INTRODUCTION Living organisms need enzymes for everyday use. (Hershey 2009). They use enzymes for eating, breaking down substances, or even with products we use daily, like carpet and kitchen cleaners. Enzymes are catalytic proteins that basically speed up chemical reactions without being used up or even permanently altered during the process. Although various enzymes use different methods, they all accomplish catalysis by lowering the free energy of activation energy for the reaction, allowing it to occur more easily. They break down molecules called substrates, which binds and reacts with the active site, the location of where catalysis actually occurs. They affect the rate of almost all chemical reactions that take place in living organisms. The shape of an enzyme also plays an important role in catalysis. Once the substrate enters the active site, it will begin a process known as induced fit, where the enzyme perfectly conforms to the molecule for a more efficient catalysis. Many enzymes have specific environmental conditions where they react the fastest at (Pack 2007). As a result, changes in conditions can severely impact enzyme catalysis in both negative and positive ways. Each enzyme has specific ranges at which it optimally functions. In general, increasing the temperature will help the reaction to a point where the protein degrades and denatures. Denaturation is the loss of its structure and when ionic and hydrogen bonds break. Salty environments, acidic environments, and alkaline environments all break ionic and hydrogen bonds (Booker 2008). This interferes with their electric charges. Heat causes movement within the molecules. This disturbs their relatively weak bonds. The pH of a solution can also affect the charge of acidic and basic amino acid side chains on a protein, affecting the interactions that lead to tertiary and quaternary protein structure. Once denatured, most proteins will not reform their original shape. Other proteins, like enzymes, will react faster near optimum temperature (Brooker, 2008). Two good examples are how the optimal temperature for human enzymes is 40 degrees Celsius, whereas the optimum temperature for enzymes from prokaryotes located in hot springs is 70 degrees Celsius.
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Enzyme concentration and substrate concentration also play a role in catalysis (Campbell 2005). The more enzymes available, the quicker the reaction will occur until the substrate is all used up. More substrates will also mean a faster reaction rate until the enzyme is fully saturated so that it cannot continue increasing its activity. Inhibitors interact with an enzyme so that its activity is altered. If a molecule decreases or stops the rate of the reaction, it is an inhibitor. These substances regulate how fast an enzyme acts to something foreign. Inhibitors work by unfolding or destabilizing bonds that denature the enzyme. Some inhibitors can block or even change the shape of the active site. The purpose of this lab is to determine how the rate of enzyme activity changes due to several different environmental conditions and how long the reaction will take. This lab focuses on one particular enzyme, catalase. Its purpose is to prevent buildup of hydrogen peroxide, a waste product of cellular activity, by deconstructing it down into two water molecules and oxygen gas.
2H2O2 + catalase
→ 2H O + O + catalase 2
2
Most enzymes are adapted to the environments that they react in. For example, pepsin is found in the stomach and is suitable to acidic pH levels. However, some enzymes have optimal levels that are sustainable for them. In order to find out we will put catalase in specific environments that will certainly affect its normal reaction rate. We will be using pH, temperature, inhibitors, enzyme concentration, and substrate concentration to see what results will come due to different quantities of each of the variables that will be used. Using different variables on catalase will inform students how enzymes work in unstable environments exposed to different substances that will denature the enzyme, speed the reaction rate, or decelerate the reaction. My hypothesis for this lab is that the when catalase concentration has been increased, the reaction rate will increase at an exponential growth, because the substrates will produce no significant change in Page 2
the reaction rate. I predict that the catalase process thousands of substrate molecules per second, so the rate will keep going up. If there was more substrate concentration in the beaker, then the reaction rate will increase to a certain point, until there is a level of carrying capacity for the active site of the catalase. I predict that so many substrates can collide with the active site so many times that the concentration increases, but the rate starts to steady off. As for temperature, I predict that there will be an optimal point for the catalase to react better than the other certain temperatures that we will use in the lab. The other temperatures will most likely break down, or denature the catalase. For the inhibitor concentration I think the reaction rate will be faster than the substrate, because it will irreversibly alter the tertiary structure of the catalase by breaking its disulphide bonds, or covalent bonds. For the pH levels, I predict that there be an optimal level that will speed up the reaction rate, just like for temperatures. The acid would mostly likely denature catalase, the neutral level would probably be the optimal point, and the base will slow the reaction down. MATERIALS Filter Paper Discs Test tube containing catalase concentration from potatoes Chest of ice 3% Hydrogen Peroxide 1% Hydrogen Peroxide Acidic Solution (Hydrochloric Acid) Basic Solution (Sodium Hydroxide) Numerous amount of 40-milliliter beakers (possibly 24) Tap Water Hole puncher Large glass pipette Pipette filler Page 3
Tweezers 8 test tubes Stop watch Measuring cylinder 10% Copper II Sulfate 5 water baths Distilled Water Paper Towels Tape METHODS General Procedure 1. Take catalase solution out of ice chest. 2. Make sure you have the catalase solution and substrate solution. 3. Pour some the substrate solution into a beaker. 4. Using the hole puncher, punch out a few small paper discs from the filter paper. 5. Using the tweezers, pick up a disc and keep it in the catalase solution for 5 seconds. 6. Remove the disc and let it drain on the paper towel for 10 seconds. 7. Place the disc at the bottom of a substrate solution. Watch as the disc floats up to the surface. This is because the oxygen made from the deconstruction of hydrogen peroxide gets trapped in the fibers of the disc, and it soon floats up. 8. Use the stop watch to measure the time it took when the disc was place at the bottom of the solution and floated to the surface. 9. Don’t forget to repeat the trials twice to calculate the average time for them. 10. Once done, make sure to put catalase solution back into ice chest.
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Part 1 – Effect of Enzyme Concentration 1. Get eight beakers with 50 milliliters and label them with tape: a. 100 units/mL b. 80 units/mL c. 75 units/mL d. 60 units/mL e. 50 units/mL f.
25 units/mL
g. 10 units/mL h. 0 units/mL 2. For each beaker put the amount of catalase solution and distilled water into each of the beakers. Make sure to make use large glass pipette and pipette filler to get precise measurements a. 100 units/mL: put 40 mL of enzyme into the beaker. There should not be any distilled water. b. 80 units/mL: put 32 mL of enzyme and 8 mL of distilled water into beaker c. 75 units/mL: put 30 mL of enzyme and 10 mL of distilled water into beaker d. 60 units/mL: put 24 mL of enzyme and 16 mL of distilled water into beaker e. 50 units/mL: put 20 mL of enzyme and 20 mL of distilled water into beaker f.
25 units/mL: put 10 mL of enzyme and 30 mL of distilled water into beaker
g. 10 units/mL: put 4 mL of enzyme and 36 mL of distilled water into beaker h. 0 units/mL: put 40 mL of distilled water into the beaker. There should not be any enzyme. 3. Start an identical set of beakers for the substrate solution. However, this time you must measure out 40 mL of 1% hydrogen peroxide solution. 4. Repeat the general procedure for each of the enzyme concentration and average the time results.
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Part 2 – Effect of Substrate Concentration 1. Get nine 40 mL beakers and label them with tape from 1 to 9 2. For each beaker put the amount of 3% hydrogen peroxide solution and distilled water into each of the beakers. Make sure to make use large glass pipette and pipette filler to get precise measurements a. Beaker 1: put 40 mL of distilled water into the beaker. There should not be any hydrogen peroxide. b. Beaker 2: put 1.3 mL of hydrogen peroxide and 38.7 mL of distilled water into beaker c. Beaker 3: put 2.7 mL of hydrogen peroxide and 37.3 mL of distilled water into beaker d. Beaker 4: put 4.0 mL of hydrogen peroxide and 36.0 mL of distilled water into beaker e. Beaker 5: put 6.7 mL of hydrogen peroxide and 33.3 mL of distilled water into beaker f.
Beaker 6: put 10.7 mL of hydrogen peroxide and 29.3 mL of distilled water into beaker
g. Beaker 7: put 13.3 mL of hydrogen peroxide and 26.7 mL of distilled water into beaker h. Beaker 8: put 26.7 mL of hydrogen peroxide and 13.3 mL of distilled water into beaker i.
Beaker 9: put 40 mL of hydrogen peroxide into beaker. There should not be any distilled water.
3. Repeat the procedure using filter paper discs. Always keep the enzyme solution in ice chest when it not in use. 4. Repeat the general procedure for each substrate concentration levels. Average the amount of time Part 3 – Reaction Rate with Enzymes and Inhibitors 1. Put 5 mL of catalase solution into a beaker 2. Add 25 drops of 10% Copper II Sulfate to 5 mL of catalase solution and let it stand for 60 seconds. 3. Prepare a control for this part, using catalase solution and 1% hydrogen peroxide solution
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4. Add 40 mL of hydrogen peroxide into another beaker. 5. Using the general procedure, measure the amount of time for each of the solutions. Part 4 – Effect of Temperature on Reaction Rate 1. Get out 5 test tubes and place 40 mL of enzyme solution into each of them 2. Put 40 mL of 1% hydrogen peroxide solution into 5 beakers 3. The five water baths should each be of different temperatures. Label each of the test tubes and put them in the classified temperatures: a. 0° Celsius b. 10° Celsius c. 22° Celsius d. 40° Celsius e. 65° Celsius 4. Use the general procedure and measure the amount of time for each of the solutions and reaction activity. Part 5 – Effect of pH on Reaction Rate 1. Put 40 mL of 1% hydrogen peroxide into 3 beakers 2. Get out 20 mL of the hydrochloric acid (acidic solution), tap water (neutral solution), and sodium hydroxide (basic solution) and put them into three individual test tubes. 3. Mix the pH solutions with 20 mL of enzyme solution in each of the 3 test tubes and wait for 60 seconds. 5. Use the general procedure and measure the amount of time for each of the solutions and reaction activity.
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RESULTS Graph 1
Time of Reaction in Seconds
Effect of Enzyme Concentration Series 1 90 80 70 60 50 40 30 20 10 0 40
32
30
24
20
10
4
0
Amount of Enzyme Concentration in Mililiters
This graph shows the results for the first trial of the enzyme experiment, or part 1.
Time of Reaction in Seconds
Graph 2
Effect of Enzyme Concentration Series 2
100 90 80 70 60 50 40 30 20 10 0 40
32
30 24 20 10 Amount of Enzyme Concentration in Milliliters
This graph shows the results for the second trial of the enzyme experiment, or part 1. Page 8
4
0
Graph 3
Time of Reaction in Seconds
Effect of Enzyme Concentration 100 80 60
Series 1
40
Series 2
20
Average
0 40
32
30
24
20
10
4
0
Amount of Enzyme Concentration in Milliliters
Graph 3 shows the averages and trials for the enzyme experiment for part 1. Notice how when there are no enzymes in the beaker and all distilled water, there is no reaction or reaction time. Table 1 – Effect of Enzyme Concentration on Enzyme Activity
Enzyme Concentration (units/mL)
Time to Float Disc (in seconds) Trial 1
Trial 2
Average
Class Average
100
18.4
15.5
16.95
11.326
80
14.2
15.8
15.0
12.122
75
14.7
17.0
15.85
12.568
60
17.9
16.6
17.25
15.555
50
20.2
19.3
19.75
20.14
25
31.2
31.8
31.5
34.20368056
10
82.2
88.8
85.5
109.965
0
Did Not Rise
Did Not Rise
None
None
This table shows the trials, averages, and class averages for the enzyme experiment for part 1.
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Graph 4
Effect of Substrate Concentration Series 1 Time of Reaction in Seconds
80 70 60 50 40 30 20 10 0 0
1.3
2.7
4
6.7
10.7
13.3
26.7
40
Amount of Substrate Concentration in Milliliters
This graph shows the results for the first trial of the substrate experiment, or part 2. Graph 5
Effect of Substrate Concentration Series 2 Time of Reaction in Seconds
80 70 60 50 40 30 20 10 0 0
1.3
2.7
4
6.7
10.7
13.3
26.7
Amount of Substrate Concentration in Milliliters
This graph shows the results for the second trial of the substrate experiment, or part 2.
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40
Graph 6
Time of Reaction in Seconds
Effect of Substrate Concentration 80 70 60 50 40 30 20 10 0
Series 1 Series 2 Average 0
1.3
2.7
4
6.7
10.7
13.3
26.7
40
Amount of Substrate Concentration in Milliliters
Graph 6 shows the averages and trials for the substrate experiment for part 2. Notice how when there are no substrates in the beaker with 40 mL of distilled water, there was no reaction time. Table 2 – Effect of Substrate Concentration on Enzyme Activity
Substrate Concentration %
Time to Float Disc (in seconds) Trial 1
Trial 2
Average
Class Average
0%
Did not rise
Did not rise
None
None
0.1%
67.8
72
69.9
101.635
0.2%
66
69
67.5
67.37875
0.3%
36.2
36.9
36.55
49.76375
0.5%
29.3
28.1
28.7
26.86
0.8%
25.4
26.1
25.75
22.083
1.0%
21.2
21.3
21.25
15.994
2.0%
11.9
11.1
11.5
10.197
3.0%
6.3
10.5
8.4
8.585
Table 2 shows the trials, averages, and class averages for the substrate experiment for part 2. Page 11
Graph 7
Time of Reaction in Seconds
Effect of Temperature Series 1 10 9 8 7 6 5 4 3 2 1 0 0°
10°
22°
40°
65°
Amount of Temperature in Celsius
This graph shows the results for the first trial of the temperature experiment, or part 4.
Graph 8
Effect of Temperature Series 2 Time of Reaction in Seconds
12 10 8 6 4 2 0 0°
10°
22°
40°
65°
Amount of Temperature in Celsius
Graph 8 shows the results for the second trial of the temperature experiment, or part 4. Page 12
Graph 9
Effect of Temperature Time of Reaction in Seconds
12 10 8 Series 1
6
Series 2
4
Average 2 0 0°
10°
22°
40°
65°
Amount of Temperature in Celsius
This graph shows the averages and trials for the temperature experiment for part 4. Notice that the filter paper did not rise at 65 degrees Celsius. Table 3 – Effect of Temperature on Enzyme Activity
Time to Float Disc (in seconds) Temperature (degrees C°)
Trial 1
Trial 2
Average
Class Average
0° C
3
5.6
4.3
4.161
10° C
4.3
4.7
4.5
4.456
22° C
7.9
6.6
7.25
4.803
40° C
8.6
10.5
9.55
10.417
65° C
Did Not Rise
Did Not Rise
None
271.125
This table shows the results and averages from the experiments for part 4. Page 13
Table 4 – Effect of an Inhibitor on Enzyme Activity
Name of Beaker
Time to Float Disc (in seconds) Trial 1 8.3
Trial 2 19
Average 13.65
Class Average 8.793
Did Not Rise
Did Not Rise
None
None
Control Copper II Sulfate
Table 4 shows the results and averages from the experiments for part 3.
Graph 10
Effect of Inhibitors 20 Time of Reaction in Seconds
18 16 14 12 10
Series 1
8
Series 2
6
Average
4 2 0 Control Beaker
Inhibitor Beaker Name of Beaker
This graph shows the results and averages for part 3, when the catalase was with the inhibitor, copper II sulfate. Notice that there are no results, or bars in the graph, for the inhibitor beaker. This means that the filter paper did not rise.
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Graph 11
Effect of pH Series 1 Time of Reaction in Seconds
60 50 40 30 20 10 0 Acid
Neutral
Base
pH Level
Graph 11 shows the results for the first trial of the pH experiment, or part 5.
Graph 12
Effect of pH Series 2 Time of Reaction in Seconds
18 16 14 12 10 8 6 4 2 0 Acid
Neutral
Base
pH Level
Graph 12 shows the results for the second trial of the pH experiment, or part 5. Page 15
Graph 13
Effect of pH on Reaction Rate Time of Reaction in Seconds
60 50 40 Series 1
30
Series 2
20
Average
10 0 Acid
Neutral
Base
pH Level
This graph shows the averages and trials for the pH experiment for part 5.
Table 5 – Effect of pH on Enzyme Activity
pH Level Time to Float Disc (in seconds) (from serial dilutions) Trial 1 Trial 2
Average
Class Average
Acid
55.0
Did Not Rise
27.5
132.75
Neutral
11.0
11.6
11.3
246.125
Base
14.1
16.8
15.45
13.3725
This table shows the trials, averages, and class averages for the pH experiment for part 5.
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Figure 1
Figure 1 shows the ideal results and graphs for the temperature (a), pH (b), and substrate concentration (c) labs. Figure 2
This figure shows the ideal results for the enzyme concentration experiment.
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Figure 3
Figure 3 shows the ideal results for the effect of inhibitors on enzymes. Because copper sulfate was an inhibitor, the enzyme catalase would have been more different than normal catalase.
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DISCUSSION In the first part of this experiment, the degradation of hydrogen peroxide was observed by using different types of catalase solution. When there was a high concentration of catalase, the filter paper disc rose up in a short amount of time. The lowest time for the high enzyme concentration was averaged out to around 17 seconds, and the as the concentration got smaller for the other beakers, the time kept rising. The beaker with some enzymes was averaged out to approximately 85 seconds. My hypothesis was supported, because enzymes, especially catalase, can break down substrates when there is a large number of them. When there is a little amount of enzymes, the reaction time increases, because there is not enough enzymes to make the reaction go faster. In the second part of the experiment, we observed how enzymes are affected when there were different types of substrate solution. Results showed that the time of reaction increased when there was a much smaller concentration of substrates to the point where the enzymes could not react with all of them. This was shown in the beaker that had 4 milliliters of hydrogen peroxide and 36 milliliters of distilled water. The reaction time was over 36 seconds, and the times for the others soon decreased when the substrate concentration increased to 40 milliliters. My hypothesis was supported, because when you increase the amount of substrate concentration, the reaction time decreases, which causes the enzymes to react even faster than smaller concentration of substrates. During the third part, the enzymes were faced with an inhibitor, the copper sulfate. Unlike parts one and two, you had to use only two beakers: the control beaker and the inhibitor beaker. The control beaker averaged out to 9 seconds, whereas the in the inhibitor beaker, the paper disc did not even rise. My hypothesis was supported, because the inhibitor blocked the active site of the enzymes, making the filter paper to stay in the solution. When the active site gets blocked or distorted, the reaction stops. For the fourth part of this experiment, we used different temperatures to see the effects it has on enzymes. Of all of the five temperatures we used, the catalase had the lowest reaction time at 0 degrees Celsius, at an average time of 4.3 seconds, and the longest reaction time was 40 degrees Celsius, with an average time of 9.55 seconds. My hypothesis supported the data I got, because all enzymes will have an optimum temperature where it
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will react the greatest on. For this experiment, catalase did better at 0 degrees Celsius because since we had to keep catalase in an ice chest, it is probably one of those enzymes that have a lower optimum temperature than most enzymes. In the fifth and final part of this experiment, we used different pH levels on the enzymes to see what the effect was. The results for this part varied, with the acidic solution with the enzymes having around 55 seconds for the first series and not rising at all for the second series. When we put the enzyme in the basic solution, the solution became somewhat cloudy and the paper disc changed its appearance to a brownish color. It was very interesting to notice, since most of the other paper disc did not do anything like this in the other parts of the experiment. However, of all of the experiments, this one really puzzled me a bit, because the reaction times for the levels varied so much. My hypothesis was correct, but I really couldn’t find out what the optimal pH level is for catalase. It would probably be somewhere between neutral and basic, since they both had the lowest reaction times during the first series. Overall there were not a lot of errors in this experiment since it was basically straight forward and to the point. The instructions were very detailed, and you basically knew what to do for each part of the experiment. However, one major source of error could have been if the potatoes were used or in extreme environments. Since the catalase that we used in this experiment was from the blending potatoes in order to extract it, we really don’t know where the potatoes are from or anything. We were given a large beaker of enzyme solution, and whenever we needed it, we just put some more in the beaker we used. The potatoes could have been in hot temperatures or even exposed to chemicals from soap when washing them that would denature the catalase and results we got when the paper discs did not float to the surface could have been inaccurate. Thought I bet that is very impossible, but that could have been a major source of error that would definitely cost some of our results. I really did not see any sources of error in this lab, because we really did not have any electronic devices that showed us inaccurate data, like the LabQuest in the first lab or even trying to wait for a couple of days to get results down. The lab was very quick and really easy to do. However, I do have some suggestions for the lab. We could have used different types of
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enzymes to compare and contrast which one had a better reaction rate and faster time. Another suggestion is that we could have done more for the third part. Instead of having just a control and an inhibitor beaker, there could have been solutions where you mixed the substrate and inhibitors to see how it affects the enzyme. There could have also been more temperatures for the fourth part. We could have used temperatures less than 0 degrees Celsius and see if the enzymes reacted faster than the other temperatures we used. CONCLUSION In conclusion, enzymes exposed to different changes in their environment can lead to both positive effects and negative effects. In a positive way, enzymes can speed up the reaction. However, in a negative way, they can also get denatured, making no reaction at all. My hypothesis has supported most of the data I collected. Though some of the results I got was varied a bit too much. For example, with most enzymes, there is usually an optimum pH where the reaction time will be the fastest at. However, with my results, all of the pH levels varied, meaning that the neutral level had the fastest time, and then in the next series, the base had the fastest time. Even though there were some errors in the data, my overall lab experience was pretty good. The instructions were self-explanatory, and the experiment was very interesting. The lab helped me to think about how enzymes are extremely important, especially when in cells and in the body. They break up substrates so there is no buildup from waste, just like how the enzyme, catalase, breaks down hydrogen peroxide into water and oxygen, two substances that are fairly harmless to a cell. This lab was very simple to understand, and most of the results I got would be something anyone can get. There are some things that could have been better in this lab, but the learning about enzymes and the effects it can be exposed to in this lab was essential to the unit that we were in.
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REFERENCES
1. Anderson, Paul. "AP Bio Lab 2 - Enzyme Catalysis." Bozemanscience. Web. 27 Oct. 2014. . 2. Brooker, Robert J, et al. Biology. New York: McGraw-Hill, 2008. 3. Campbell, Neil A. 2005. Biology/Neil A. Campbell, Jane B. Reece. 7th edition. San Francisco, CA: Pearson Education, Inc. p. 80-5, 150-7. 4. Goldberg, Deborah T. 2007. Barron’s AP Biology. 2nd edition. New York, NY: Barron’s Educational Series, Inc. p 409. 5. Hershey, James, et al. Biology 110 Laboratory Textbook. New York: Pearson, 2009. 6. "Enzymes." LabBench. Pearson Education. Web. 27 Oct. 2014. . 7. Lavríková, Petra. "Enzymes." Functions of Cells and Human Body. Charles University in Prague, Web. 27 Oct. 2014. . 8. Pack, Phillip E. 2007. Cliffs AP Biology. 3rd edition. Hoboken, NJ: Wiley Publishing, Inc. p. 260. 9. Vesprey, Deryce. “Oh Biochem!!” WordPress. Web. 27 Oct. 2014. . 10. "Enzymes." Chemistry for Biologists. Royal Society of Chemistry. Web. 26 Oct. 2014. .
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