˚C will have the
largest reaction height. The liver in 100˚C will have the smallest reaction height, followed by the
liver in 0˚C, then 7˚C, and then 19˚C.
This prediction is based on the concept of denaturation. As Pearson Baccalaureate:
Standard Level Biology Developed Specifically for the IB Diploma states, “Reactions which use
enzymes do have an upper limit. That limit is based on the temperature at which the enzyme (as
a protein) begins to lose its three-dimensional shape due to intramolecular bonds being stressed
and broken. When an enzyme loses it shape, including the shape of the active site, it is said to be
denatured” (75). Due to denaturation, the liver placed in 100˚C will have the smallest reaction
height because at this temperature catalase will begin to denature. Because “reactions with or
without enzymes will increase their reaction rate as temperature (and thus molecular motion)
increases”, the liver placed in 0˚ C, 7˚C, and 19˚C will have small reaction heights, but the
heights will increase as temperature increases (75). The liver placed in 37˚C will have the
greatest reaction height because “human catalase works at an optimum temperature of 37˚C,
which is approximately the temperature of the human body”. Although this lab is using beef
liver instead of a human liver, the optimum temperature for beef liver should be similar to that of
humans.
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+
Height (mm)
-
-
Temperature (˚C)
+
This predictive graph represents how as the temperature of beef liver originally increases,
the height of the reaction increases as well due to an increase in molecular collisions. At a
certain temperature, the catalase will reach its optimum temperature and have the greatest height
of reaction. However, as the temperature continues to increase the enzyme will begin to lose its
shape and denature, so the height of the reaction will decrease.
Independent Variables
The independent variable is the temperature of the liver, and it will be measured in ˚C.
The different temperatures used will be 0˚C, 7˚C, 19˚C, 37˚C, and 100˚C.
Dependent Variables
The dependent variable is the height of the reaction, and it will be measured in
millimeters.
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Control Variables
Variable
Size of
Liver
pH
Time
Effect
Could increase or decrease the height
of the reaction. A larger piece of liver
means more catalase, which could
break down the H2O2 at a more
efficient rate than smaller pieces of
liver.
pH has an effect on enzymes and each
enzyme has an optimal pH. By
making the liver more acidic, basic, or
neutral the height of the reaction could
increase or decrease as it deviates
from its optimal pH.
If some pieces of liver are kept in their
specific temperatures longer than
others, the height of the reaction could
be greatly affected. By keeping the
liver in its temperature for a shorter
period of time, the liver itself has less
time to change temperature which
could greatly alter the results.
Control
Cut every piece of liver the same size by
weighing each piece on an electric beam
balance. Each piece of liver is
approximately 1.4g.
Do not change the pH of any of the
solutions. This lab is only investigating
the effect of temperature on enzyme
activity, not the effect of pH on enzyme
activity, so nothing should be added to the
liver that would increase or decrease the
pH.
Keep all of the pieces of liver in their
specified temperatures for five minutes.
Design Aspect 2: Method
1. Prepare an ice bath by placing ice into a container, and place a thermometer into the ice
bath. Wait until the temperature has reached 0˚C.
2. Prepare a hot water bath by placing a 250mL beaker filled with water on a hot plate, and
place a thermometer into the water. Wait until the temperature has reached 100˚C.
3. Prepare a warm water bath by placing a 250mL beaker filled with water on a hot plate,
and place a thermometer into the water. Wait until the temperature has reached 37˚C.
4. Obtain beef liver and cut the liver into 10 slices which are approximately the same size.
Weigh each slice on an electronic beam balance to ensure they are the same size.
5. Obtain 10 test tubes and place them into a test tube rack. Label 5 test tubes with a
number from 1-5, and repeat for the remaining five test tubes.
6. Obtain 15mL of hydrogen peroxide and a graduated cylinder.
7. With tweezers, place a piece of liver into each of the test tubes.
8. After the liver has been placed in the test tubes labeled 3, place a thermometer into the
test tube rack and wait for 5 minutes. After 5 minutes, pour 2mL of hydrogen peroxide
into each of the test tubes, observe the reaction, and label the height of the reaction with a
Sharpie.
9. Remove both of the test tubes labeled 1 from the test tube rack, place them in the ice
bath, and wait for 5 minutes. After 5 minutes, remove the test tubes from the ice bath,
add 2mL of hydrogen peroxide into each of the test tubes, observe the reaction, and label
the height of the reaction.
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10. Remove both of the test tubes labeled 2 from the test tube rack, place them in a different
test tube rack and place this rack into the fridge. Place a thermometer into the fridge as
well. Wait for 5 minutes, and then read the temperature of the fridge and remove the test
tubes. Pour 2mL of hydrogen peroxide into each of the test tubes, observe the reaction,
and label the height of the reaction.
11. Remove both of the test tubes labeled 4 from the test tube rack, place them in the warm
water bath, and wait for 5 minutes. After 5 minutes, remove the test tubes from the warm
water bath, add 2mL of hydrogen peroxide into each of the test tubes, observe the
reaction, and label the height of the reaction.
12. Remove both of the test tubes labeled 5 from the test tube rack, place them in the hot
water bath, and wait for 5 minutes. After 5 minutes, remove the test tubes from the hot
water bath, add 2mL of hydrogen peroxide into each of the test tubes, observe the
reaction, and label the height of the reaction.
13. With a ruler, measure from the bottom of the test tube to the mark which labels the
maximum height of the reaction. Repeat this for all 10 test tubes, and record the
measurements in your data table.
14. Pour the liver from each test tube into a waste beaker, clean each of the test tubes out, and
put all materials away.
15. Combine the data that your own group obtained with the data from two other groups.
This will allow for six trials worth of data, and once you obtain this sufficient amount of
data, calculate the average height of the reaction for each of the five temperatures over
the six trials, and then calculate the standard deviation for each of the five temperatures
over the six trials as well.
Equipment Used:
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10 Test Tubes
1 Package of Beef Liver
2 250mL Beakers
2 Test Tube Racks
Tweezers
1 Hot Plate
Ice
1 Plastic Bin
Hydrogen Peroxide
1 100 mL Beaker
1 10mL Graduated Cylinder
5 Thermometers
1 Sharpie
1 Electronic Beam Balance
1 Knife
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Variable
Temperature
Height
Unit of Precision
˚C
mm
Error/Uncertainty
+/- 0.5˚C
+/- 0.5mm
In the procedure, the maximum height of the reaction will be determined by marking the
position of the tallest bubble that results from the reaction.
Design Aspect 3: Sufficiency of Data
This lab will be investigating the effect of temperature on the activity of the enzyme
catalase. In this lab, five different temperatures will be investigated (0˚C, 7˚C, 19˚C, 37˚C,
100˚C). Each temperature will have six trials, and this will ensure the reliability of the data.
With the sufficient data, the standard deviation and mean will be calculated for each temperature.
DPP Aspect 1: Results – Raw Data
The Effect of Temperature on Catalase Activity
Height of Reaction (mm +/- 0.5mm)
Trial Trial Trial Trial Trial Trial
Temperature (˚C +/- 0.5˚)
1
2
3
4
5
6
0.0
78.0 82.0 81.5 82.4 94.0 85.0
7.0
84.0 90.0 84.5 83.5 98.0 85.0
19.0
92.0 96.0 88.2 88.1 103.0 99.0
37.0
87.0 93.0 100.1 92.5 99.0 100.0
100.0
69.0 18.0 41.2 53.2 17.0 86.0
Note: The highlighted data is the data obtained from my group.
Qualitative Observations:
After adding the H2O2 to the liver in test tube #1, there were large bubbles, the liver
became a more dull red color, the bubbles rose fairly high, and the liquid in the test tube turned a
reddish color. After adding the H2O2 to the liver in test tube #2, there was a slower reaction,
small bubbles that were more like fizz, and the majority of the fizz was surrounding the liver.
After adding the H2O2 to the liver in test tube #3, the bubbles were smaller and more compact
than the previous two test tubes, there was a lot of fizz at the bottom near the liver, and the
reaction was very quick. After adding the H2O2 to the liver in test tube #4, there was a very fast
reaction, there was mostly fizz at the start of the reaction, but the fizz soon turned to bubbles, and
the liver turned a bright red color. After adding the H2O2 to the liver in test tube #5, there were
small bubbles that looked mostly like fizz, the reaction was very slow and not very tall, the liquid
turned a yellowish color, the liver shriveled up and turned a gray color, and the height of the
reaction rose slowly after the H2O2 was added, but the reaction had a very slow start.
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DPP Aspect 2: Processing Data
Temperature
(˚C +/- 0.5˚)
0.0
7.0
19.0
37.0
100.0
The Effect of Temperature on Catalase Activity
Height of Reaction (mm +/- 0.5mm)
Trial Trial
Trial
Trial
Trial
Trial Average Height of
1
2
3
4
5
6
Reaction (mm +/0.5mm)
78.0
82.0
81.5
82.4
94.0
85.0
83.8
84.0
90.0
84.5
83.5
98.0
85.0
87.5
92.0
96.0
88.2
88.1 103.0 99.0
94.4
87.0
93.0 100.1 92.5
99.0 100.0
95.3
69.0
18.0
41.2
53.2
17.0
86.0
47.4
Sample Calculations:
Average Height of Reaction =
=
=
Standard Deviation = Calculated on Excel
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= 83.8mm
Standard Deviation
(mm +/- 0.5mm)
5.5
5.7
6.0
5.3
27.6
DPP Aspect 3: Presentation of Processed Data
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The Effect of Temperature on Catalase Activity
Average Height of Reaction (mm +/- 0.5mm)
100
80
60
40
20
0
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
Temperature (˚C +/- 0.5˚)
CE Aspect 1: Conclusion
The results obtained from this lab support my hypothesis. My hypothesis was: If liver is
placed in different temperatures of 0˚C, 7˚C, 19˚C, 37˚C, and 100˚C and hydrogen peroxide is
added to each piece of liver, then the liver placed in 37˚C will have the largest reaction height.
By viewing the average height of the reaction, these results are supported. The liver placed in
37°C had an average reaction height of 95.3mm, the liver in 19°C had an average reaction height
of 94.4mm, the liver in 7°C had an average reaction height of 87.5mm, the liver in 0°C had an
average reaction height of 83.8mm, and the liver placed in 100°C had an average reaction height
of 47.4mm. This data coincides with the predictions stated in the hypothesis, where I stated
“The liver in 100˚C will have the smallest reaction height, followed by the liver in 0˚C, then 7˚C,
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120.0
and then 19˚C.” By observing the constructed graph, it is clear that the average height of the
reaction initially increased as the temperature increased, but when temperatures increased past
37°C, the average height of the reaction began to decrease, so the liver placed in 100°C has the
smallest average reaction height. The graph constructed from the obtained data was also very
similar to the predictive graph included in the hypothesis. These results outline the effect of
temperature on enzyme activity, and it is apparent that changes in temperature do have an effect
on the enzyme catalase. The results obtained give increased insight into the role of enzymes in
the human body. The beef liver closely modeled the human liver, and it is clear that catalase
works at an optimum temperature of 37°C. This is important because humans maintain a stable
body temperature of 37°C, and with the aid of enzymes this temperature provides enough
activation energy for metabolic reactions, in this case the breakdown of hydrogen peroxide into
oxygen gas and liquid water. The liver was placed in 100°C had the smallest average reaction
height because at this temperature the enzyme had denatured and could no longer effectively
breakdown the hydrogen peroxide. This was also apparent in the qualitative observations where
it was observed that the liver placed in 100°C “shriveled up and turned a grayish color”.
CE Aspect 2: Evaluation and Improvements
While the results obtained from this lab clearly support my hypothesis, my examining the
calculated standard deviation for each temperature it is clear that there was a large range of data
in this lab, and this range is due to errors that may have occurred throughout the lab. The liver
placed in 0˚C had a standard deviation of 5.5mm, the liver placed in 7˚C had a standard deviation
of 5.7mm, the liver placed in 19˚C had a standard deviation of 6.0mm, the liver placed in 37˚C
had a standard deviation of 5.3mm, and the liver placed in 100˚C had a standard deviation of
27.6mm. The standard deviations of the liver placed in 0°C, 7°C, 19°C, and 37°C all had similar
standard deviations, as seen by observing the error bars on the constructed graph. However, the
liver placed in 100°C had a very large standard deviation, and by observing the graph it is clear
that there is a large range in the class data. The larger the standard deviation, the less reliable the
data, so there are multiple errors that may have accounted for the large standard deviation for the
liver in 100°C. As noted in the qualitative observations, when the hydrogen peroxide was added
to the liver in 100°C the reaction was very slow to begin with, and the height was very small.
However, the reaction continued to grow as time passed, so one possible error is that some
groups may have not noticed this slow reaction growth. This could account for the small
measurements of 17.0mm and 18.0mm. Another error that occurred in this lab deals with the
timing of the procedure. Because each group simultaneously placed two test tubes in each
temperature, it is difficult to obtain an accurate measurement. Both test tubes were removed
from their temperatures at the same time, but only the hydrogen peroxide was added to one test
tube at a time. As my partner and I were conducting the experiment with one test tube, the other
test tube was unattended to and had the time to either cool off or warm up, and this affected the
reaction rate and the accuracy of the measurements. This error accounts for the different
measurements for each trial. Because some of the data varied so significantly from one group to
the next, there may have been an error with the liver itself. While each piece of liver was
approximately the same size, not all pieces were cut from the same section of the liver. It is
possible that each part of the liver varied slightly, affecting the reaction rate once the hydrogen
peroxide was added.
By improving the lab, some of the errors mentioned above can be avoided. One
improvement is to make the procedure more specific. The procedure should specify a length of
time for observing the reactions, such as five minutes. If each group observed the reaction rate
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for a full five minutes after adding hydrogen peroxide to the liver, they would not overlook the
reactions that occur at a slower rate. Another improvement to the procedure would be to stagger
the timing of the lab. It would be helpful if each group only worked with one test tube at a time
instead of two. This way, the unattended test tube would not have time to heat up or cool down,
allowing for more accuracy in the results. An additional improvement to the lab would be to
conduct the trials at different temperatures. Many of the temperatures used in this lab were
similar to each other (0°C and 7°C), and we would yield more interesting results if we added
additional temperatures. It would have been interesting to see how liver reacted in a temperature
between 37°C and 100°C.
References
Damon, Alan, Randy McGonegal, Patricia Tosto, and William Ward. Pearson Baccalaureate:
Standard Level Biology for the IB Diploma (Pearson International Baccalaureate
Diploma: International Editions). n/a: Imprint Unknown, 2008. Print.
“Effect of Catalase on Hydrogen Peroxide.”
http://www.sciencegeek.net/Biology/biopdfs/Lab_Catalase.pdf. (26 Jan. 2011).
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