Enzyme Catalysis Lab
**NOTICE: All Procedures are paraphrased, or directly quoted from the “Lab 2 Enzyme Catalysis” Worksheet
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Exercise 2A: Test of Catalase Activity
This exercise investigates the effects of temperature and the presence of living tissue on the enzymatic activity of breaking down H2O2.
Procedure:
1. To observe the reaction to be studied, transfer 10 mL of 1.5% (0.44 M) H2O2 into a 50-mL glass beaker and add 1 mL of the freshly made catalase solution. The bubbles coming from the reaction mixture are O2, which results from the breakdown of H2O2 by catalase. Be sure to keep the freshly made catalase solution on ice at all times.
2. To demonstrate the effect of boiling on enzymatic activity, transfer 5 mL of purified catalase extract to a test tube and place it in a boiling water bath for five minutes. Transfer 10 mL of 1.5% H2O2 into a 50-mL glass beaker and add 1 mL of the cooled, boiled catalase solution.
3. To demonstrate the presence of catalase in living tissue, cut 1 cm^3 of potato or liver, macerate it, and transfer it into a 50-mL glass beaker containing 10mL of 1.5% H2O2.
Analysis:
This experiment demonstrates how alterations in the environment, such as temperature, can effect an enzyme’s rate of reaction. Even though we did not physically perform this experiment, we can concur that putting an enzyme in any extreme environment (such as a temperature or pH that does not comply with that specific enzyme’s optimal condition needs), can cause the enzyme to refrain from working efficiently, unable to function, and ultimately denature it. From this experiment, we can also conclude that living tissues contain their own specific enzymes. If catalase is put in the presence of living tissue, this reaction may take place more quickly.
Exercise 2B: The Base Line Assay
This exercise assists us in determining the amount of H2O2 that is initially present in a 1.5% solution in order to have a base line to compare other values to.
Procedure:
1. Put 10 mL of 1.5% H2O2 into a clean cup.
2. Add 1 mL of H2O (instead of enzyme solution)
3. Add 10 mL of H2SO4 (1.0 M)
4. Mix well.
5. Remove a 5-mL sample. Place this 5-mL sample into another cup and assay for the amount of H2O2. Use a burette to add KMnO4, a drop at a time, to the solution until a persistent pink or brown color is obtained.
Analysis:
Establishing this baseline value is necessary to determine how much H2O2 is present in a 1.5% solution. By having this value, we are now able to calculate the efficiency of a catalase on the decomposition of H2O2 by measuring how much H2O2 is initially present before the introduction of an enzymatic solution.
Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition
The intent of this exercise was to observe the rate of decomposition in an uncatalyzed reaction, and to compare the results to the rate of decomposition of a catalyzed reaction.
Procedure:
1. Leave a small quantity of 1.5% H2O2 uncovered at room temperature for approximately 24 hours.
2. Add 1 mL of H2O (instead of enzyme solution)
3. Add 10 mL of H2SO4 (1.0 M)
4. Mix well.
5. Remove a 5-mL sample. Place this 5-mL sample into another cup and assay for the amount of H2O2. Use a burette to add KMnO4, a drop at a time, to the solution until a persistent pink or brown color is obtained.
6. Record your findings in the chart below.
Analysis:
From this exercise, we were able to observe that, when left out overnight and uncovered, the amount of H2O2 decomposed is about half of the baseline value. Since it was exposed to oxygen and light for a period of 24 hours, we can conclude that light causes H2O2 to spontaneously decompose, since most of the H2O2 was already decomposed spontaneously overnight before adding KMnO4. (This also explains why H2O2 is usually stored in a dark bottle in order to preserve the substance.) Since H2O2 is able to spontaneously decompose, we can conclude that the decomposition of H2O2 is an exergonic reaction.
Exercise 2D: An Enzyme Catalyzed Rate of H2O2 Decomposition
This exercise is intended use the amount of H2O2 used in the timed reaction to demonstrate the relationship between the rate of reaction over time.
Procedure:
1. 10 seconds
a. Put 10 mL of 1.5% H2O2 in a clean 50-mL glass beaker.
b. Add 1 mL of catalase extract.
c. Swirl gently for 10 seconds.
d. At 10 seconds, add 10 mL of H2SO4 (1.0 M)
2. 30, 60, 90, 120, 180, and 360 seconds
Each time, repeat Steps 1-4. However each time, allowing the reaction to occur for 30, 60, 90, 120, 180, and 360 seconds, respectively.
Note: Each time, remove a 5-mL sample and assay for the amount of H2O2 in the sample. Use a burette to add KMnO4.
3. Record your data in the table below (Table 2.1)
4. Graph the data for enzyme catalyzed H2O2 decomposition.
Analysis:
From the data we collected, we can observe how enzymatic activity impacts the decomposition of H2O2 over specific intervals of time. We can see, by looking at the graph above, that, initially, the H2O2 begins to decompose rapidly. While the amount of H2O2 used over time does continue to increase, our rate of reaction starts to slow down, eventually beginning to plateau. This is because, after a certain amount of time, all of the enzyme will be used up and there will be nothing left to keep reacting.