Enzyme Lab

Introduction:

This lab observes the process by which Hydrogen Peroxide [H₂O₂] breaks down into water and oxygen with the help of the enzyme catalase. The amount of oxygen released as a product of this reaction, and the rate of the enzymatic reaction, will also be measured and calculated.

Exercise 2A: Test of Catalase Activity

Purpose:

This exercise investigates the effects of temperature and the presence of living tissue on the enzymatic activity of breaking down H₂O₂.

Procedure & Methods:

1. To observe the reaction, 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.
3. Transfer 10 mL of 1.5% H₂O₂ into a 50 mL glass beaker and add 1 mL of the cooled, boiled catalase solution.
4. To demonstrate the presence of catalase in living tissue, cut 1 cm³ of potato or liver, macerate it, and transfer it into a 50 mL glass beaker containing 10 mL of 1.5% H₂O₂.

Analysis:

Our observations in such an experiment would lead us to understand that enzymes react at different rates depending on the temperature of the environment. Extreme heat can denature enzymes, making them inactive and unable to function. Also, from this experiment, we can understand that living tissues have enzymes of their own, so if catalase is put in the presence of living tissue, this reaction may take place more quickly.

Exercise 2B: The Baseline Assay

Purpose:

This exercise determines the amount of H₂O₂ that is initially present in a 1.5% solution. This value can be used as a baseline to compare other values to.

Procedure & Methods:

1. Put 10 mL of 1.5% H₂O₂ into a cup.

2. Add 1 mL of H₂O [instead of enzyme solution].
3. Add 10 mL of H₂SO₄ [1.0 M] and mix well.
4. Remove a 5 mL sample. Place this 5 mL sample into another cup and assay for the amount of H₂O₂.
5. Use a burette to add KMnO₄, a drop at a time, to the solution until a persistent pink or brown color is obtained.

Data:

Analysis:

The purpose explained the use of this baseline value, but to reiterate, this number is needed to determine how much H₂O₂ is present in a 1.5% solution. With this value, we can calculate the effectiveness of a catalase on the decomposition H₂O₂ by measuring how much H₂O₂ is present after the enzymatic reaction takes place.

Exercise 2C: The Uncatalyzed Rate of H₂O₂

Purpose:

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 & Methods:

1. Leave a small amount of 1.5% H₂O₂ uncovered at room temperature for 24 hours.
2. Add 1 mL of H₂O [instead of enzyme solution].
3. Add 10 mL of H₂SO₄ [1.0 M] and mix well.
4. Remove a 5 mL sample. Place this 5 mL sample into another cup and assay for the amount of H₂O₂.

5. 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 findings in the chart.

Data:

Analysis:

From this exercise, we were able to conclude that about half of H₂O₂ decomposes on it’s own from exposure to oxygen and light over a period of 24 hours [This is why H₂O₂ is stored in dark bottles, as light causes H₂O₂ to spontaneously decompose]. Spontaneous reactions occur without the help of catalase, so this was a helpful value to compare with the values from 2D just to see how a catalyst may impact decomposition.

Exercise 2D: An Enzyme-Catalyzed Rate of H₂O₂ Decomposition

Purpose:

In this exercise, the amount of H₂O₂ used in timed reactions, compared to the baseline value from Exercise 2B, demonstrates the relationship between the rate of a reaction over a certain amount of time.

Procedure & Methods:

1. 10 seconds
1. Put 10 mL of 1.5% H₂O₂ in a clean 50 mL glass beaker.
2. Add 1 mL of catalase extract.
3. Swirl gently for 10 seconds.
4. At 10 seconds, add 10 mL of H₂SO₄ [1.0 M]
2. Repeat Steps 1a-1d, substituting the time value for 30, 60, 90, 120, 180, and 360 seconds, respectively. For every reaction, remove a 5 mL sample and assay for the amount of H₂O₂ in the sample. Use a burette titration to add KMnO₄.

3. Record data in Table 2.1.

Data & Graph:

Analysis:

From the data we collected in this experiment, we were able to observe how enzymatic activity impacted decomposition over various amounts of time. From the graph, we can conclude that while the amount of H₂O₂ used may keep increasing, this value will eventually plateau and stay the same as the trend we see is beginning to slow down and reach this point. This is because after a certain amount of time, all the enzyme will be used up, and there is nothing left to keep reacting.

References:

Diana, Amanda. "Failure To Thrive." Failure To Thrive. Amanda Diana, 02 Nov. 2015. Web. 04 Nov. 2015. <http://amandadoesscience.tumblr.com/>.

Rembac, Justine. "Catalase and Hydrogen Peroxide Experiment." Catalase and Hydrogen Peroxide Experiment. Education.com, 18 Nov. 2013. Web. 04 Nov. 2015. <http://www.education.com/science-fair/article/activator/>.