Biology Lab Section I/Class Section I
Martina Zucchini/Judy Lonsdale
The Balancing Act: pH and Its Affect on Enzymes
Enzymes are present in all living things. These specialized proteins speed up chemical reactions fast enough to sustain life (Johnson and Losos, 53). Since enzymes are a huge topic to even begin to cover, we tested a hypothesis using the enzyme “catalase” from a cow’s liver as the basis of our enzyme research. This research project tested the hypothesis that extreme levels of pH will affect or denature the catalase (Boise State University and Lonsdale, 27-33).
Before we can test the effects of “lower” or “higher” pH levels, we must first determine what constitutes these values. In the book “The Living World” by George Johnson and Jonathan Losos, pH levels are determined by a ruler of numbers, from 0 being the most acidic (Hydrochloric acid) to 14 being the most basic (Sodium hydroxide). The level of Hydrogen ions present characterizes these numbers (Johnson, and Losos, 114). The average human has a pH level of approximately 7, which is neutral on the pH scale (Johnson, and Losos, 53). So the higher and lower values will be set from the median of 7.
Catalase is an organic catalyst, which is by definition a “Substance that increases the rate of a reaction without itself being altered” (Zucchini, 2010). The liver providing the catalase we tested with are from a cow, so the enzymes shouldn’t be too much different from a human’s. The function of catalase in its most simple form is to break down H2O2 (Hydrogen Peroxide). Catalase breaks H2O2 down into two elements, H20 (water) and O2 (Oxygen), which is the bubbling that takes place when you pour hydrogen peroxide on a cut (Boise State University and Lonsdale, 29).
An identifying factor of enzymes is their malleability, in order for enzymes to do their job they must bind to a specific molecule and stress the molecular bonds of that molecule in order to make the objective result more likely (Johnson and Losos, 65). “An enzyme is specific for a particular reactant, or substrate, because the enzyme surface provides a mold that very closely fits the shape of the desired reactant. Other molecules that fit less perfectly simply don’t adhere to the enzyme’s surface. The site on the enzyme surface where the reactant fits is called the active site. The site on the reactant that binds to an enzyme is called the binding site” (65). After the components are bound to the enzyme, the enzyme stresses key covalent bonds, resulting in a chemical reaction that forms the product, which then diffuses away from the enzyme. The enzyme is now free to work again (Johnson and Losos, 113). Now why are pH levels so important to this project? In order to fully understand enzyme activity, we need to study the effects of different levels of pH on enzymes.
When an enzyme is denatured, it can unfold and lose its shape leaving it useless (Zucchini, 2010). And so if my hypothesis is correct, then when the Hydrogen Peroxide is added to the liver, with the liver mixed with significantly lower or higher levels of pH, the enzyme catalase should decrease its activity/denature. If the enzyme is affected, this will help support that different levels of hydrogen ion (H+) concentration are a factor that affects enzyme activity (Johnson, and Losos, 114). My prediction on the results of this experiment is that extreme levels of pH will denature the catalase, because the human body cannot consume substances that are extremely low or high on the pH scale.
Preparing the Equipment and Substances
I began the experiment by organizing the materials I needed to conduct the testing of my hypothesis. I had 3 dropper bottles, containing solutions of pH 2, 8 and 14 respectively, using water as the control, and Hydrogen Peroxide (H2O2) to engage the catalase. The effet of pH on catalase activity will be displayed by the number of mililmeters of foam which are consequent of catalase converting the Hydrogen to water and oxygen. In four separate test tubes, I dropped 6 droplets of shredded cow liver (which contains catalase) in each. The purpose of this is to test the affect of different pH levels on enzyme activity, so I next added the control, water, and the pH solutions of 2, 8 and 14 each separately to the test tubes with the shredded cow liver. Finally, I added the Hydrogen Peroxide (H2O2) and recorded the results after fifteen minutes.
Mixed Up, Time to Confirm or Make Null my Hypothesis
After adding Hydrogen Peroxide to the liver/pH solutions, the level of foam put off by the catalase converting the H2O2 into carbon dioxide and oxygen was instantly telling of whether the pH levels had any effect on enzyme activity. The first result from the control (water) came in at 130 millimeters (mm), water has a pH level of 7, making it neutral- a very weak base and a very weak acid. The second result came from a solution of pH 2, which is very acidic. As illustrated in Figure 1, this acidic of a solution would appear to have nearly completely denatured the catalase. The results of the water solution increased my confidence in the possibility of better success with the next solution, a pH level of 8. This came out to be partly true, the catalase definitely performed better than when it was in a pH solution of 2, however not as well as when it was in water. The total amount of foam was 102mm.. Finally to the last part of the experiment, I mixed the H2O2 into the solution with a pH of 14. Back on the “Lunatic Fringe” of the pH scale- this time on the opposite side- I expected similar results, and the pH didn’t let me down. The foam level was even smaller than the lowest acidic level, less than 1mm. As a partially extraneous thought, after conducting this experiment I formulated a hypothesis- that catalase in mammals can function into farther levels of the pH scale into acidity (lower) than in more basic substances (higher).
Moment of Truth: Do Extreme pH levels Affect Enzyme Activity?
After exposing the enzyme “catalase” to extreme levels of pH, the results were indicative of the obvious damage levels above or below the native level 7 on the pH scale have on catalase (Johnson and Losos, 53). I was then moved to confirm my hypothesis that “extreme levels of pH will affect or denature the catalase”.
When the catalase from cow’s liver was exposed to extreme levels of pH, the enzyme was either damaged or denatured. This was signified by the results of the levels of foam (in mm) which were recorded at different levels when H2O2 was added to the enzyme with different levels of pH. When the enzyme was added to a mixture with a pH level of 2, the result was minimal at 4mm. When the enzyme was mixed with a combination with a pH level of 14, the result was even smaller at less than 1mm. The results I was looking for were confirmed when the enzyme was mixed with a pH level of 8, and the foam rose to a level of 102mm. This confirms the hypothesis that extreme levels of pH effect enzyme activity.
According to “The Living World” by George Johnson and Jonathan Losos, enzymes “function within an optimal pH range, because the shape-determining polar interactions of enzymes are quite sensitive to hydrogen ion (H+) concentration (115)” Since the shape of the enzyme is vital to its adherence to the necessary particles, pH levels are strong factors in the efficacy of enzyme activity. “Enzymes catalyze chemical reactions within cells. Sometimes enzymes are organized into biochemical pathways. Enzymes are sensitive to temperature and pH, because both of these variables influence enzyme shape (Johnson and Losos, 114-115).”
However, these results can be prone to human error, such as miscalculating the amount of pH added to each mixture of cow liver and H2O2, or simply mislabeling or misreading an amount. The study we conducted also was on a small scale, with only one reading of each pH level. The readings would be far more accurate if the mixtures were done multiple times for each reading, then you would be able to assess an average.
The results from this experiment spark a very large hypothesis; that extreme levels of pH affect all enzymes. If this is true, then the level of pH in humans will directly affect their chemical reaction processes. But this leads one to think there must be a way our bodies regulate all this. And there is, cells control enzymes. “Many enzymes have shapes that can be altered by the binding of “signal” molecules to their sufaces.” Such enzymes are called “allosteric”. Enzymes are also usually regulated by a process called “feedback inhibition”, in this case the product of the reaction acts as the repressor (Johnson and Losos, 115).
To further test this hypothesis, one could test actual human liver and see if it functions best at its native pH level, if this also holds true, the results could be the basis for further research into enzyme function and activity in humans and animals.
Johnson, George, and Jonathan Losos. The Living World. 6th ed. New York, NY: McGraw-Hill, 2010. Print.
Boise State University, and Judy Lonsdale. “Enzyme Function and Activity.” Concepts of Biology Laboratory Manual. 5th ed. Dubuque, IA: Kendall Hunt, 2009. Print.
Zucchini, Martina. (2010). Lab lecture.