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In this enzyme catalysis lab, we investigated the impact of varying catalase concentrations, temperature, and hydrogen peroxide (H2O2) concentrations on the rate of the catalase-catalyzed reaction. We found that the 33% catalase concentration, 25°C temperature, and a 30% H2O2 concentration without inhibitor resulted in the highest reaction velocity. When sodium cyanide was introduced as an inhibitor, the reaction velocity decreased significantly, indicating its inhibitory effect. Through Michaelis-Menten and Lineweaver-Burk plots, we calculated kinetic parameters such as Km and Vmax, providing insights into the enzyme-substrate interactions and the impact of inhibition.
Enzymes play a critical role in facilitating biochemical reactions by accelerating the conversion of substrates into products.
Catalase, a common enzyme found in organisms, catalyzes the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). This reaction is essential for preventing the buildup of toxic H2O2 in cells. In this lab, we aimed to explore the factors affecting the rate of catalase-catalyzed reactions, including catalase concentration, temperature, and substrate concentration.
Before conducting any experiments, we extracted and juiced potato skins, which served as the source of catalase.
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The potato juice was prepared by adding 4 mL of 7.2 pH citrate buffer to the potato peels and then filtering the solution through cheesecloth to obtain 20 mL of clear juice. Safety goggles were worn throughout the experiment to protect against contact with 8.8 M H2O2.
A eudiometer was set up by placing an inverted burette in water, connected to a flask with a rubber stopper.
H2O2 was introduced into the flask using a 1.0 cc syringe. A 33% catalase solution, prepared by mixing potato juice and citrate buffer, was used for most experiments after determining its efficiency in the preliminary experiment.
The initial experiment aimed to identify the optimal catalase concentration. Four flasks were prepared with different catalase concentrations, and 1 mL of H2O2 was added to each flask. Data was recorded every 15 seconds for 2 minutes for each flask.
Subsequently, a temperature experiment was conducted using five different temperatures (5°C, 20°C, 25°C, 30°C, and 40°C). Each flask contained catalase solution and buffer, and 1 mL of H2O2 was added. Data was collected at 15-second intervals for 2 minutes at each temperature.
The third experiment examined how varying H2O2 concentrations influenced reaction velocity. Six flasks were prepared with different H2O2 concentrations, and data was collected similarly to the previous experiments.
The final experiment repeated the variation in H2O2 concentration but included a sodium cyanide inhibitor. Six flasks were created with the appropriate catalase stock solution, and sodium cyanide was added to inhibit the reaction. Data was collected as in the previous experiments.
Data collection involved recording the meniscus value every 15 seconds for 2 minutes, representing the amount of oxygen gas generated. Various graphs, including volume of oxygen gas evolved versus time and Michaelis-Menten plots, were created to analyze the data and calculate kinetic parameters such as Km and Vmax.
The preliminary experiment determined the most efficient enzyme concentration to be 33% catalase, as it produced the highest amount of oxygen gas without the eudiometer emptying completely. The temperature experiment showed that 25°C yielded the highest reaction velocity, while 5°C and 40°C performed poorly, producing minimal oxygen gas. In the experiment examining the impact of hydrogen peroxide concentration on reaction velocity, a positive linear correlation was observed between H2O2 concentration and velocity, with the highest velocity at 30% H2O2 concentration.
| H2O2 Concentration (%) | Initial Velocity (mL/s) |
|---|---|
| 30% | 1.5 |
| 1/V0 (mL/s) | 1/[S] (M) |
|---|---|
| 0.67 | 0.32 |
| H2O2 Concentration (%) | Initial Velocity (mL/s) |
|---|---|
| 22.5% | 1.1 |
| 1/V0 (mL/s) | 1/[S] (M) |
|---|---|
| 0.91 | 0.8 |
The experiment with sodium cyanide as an inhibitor showed a decreased reaction velocity, with the most significant impact observed at a 22.5% H2O2 concentration.
The results indicate that the 33% catalase concentration was the most efficient for the catalase-catalyzed reaction, as it produced the highest amount of oxygen gas. This is consistent with the enzyme-substrate interaction principle that enzyme activity increases with substrate concentration up to a point.
Temperature had a significant impact on the reaction velocity, with 25°C being the optimal temperature for the catalase-catalyzed reaction. Extreme temperatures (5°C and 40°C) adversely affected the reaction, likely due to denaturation of the enzyme.
The experiment on hydrogen peroxide concentration revealed a positive correlation between substrate concentration and reaction velocity. The Michaelis-Menten and Lineweaver-Burk plots further confirmed this relationship and provided kinetic parameters Km (3.14) and Vmax (4.82) for the reaction without inhibitor.
In the presence of sodium cyanide as an inhibitor, the reaction velocity decreased, as indicated by the Michaelis-Menten and Lineweaver-Burk plots. This suggests that sodium cyanide interferes with the catalase-catalyzed reaction, reducing the enzyme's ability to convert H2O2 to water and oxygen.
In conclusion, our experiments revealed the importance of catalase concentration, temperature, and substrate concentration in influencing the rate of the catalase-catalyzed reaction. A 33% catalase concentration and a temperature of 25°C were found to be optimal for maximizing reaction velocity. Additionally, the experiments with varying H2O2 concentrations and the presence of a sodium cyanide inhibitor highlighted the impact of substrate concentration on the reaction rate and the inhibitory effect of sodium cyanide. These findings contribute to our understanding of enzymatic reactions and their regulation.
Further research could explore additional factors affecting catalase activity, such as pH levels and the presence of other inhibitors or cofactors. Additionally, investigating the application of catalase in various industries, such as food preservation and biotechnology, could yield valuable insights into its practical use.
Enzyme Catalysis Lab Report. (2021, Sep 20). Retrieved from https://studymoose.com/document/experiment-with-potato-and-enzyme
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