Catalase Lab

Title: The Effect of Low pH on the Rate of Catalase Catalyzed Reactions

Purpose: The purpose of this experiment is to test how low pH or acidity affects the rate of reactions catalyzed by catalase.

Background: Enzymes are catalysts that speed up the rate of chemical reactions by lowering the activation energy, or the energy needed to start a reaction. The baseline of a reaction is the standard for the reaction, which includes rate, color, and other variables. Enzymes are not consumed by reactions, but are instead recycled. A particular enzyme only has an active site that will suit a particular molecule. Catalase is an enzyme that specifically participates in the breakdown of hydrogen peroxide, which is produced naturally by cellular metabolic processes, into oxygen gas and water. This reaction is represented as: 2 H2O2 → 2 H2O + O2. Hydrogen peroxide is harmful to cells, which is why this reaction occurs. The optimal pH for catalase in humans is 7, or neutral, and the optimal temperature is 37 °C. Catalase is found the peroxisomes of plant and animal cells; the livers of humans and animals have a great supply of catalase.

Hypothesis: If catalase is added to a solution of lemon juice and hydrogen peroxide with a pH of 3.4, then the reaction will occur at a slower rate than when catalase is added to hydrogen peroxide with a pH of 4.7 and at a faster rate than when catalase is added to lemon juice with a pH of 2.5.

Materials (for the test when catalase is added to hydrogen peroxide with a pH of 4.7):
• Two graduated tubes connected to a piece of plastic tubing [one tube has a hole in the cap (graduated tube 1) and the other has a hole in the cap and an extension at the bottom of the tube that connects the tube to the plastic tubing (graduated tube 2)]
• Two binder clips
• Two 400mL beakers
• 10 mL of hydrogen peroxide with a pH of 4.7
• 400 mL of distilled water
• One P1000 micropipette with tips
• 100 μL of animal liver tissue containing catalase (referred to as just catalase in this lab for simplicity)
• Timer

Procedure (for the test when catalase is added to hydrogen peroxide with a pH of 4.7):
1) Clip the plastic tubing with the binder clips close to where the tubes connect so no oxygen gas flows through the tube.
2) Fill one of the beakers with 400 mL of distilled water and place graduated tube 2 in the beaker with the hole in the cap pointed downwards. Record the initial presence of oxygen gas in the tube by reading the water level.
3) Take the cap off of graduated tube 1 and add 10 mL of hydrogen peroxide to the tube.
4) Using a micropipette, add 100 μL of catalase to graduated tube 1. Quickly screw the cap back on and place the tube into the empty beaker.
5) Remove the binder clips and start the timer. Record the water level at each time interval to determine the amount of oxygen gas produced.
6) Clean all the materials and preform additional trials (three recommended) and choose the most accurate to include as data.

Lab Setup x

Data:

Table

The Rate of Catalase Catalyzed Reactions at Different pHs

Screen Shot 2013-10-06 at 1.21.13 PM

Graph

The Rate of Catalase Catalyzed Reactions at Different pHs

Lab graph final

Lab Setup y

Analysis: As the pH of the catalase catalyzed reactions decreased, the amount of oxygen produced by the reactions decreased. The reaction that occurred at a pH of 4.7 produced the most oxygen. The reaction that occurred at a pH of 3.4 produced less oxygen than the reaction that occurred with a pH of 4.7, but more than the reaction that occurred at a pH of 2.5. The reaction that occurred at a pH of 2.5 produced no oxygen at all. The evidence that the reaction that occurred at a pH of 2.5 produced no oxygen is that there was no difference in water level in graduated tube 2 over time, therefore, the water level was constant and was not decreased by the pressure of oxygen on the water.

Conclusion:
The data reveals that low pH or acidity affects the rate of catalase catalyzed reactions. The data shows that the rate of the reaction that occurred at a pH of 3.4 was greater than the rate of the reaction that occurred at a pH of 2.5, but less than than the rate of the reaction that occurred at a pH of 4.7. The hypothesis is, therefore, supported by the data. As the pH became more acidic, the catalase catalyzed reactions occurred at a slower rate. The reaction that occurred at a pH of 2.5 produced 0 mL of oxygen gas, evident by the constant water level in graduated tube 2. If no oxygen was being produced, then the rate of the reaction was either extremely slow or the reaction was not occurring. The reaction that occurred at a pH of 3.4 produced 0 mL of oxygen up until 80 seconds. Then, the amount of oxygen produced was constantly 0.25 mL up until 160 seconds. Then, the amount of oxygen produced continued to increase, as the amount of oxygen produced at 200 seconds was 0.625 mL, at 300 seconds was 0.875, and at 400 seconds was 1 mL. The rate of the reaction continuously increased over time. The reaction that occurred at a pH of 4.7  also increased continuously over time.  The reaction began increasing a slow rate and later on, between 60 seconds and 100 seconds, the amount of oxygen produced began increasing at a greater rate. The amount of oxygen produced at 60 seconds was 1.58, at 80 seconds was 2.48, and at 100 seconds was 3.48; the water level difference is greater to or equal to 1 mL. After 100 seconds occurred, the rate of the reaction began to slow down, evident by the lower water level difference after 100 seconds. At 120 seconds, the amount of oxygen produced was 4.3 mL, at 140 seconds was 5 mL, and at 160 seconds was 5.5 mL; the water level differences for these times are less than 1 mL. 

The baseline control of this experiment was the reaction that occurred when catalase was added to the hydrogen peroxide with a pH of 4.7. This set the standard rate of the breakdown of hydrogen peroxide into oxygen gas and water for the experiment. When catalase was added to the solution of lemon juice and the hydrogen peroxide, the high pH must have denatured some individual enzymes, but not all, so the reaction occurred at a slower rate than the baseline reaction. Denaturation changes the shape of an enzyme, including the enzyme’s active site, so a particular cannot bind to the enzyme anymore. This supports the idea that enzymes are specific to the molecules enzymes bind to. Since the baseline reaction occurred under a more acidic environment than the optimal pH for catalase, the rate of the breakdown of hydrogen peroxide into oxygen gas and water will be faster under a neutral pH. A possible error that could have occurred in this experiment could include misreading the water level. If the water levels were incorrectly read, then the amount of oxygen produced by each reaction recorded in the data chart are inaccurate. As a result, the rate of the reactions could have been faster or slower. This is why  multiple trials were preformed and the most accurate was chosen to graph.

References:

“Catalase.” GMO Compass. 7 July 2010. Web. 3 Oct. 2013. <http://www.gmo-compass.org/eng/database/enzymes/89.catalase.html&gt;.

Enzymes. Perf. Paul Anderson. YouTube. YouTube, 26 Nov. 2011. Web. 3 Oct. 2013. <http://www.youtube.com/watch?v=ok9esggzN18&gt;.

“Peroxisomes.” BSCB: The British Society for Cell Biology :: www. bscb.org. BSCB, 2013. Web. 3 Oct. 2013. <http://www.bscb.org/?url=softcell/peroxi&gt;.

Reece, Jane B., Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, and Robert B. Jackson. Campbell Biology. 9th ed. San Francisco: Pearson Benjamin Cummings, 2011. Print.

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