Enzyme Lab Report
Introduction: Enzymes are biological catalysts that permit cells to carry out the many functions that are required in a living system. Every enzyme has a specific substrate and a specific function. Enzymes alter substrates one of three ways: by adding something to the substrate, removing something from the substrate, or by changing its conformation, otherwise known as its shape. The structure of an enzyme and its ability to function exists because it binds to an active site, thus increasing the reactivity of the enzyme and allowing for the reaction to speed up more quickly. However, the regulation of enzyme activity, which is catered towards attention to sensitivity in changes to catalysis as well speed of a catalyzed reaction, is monitored
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by factors that affect the ability of an enzyme to work such as pH and temperature. Thus, the purpose of this experiment is to primarily isolate the enzyme polyphenol oxidase from a potato extract. When the enzyme and oxygen are exposed to compounds called phenols that are present in the tissues of potatoes, the phenols are converted to other compounds called quinones. The quinones rapidly react with proteins in the potato to make the brown-colored compounds that one can see on the surface of potatoes. The brown bruise is caused by the impact rupturing cells beneath the surface, using oxygen that is available in the cells. The exposure of air to the potato extract results in the enzyme polyphenol oxidase to initiate a reaction. The substrate that the enzyme is changing is the phenotic compound in the potato. The enzyme changes the substrate’s chemical structure causing the potato to inherently change color. Polyphenolic compounds are often found in many fruits and vegetables such as apples, mangos, and potatoes. These polyphenolic compounds also is a factor in the determination of color, aroma, and bitterness of the fruit or vegetable. Since polyphenol oxidase catalyzes the reaction of dopa to form dopaquinone resulting in the immediate oxidation to dopachrome. Thus, the conversion of dopa to dopachrome, an orange colored o-quinone with an absorption at 475 nm, will be imperative for this experiment in order to isolate the polyphenol oxidase to measure the rate of the catalyzed reaction. A 5 mM dopachrome solution provided that has already been oxidized by a 5 mM dopa solution will be the substrate for the experiment. To determine the rate of the catalyzed reaction, application of enzyme kinetics is vital.
The purpose of enzyme kinetics is to determine how fast or efficient an enzyme is as well as how specific an enzyme is. This assists in determining whether a certain factor or treatment affects enzyme efficiency which is inclusive of identification of enzymes and prediction of reactions. Kinetic experiments examine the concentration of a product formed or a substrate consumed per unit of time. Plotting the variation of initial velocity with the substrate often results in a linear graph allowing us to determine the maximum velocity at which it can catalyze the reaction. Therefore, it will be presumed that the result of this experiment by isolating the polyphenol oxidase within the potato extract through the oxidation of 5mM dopa solution with highly concentrated enzymatic preparation of polyphenol oxidase with a buffer as the diluent will indicate a rapid increase in the rate of the reaction as well as the amount of activity present in the potato extract. Proposing that Vmax is proportional to enzyme concentration, a linear relationship should be present and enzymatic activity at varying enzyme and substrate concentrations can be
determined. Methods and Materials: In analyzing the polyphenol oxidase enzyme found in potatoes, PPO activity has to be measured which is done by the instructor by grinding up potatoes to create extract and then performing partial purification in order to remove the endogenous PPO substrates such as tyrosine. After completion of the preparation of the polyphenol oxidase extract, a qualitative PPO enzyme assay has to be completed. Therefore, 1.6 mL of Frozen potato extract was extracted and mixed completely by tapping tube to help thaw and was kept cold at all times. 8 mL of a 1:5 dilution was prepared for both PPO assays and the amount of buffer and enzyme that is serially diluted for each test tube was calculated and recorded. Three test tubes were prepared and only the first three solutions were added which excluded the PPO extract.
Then, enzyme PPO extract was added and a color change occurred. A serial dilution was made and after adding the enzyme extract, each tube was mixed well before a new transfer was made allowing for each tube volume to be 5 mL. The Spec 20 was turned on and warmed up for a duration of 15-20 minutes as the absorbance was set for a value at 475 nm. After measures were recorded from the Spec 20, the dopa was then added to each test tube which initiated the reaction. The rate of reaction was then measured by increase in absorbance over a 4 min. period and the absorbance values were then converted to dopachrome concentrations by subtracting the control tubes from the experimental tubes.
Then, dopachrome concentration was converted to dopachrome amount within each 5mL reaction and dopachrome produced was plotted as a function of time, which created four independent assays. After, the rate of the reaction was determined and the dilution and initial velocity graphed, the enzyme activity for each reaction was determined using the formula activity = product produced/time/volume. Conclusively, the enzyme activity of the undiluted potato extract was determined as well as the amount of PPO present in the potato.
Results:
According to Table 1 of the Qualitative Polyphenol Oxidase Test, the color change present from Tube 1-3 was negative, positive, and negative. The final dopa concentrations varied from 0 mM and 4 mM based on whether buffer volume or 40 mM dopa volume was used due to one test tube being kept as a control and the other as experimental. The mean absorbances based on the standard curve were reported to be -.47, 0.68, 1.07, and 1.44 with absorbances increasing as dopachrome concentrations increased. According to Figure 1 of the standard curve of dopachrome concentrations, the R2 value was equivalent to 0.8495 and it did not heed or produce any negative values. According to Table 1 of absorbance data from time course, the averages of control tubes 1, 3, 5, and 7 were 0.019, 0.032, 0.059, and 0.112. According to Table 2 of the Corrected Absorbance Data, each control tube was subtracted from the experimental tube and then converted to dopachrome amount in millimolars.
According to Figure 2 Enzyme Kinetics: Dopachrome vs time, all dilution series are linear and the series with the greatest R2 value is the 1/10 series dilution with a R2 of 0.9984 and an equation of 0.0002x – 0.0004, with a Vi of 0.002. This equation will then be used to determine the initial velocity of the other series dilutions when plotted creates figure 3 displaying total enzyme concentration vs Vmax as the R2 value of this graph is 0.9725. The average activity of potato extract stock shown on Table 6 found was 13.2 mmol/min/mol and the activity per weight of potato given 3750 mg of potato to make 1 mL of potato extract was found to be 0.05 mmol/min/mL.
Discussion:
Although the initial velocity tends to increase with increasing substrate concentration, it eventually reaches a plateau where even if substrate increases, the enzyme cannot work faster, resulting in the enzyme to become a limiting factor.
by factors that affect the ability of an enzyme to work such as pH and temperature. Thus, the purpose of this experiment is to primarily isolate the enzyme polyphenol oxidase from a potato extract. When the enzyme and oxygen are exposed to compounds called phenols that are present in the tissues of potatoes, the phenols are converted to other compounds called quinones. The quinones rapidly react with proteins in the potato to make the brown-colored compounds that one can see on the surface of potatoes. The brown bruise is caused by the impact rupturing cells beneath the surface, using oxygen that is available in the cells. The exposure of air to the potato extract results in the enzyme polyphenol oxidase to initiate a reaction. The substrate that the enzyme is changing is the phenotic compound in the potato. The enzyme changes the substrate’s chemical structure causing the potato to inherently change color. Polyphenolic compounds are often found in many fruits and vegetables such as apples, mangos, and potatoes. These polyphenolic compounds also is a factor in the determination of color, aroma, and bitterness of the fruit or vegetable. Since polyphenol oxidase catalyzes the reaction of dopa to form dopaquinone resulting in the immediate oxidation to dopachrome. Thus, the conversion of dopa to dopachrome, an orange colored o-quinone with an absorption at 475 nm, will be imperative for this experiment in order to isolate the polyphenol oxidase to measure the rate of the catalyzed reaction. A 5 mM dopachrome solution provided that has already been oxidized by a 5 mM dopa solution will be the substrate for the experiment. To determine the rate of the catalyzed reaction, application of enzyme kinetics is vital.
The purpose of enzyme kinetics is to determine how fast or efficient an enzyme is as well as how specific an enzyme is. This assists in determining whether a certain factor or treatment affects enzyme efficiency which is inclusive of identification of enzymes and prediction of reactions. Kinetic experiments examine the concentration of a product formed or a substrate consumed per unit of time. Plotting the variation of initial velocity with the substrate often results in a linear graph allowing us to determine the maximum velocity at which it can catalyze the reaction. Therefore, it will be presumed that the result of this experiment by isolating the polyphenol oxidase within the potato extract through the oxidation of 5mM dopa solution with highly concentrated enzymatic preparation of polyphenol oxidase with a buffer as the diluent will indicate a rapid increase in the rate of the reaction as well as the amount of activity present in the potato extract. Proposing that Vmax is proportional to enzyme concentration, a linear relationship should be present and enzymatic activity at varying enzyme and substrate concentrations can be
determined. Methods and Materials: In analyzing the polyphenol oxidase enzyme found in potatoes, PPO activity has to be measured which is done by the instructor by grinding up potatoes to create extract and then performing partial purification in order to remove the endogenous PPO substrates such as tyrosine. After completion of the preparation of the polyphenol oxidase extract, a qualitative PPO enzyme assay has to be completed. Therefore, 1.6 mL of Frozen potato extract was extracted and mixed completely by tapping tube to help thaw and was kept cold at all times. 8 mL of a 1:5 dilution was prepared for both PPO assays and the amount of buffer and enzyme that is serially diluted for each test tube was calculated and recorded. Three test tubes were prepared and only the first three solutions were added which excluded the PPO extract.
Then, enzyme PPO extract was added and a color change occurred. A serial dilution was made and after adding the enzyme extract, each tube was mixed well before a new transfer was made allowing for each tube volume to be 5 mL. The Spec 20 was turned on and warmed up for a duration of 15-20 minutes as the absorbance was set for a value at 475 nm. After measures were recorded from the Spec 20, the dopa was then added to each test tube which initiated the reaction. The rate of reaction was then measured by increase in absorbance over a 4 min. period and the absorbance values were then converted to dopachrome concentrations by subtracting the control tubes from the experimental tubes.
Then, dopachrome concentration was converted to dopachrome amount within each 5mL reaction and dopachrome produced was plotted as a function of time, which created four independent assays. After, the rate of the reaction was determined and the dilution and initial velocity graphed, the enzyme activity for each reaction was determined using the formula activity = product produced/time/volume. Conclusively, the enzyme activity of the undiluted potato extract was determined as well as the amount of PPO present in the potato.
Results:
According to Table 1 of the Qualitative Polyphenol Oxidase Test, the color change present from Tube 1-3 was negative, positive, and negative. The final dopa concentrations varied from 0 mM and 4 mM based on whether buffer volume or 40 mM dopa volume was used due to one test tube being kept as a control and the other as experimental. The mean absorbances based on the standard curve were reported to be -.47, 0.68, 1.07, and 1.44 with absorbances increasing as dopachrome concentrations increased. According to Figure 1 of the standard curve of dopachrome concentrations, the R2 value was equivalent to 0.8495 and it did not heed or produce any negative values. According to Table 1 of absorbance data from time course, the averages of control tubes 1, 3, 5, and 7 were 0.019, 0.032, 0.059, and 0.112. According to Table 2 of the Corrected Absorbance Data, each control tube was subtracted from the experimental tube and then converted to dopachrome amount in millimolars.
According to Figure 2 Enzyme Kinetics: Dopachrome vs time, all dilution series are linear and the series with the greatest R2 value is the 1/10 series dilution with a R2 of 0.9984 and an equation of 0.0002x – 0.0004, with a Vi of 0.002. This equation will then be used to determine the initial velocity of the other series dilutions when plotted creates figure 3 displaying total enzyme concentration vs Vmax as the R2 value of this graph is 0.9725. The average activity of potato extract stock shown on Table 6 found was 13.2 mmol/min/mol and the activity per weight of potato given 3750 mg of potato to make 1 mL of potato extract was found to be 0.05 mmol/min/mL.
Discussion:
Although the initial velocity tends to increase with increasing substrate concentration, it eventually reaches a plateau where even if substrate increases, the enzyme cannot work faster, resulting in the enzyme to become a limiting factor.