The Effect of pH on the Rate of Enzyme Catalysis of Catalase

Objectives: 

The objective of this lab was to develop a protocol to investigate the effect of an environmental variable on the catalytic function of an enzyme.  More specifically, the objective was to perform an experiment in order to test the effect of pH on the function of the enzyme catalase.

Introduction:

        Enzymes are proteins that act as catalysts for reactions.  This simply means that enzymes lower the activation energy required for a reaction to take place, allowing a particular reaction to take place much quicker and easier.  Specific enzymes only lower the activation energy for specific reactions, and enzymes are shape-specific.  The unique folds of the amino acid chains that make up an enzyme result in the formation of a specifically shaped active site.  When the reactants of a reactions, called substrates, fit perfectly into the active site of an enzyme, the enzyme is able to catalyze the reaction.  The activity of enzymes is affected by both the concentrations of enzymes present and the concentration of substrate present.  As the amount of enzyme present increases, the rate of reaction increases.  Furthermore, as the amount of substrate increases, the rate of reaction will initially increases.  Most enzymes require specific environmental conditions to be met in order for them to function properly and efficiently.  These conditions include temperature, then concentration of salt, and the pH level.  If the optimum conditions for an enzyme are altered, the enzyme may denature, or change its shape, and deactivate.  As a result, the enzyme would no longer to be able to catalyze the reaction, and the reaction rate would significantly decrease ("Worthington Biochemical Corporation").

        Catalase is found in all organisms that use oxygen for their metabolism.  The enzyme is found in high concentrations in a organelle in cells called the peroxisome.  One of the functions of catalase is to prevent a toxic accumulation of hydrogen peroxide (H2O2) in cells.  It catalyses the conversion of hydrogen peroxide to water and molecular oxygen.  Hydrogen peroxide is a by product of metabolic processes.  It is usually produced in peroxisomes when they partially oxidize fatty acids.   When catalase is absent, the reaction it catalyzes is spontaneous, but at very low rates that are not able to reduce the harmful effects of hydrogen peroxide (Crook).

Research Question and Hypothesis:

Question:  How will altering the pH level of a solution affect the rate of enzyme catalysis of the enzyme catalase?

Hypothesis:  If the enzyme catalase is placed in different solutions containing different pH levels, the enzyme will function most effectively at a neutral pH, or a pH of 7.  As the pH of the solutions are lowered, catalase will begin to work less effectively, and the rate of reaction will begin to decrease.  Catalase, like most enzymes, works best in specific environmental conditions.  The reason that catalase will begin to work less effectively is because as the pH decreases, and the environmental conditions are changed, the change in pH will begin to denature and deactivate the enzymes.  As more and more enzymes become deactivated, the rate of reaction decreases.

Materials:

• Hydrogen Peroxide (1% H2O2 solution)
• Catalase (well-blended/strained potato extract)
• Sulfuric Acid
• Buffers (pH of 7, 6, 5, 3)
• Potassium Permanganate
• Water
• 10 mL Syringes
• Plastic Cups
• Beakers
• 10 mL graduated cylinders
• Gloves
• Goggles

Procedure:

1. Before the experiment began, a baseline was determined.
2. Five 50 mL beakers were labelled with the pH of the buffer that was being tested (Control, 6, 5, 3).  The pH of the solution was the independent variable.
3. 10 mL of 1% hydrogen peroxide solution was added to each beaker.
4. 10 mL of the correct buffer solution was added to the first corresponding beaker.
5. 10 mL of the extracted catalase was added to the same beaker, which initiated the reaction.
6. The reaction was timed for 180 seconds, or two minutes.  Once the two minutes had passed, it was time to stop the reaction.
7. In order to stop the reaction, 10 ml of hydrochloric acid was added to the beaker.  With a pH of 2, the sulfuric acid is very acidic and was sure to denature the enzyme.
8. After the reaction was stopped, the amount of substrate (H2O2) remaining in the beaker had to be measured. To measure this quantity, potassium permanganate (KMnO4) was used.
9. 5 mL of the solution was removed from the beaker.  This 5 mL sample was placed into another small plastic cup and the amount of hydrogen peroxide was determined as follows. A syringe was used to add potassium permanganate, one drop at a time, to the solution until a persistent pinkish brown color was obtained.  The solution was gently swirled as drops were added.  All data was recorded (the initial reading on syringe and final reading on the syringe was recorded).
10. Steps 3-8 were repeated for each pH level so that there was data for a pH of 6, 5, 4, and the control.
11. Once the amount of H2O2 used in the reaction had been calculated, the reaction rate at each pH level was calculated by dividing the total amount of H2O2 used by the time (two minutes).  The rate of reaction was the dependent variable of the experiment.

Determining the Baseline:

1. 10 mL of hydrogen peroxide was put into a small plastic cup.
2. 1 mL of water was added (instead of enzyme solution).
3. 10 mL of 1.0 M sulfuric acid was added.
4. The solution was mixed well.
5. A 5 mL sample was removed.  This 5 mL sample was placed into another small plastic cup and the amount of hydrogen peroxide was determined as follows.  A syringe was used to add potassium permanganate, one drop at a time, to the solution until a persistent pinkish brown color was obtained.  The solution was gently swirled as drops were added.  The amount of potassium permanganate used is proportional to the amount of hydrogen peroxide present, and was therefore used as the baseline.

Data:

Table 1: Determining the amount of H2O2 consumed in each reaction.

Figure 1: Amount of H2O2 consumed at each pH level.

Table 2: The reaction rate for each pH level.

Figure 2: Reaction rate when catalase is present at each pH level.

Conclusion:

        The data from the experiment supports the hypothesis that catalase functions the most efficiently at a neutral pH of 7.  Table 1 shows that catalase helped consume 3 mL of hydrogen peroxide in the solution with a pH of 7, more than any other solution.  As the pH of the solutions decreased, so did the amount of hydrogen peroxide consumed.  Furthermore, after calculating the reaction rates, it is shown that the reaction rate in the solution with a pH of 7 (1.5 ml/min) was higher than any other solution (Table 2).  By looking at figure 2, one can see that as the pH of the solution rose to a pH of 7, catalase became more efficient and was able to better carry out its function.  These results help support the idea that as a solution becomes more acidic than the optimum pH of an enzyme, the enzymes present in the solution will denature, and in turn will not be able to function properly.  This will result in lower reaction rates, which is shown in figure 2.

        The information gathered throughout this experiment is very useful for the future.  This experiment has shown that enzymes must have certain environmental conditions present in order for them to function properly.  With this knowledge, one can successfully perform experiments using enzymes in the future by making sure that the environmental conditions present are optimum for the enzyme that is being used.

        A limitation of the procedure was that we were unable to test for the presence of catalase in the extract before beginning the experiment.  If we were able to test for the presence of catalase in the extract, we could have ensured that the decomposition of hydrogen peroxide resulted from enzyme catalysis and not from the natural spontaneous decomposition of the chemical.  Instead, we were forced to assume that catalase was present in the extract, an assumption that may, or may not have, been correct.

        One source of error was that there was not enough potato extract present at the beginning of the experiment in order to complete it.  In order to get the amount of catalase needed, we were forced to manually crush apple slices and extract the juice from the slices.  Without the use of a blender and a filter in order to correctly extract the catalase, this process may not have provided the necessary amount of catalase needed in order for the experiment to run properly.  If this was the case, then the data would not be valid, as the change in hydrogen peroxide would have been due to the natural spontaneous reaction that already takes place.  Another source of error was that it was difficult to tell when a persistent pinkish brown color had been obtained during the potassium permanganate titration.  As a result, we may have added too much, or too little, potassium permanganate to the hydrogen peroxide solution.  If this did happen, then this would have altered the amount of hydrogen peroxide consumed during the reaction, affecting the calculated rate of reaction.  A third source of error was that an unequal amount of catalase may have been distributed to each solution.  Since we were forced to extract the catalase from the apple slices while we were performing the experiment, more catalase may have been put into the solutions with a pH of 7 and 6, than the solutions with a pH of 5 and 3.  If this happened, then the higher concentration of catalase would have resulted in the rate of reaction increasing.  This, not the change in pH, would account for the differences in the amount of hydrogen peroxide consumed.

Evaluation of Procedure:

        Although the procedure basically worked like I thought it would, there were some times that I felt stressed or under pressure.  While performing the experiment, I had to make sure to always be one step ahead of what I was actually doing, otherwise I would risk falling behind and potentially causing invalid results to be received.  In addition, it was stressful to have to try to extract catalase from the apple slices and perform the experiment at the same time.  In order to solve this problem, it will have to be ensured that there is an excess of potato extract present before the experiment is started.  Other than these few challenges, the procedure did run relatively smoothly, and the experiment was a success.

        One thing that could be added to the procedure in order to improve it is to test the function of catalase in solutions that are basic, with pH levels of 8 and higher.  This could be done in order to show that a neutral pH of 7 is really the optimum pH for the function of catalase, and not a pH that is more basic than 7.  This would also further show how altering the pH of a solution, either lowering it or raising it, will affect the function of an enzyme.  Another thing that could be done in order to improve the procedure would be to test multiple solutions with the same pH level.  This could be done in order to ensure that the data received is accurate and is not flawed.  If conflicting results are received, however, then that means that something may have gone wrong during the experiment.  In order to have a better lab experience, we could be more prepared.  For example, we could ensure that enough potato extract was present before the experiment began.  In addition, we could practice titrating solutions so that we have experience before we begin.

Works Cited:

1. Crook, James. "Catalase - An Extraordinary Enzyme."Catalase Website. N.p., 05 Jul 2003. Web. 3 Mar 2012.
2. "Introduction to Enzymes." Worthington Biochemical Corporation. N.p., 27 Feb 2012. Web. 3 Mar 2012.