The research performed through these experiments supports the hypothesis that a change in pH value or substrate concentration will change the working efficiency of catalase, as was depicted through the changes in its observed reaction rate. This hypothesis was tested through two similar experiments which assayed the enzymatic activity of the decomposition of hydrogen peroxide to water and molecular oxygen. By collecting the oxygen gas synthesized by the reaction over time and displaced from the apparatus into the graduated cylinder, an assay of the current reaction rate can help determine the current enzymatic activity of catalase. For each experiment, a control group was set up in a flask with 6.0 mL of pH 7.0 buffer solution and 3.0 mL of 3% HβOβ solution, where a basic enzyme assay was performed. Then, four experimental groups were assayed per each experiment. One experiment assayed four groups in 4.0, 5.0, 7.0, and 8.0 mL of pH 7.0 buffer solution, while the volume of 3% HβOβ in the solution was changed to 5.0, 4.0, 2.0, and 1.0 mL respectively. The setup of each experimental group changed the final concentration of hydrogen peroxide present in each run. The other experiment assayed four groups in 3.0 mL of 3% HβOβ solution while the pH of the buffer solution used was changed to pH 11, 9, 5, and 3. The data from each group, including the control groups, produced different values for the initial reaction rate.
When only the concentration of the substrate, the hydrogen peroxide, was altered, the data showed that the substrate concentration was proportional to the volume of oxygen gas produced. Tables 7 through 9 show that during the first three-second interval recorded, the volume of water displaced while using both the solutions of 1.2% and 1.5% HβOβ concentration was higher than the volume of water displaced through the control group: there was a change in volume of 21.5 and 22.0 mL of water respectively compared to a change of 14.5 mL in the control. Conversely, tables 10 and 11 show that the volume of water displaced while using both the solutions of 0.6% and 0.3% HβOβ, a smaller volume of water was displaced: 7.0 and 4.5 mL of water per respective experimental group. Assuming the data and reasoning of the experiment is valid, the expulsion of oxygen gas from the flask and tubing was the cause of any observed displacement of water (Naganuma & Roffey, 2018). This data would show the rate of synthesis of molecular oxygen produced by the catalyzed decomposition of the substrate was dependent on the substrate concentration; or, as the substrate concentration increased, the rate of the catalyzed reaction increased.
The increase in substrate concentration is reasonable causation for the increase in the rate of reaction. An enzyme must collide with a molecule of its appropriate substrate to bind and catalyze the substrate into an enzyme-substrate complex, a state that allows the enzyme to transform the substrate into the intended product (Urry, et al., 2020). The enzyme only recognizes and will bind to its target enzyme; by increasing the concentration of substrate in the stirred solution, the amount of molecules of substrate is increased, and the stirring will increase the frequency of collision between the enzyme and its substrate (Naganuma & Roffey, 2018). This trend was reflected between the ranges of 0.3% to 0.9% concentration of HβOβ; as the concentration of hydrogen peroxide increased, the reaction rate increased from 1.4 mL of Oβ produced per second to 4.25 mL of Oβ produced per second (Table 12). However, this rate is limited by the amount of enzyme in solution; an enzyme can bind and form a complex with a finite amount of substrate, and the reaction rate will stagnate once that limit is exceeded (House, 2007). This limit is created by the number of active sites present: small regions specifically shaped by the tertiary structure of the enzyme (Urry, et al., 2020). This phenomenon was observed on Table 12 and Figure 6, which summarized the effect of the concentration of hydrogen peroxide on the beef liver catalase. The reaction rate exponentially increased between the run of 0.3% to 0.6% HβOβ concentration from 1.4 to 2.42 mL of Oβ produced per second. Once it reached 1.2% concentration, the graph curved off, and as the concentration approached 1.5%, the reaction rate increased from 6.4 to 7.0 mL of Oβ produced per second. The beef liver catalaseβs active sites were likely reaching maximum capacity; if not all of the sites had been binded, the active sites free on the catalase were likely few and spread out, making it harder for the substrate to find a site to bind to.
When only the pH of the buffer in solution was altered, a graph of the data showed one peak where the most oxygen gas was produced, and as the pH value of the solution approached an acidic or basic extremity, less oxygen gas was produced. As recorded in Table 4, at pH 9, 8.5 mL of water was displaced within the first three-second interval: the highest volume displaced through the five experiments which changed the pH of the solution. 33.5 mL of water was displaced in 33 seconds during the pH 9 run before the reaction slowed down. Table 5 shows the second highest volume of water displaced was at pH 11, where 5 mL was displaced in the first interval and 38.8 mL was displaced in 33 seconds before the reaction slowed. The control group (Table 3) displaced the third largest volume of water, and the runs with pH 5 and 3 in solution displaced the fourth and fifth largest. In these runs, 30.5 mL (Table 2) and 5.8 mL of water (Table 1) had been displaced when 33 seconds had elapsed. The same assumption imposed on the experiments on substrate concentration can be applied to the experiments on pH. The displacement of water in the graduated cylinder was related to the production and displacement of oxygen gas through the tube; at more acidic or basic pHs, such as pH 3 or pH 11, the production of oxygen gas decreases, while its production reaches an optimum at around pH 7. These results are reflected in the summary of reaction rates in Table 6, where pH 9 had the highest rate of production, 2.8 mL of Oβ per second, while pH 3 had the lowest, 0.37 mL of Oβ.
The change in reaction rate of the catalase can also be connected to the change in pH. An enzyme is a protein, and a proteinβs structure can be affected by changes in pH; by that logic, changing the pH value of the solution the enzyme is in will affect its enzymatic performance (Naganuma & Roffey, 2018). Different enzymes have different pH values at which they perform optimally; they may perform suboptimally at different pH values, but will begin to denature as the distance from optimal pH value increases, and they will lose the structure of the active sites necessary to bind to substrates in the process (Urry, et al., 2020). Less enzyme-structure complexes are able to be formed, if any at all depending on how far the protein has denatured. This phenomenon was observed through the five runs in the pH experiment; at the most acidic and basic pH levels tested, pH 3 and pH 11, the volume of oxygen gas produced fell in comparison to the maximum of the summary graph, depicted at pH 9. The experimental group with pH 3 solution was only able to displace 5.8 mL of water before the trial ended (Table 1), while the group with pH 11 produced 0.76 less mL of Oβ compared to the pH 9 group. It is likely that the catalase has an optimal pH around pH 9 and would denature in acidic solutions. Because the catalase produced the least amount of oxygen gas when it was placed in a pH 3 solution, the pH was likely too acidic and very few, if any, active sites were present after 33 seconds; the enzyme had lost its structure, and any active site that did not bind to a substrate quickly was lost as it denatured.
One notable result from the experiments in pH was the result of running with a pH 7 buffer in solution. The rate of reaction calculated from the pH 7 control group was 1.17 Oβ/sec; this value was less than the values obtained from both a pH 5 and a pH 9 buffer in solution (Table 6). Based on the trend of the four experimental groups, which formed a bell curve on the summary graph, and the ability of a protein to perform more efficiently when the pH of its environment approaches its optimum pH, the reaction rate from the pH 7 buffer should have been higher than the pH 5 groupβs reaction rate, but still lower than the pH 9 reaction rate. The reaction rate was thus lower than expected from the control group. The unexpected result may have been caused by the reagents used in the experiment. Each pH buffer was a stock solution with an unknown composition and created an unknown amount of time prior to the experiments. It is possible that the components used to create the pH 7 stock solution were inhibitors of catalase, which would have caused the catalase to decompose less hydrogen peroxide, or the stock solution had been contaminated over time, causing it to be diluted and the pH to drop below the stated value.
The information gathered from these experiments shows the importance of the environment on proteins and enzymes. Enzymes are a key element in metabolism, the sum of all chemical reactions in an organism (Urry, et al., 2020). These reactions use and release chemical energy which fuels the ability of molecules to do work and allow important life processes like movement and digestion, which all organisms require to survive. Enzymes help catalyze metabolic reactions that reach their transition states, points at which the reactants have the energy required to form the products in a reaction, too slowly to maintain the necessary processes of life. All enzymes function differently in various conditions, possess an optimal environment where they perform most efficiently, and can be affected by physical and chemical changes like other proteins, such as pH, concentration, and temperature. By studying these variables and more with different enzymes, researchers can identify enzymes that are adaptable, survive and perform efficiently in more environments or extreme environments. These enzymes can be used further in biotechnical and industrial fields to advance developments in food cultivation and medicine (Pandey, et al., 1999).
Future researchers aiming to replicate these experiments should make sure to carefully read through and follow the methods outlined. The experiments analyzed in this report require precision in timing, observation, and execution. The solutions of hydrogen peroxide and pH 7 buffer added to the flask in the experiment that changes the concentration of the substrate should be as precise as possible to avoid inaccuracies in the amount of oxygen gas produced: based on the results, the production of oxygen gas should increase with increases in concentration until the enzymes have been saturated by the substrate. Researchers should also handle these reagents with care, using latex gloves in case of spills of beef liver or pH buffers, and avoid ingesting any buffers, especially the pH 11 solution. When performing the experimental runs, the injection of beef catalase into solution should be coordinated with the recording of data to avoid mistiming. The researcher stirring the flask should also ensure they stir at a constant speed with their hand on the neck of the flask and not the base; this will ensure the observed rate of reaction is steady as the catalase binds to the hydrogen peroxide in solution and that the temperature of the flask and internal reaction remains constant.