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Before we were able to begin with any experiments, the skin of several potatoes had to be extracted and then juiced. To juice the potatoes, about 4 mL of 7.2 pH citrate buffer had to be added to the potatoes peels in the juicer. After the peels and buffer were relatively liquified, the solution was filtered using cheesecloth until we were left with 20 mLs of juice with no organic matter. From this point forward everyone should have been wearing goggles to protect skin from contacting the 8.
8 M H2O2 that we would be using in the remainder of the procedure.
A eudiometer was created by placing a burette upside down suspended in water, with a tube going from the rubber stopper on a flask to inside the burette. H2O2 was plunged into the flask through the stopper by using a 1.0 cc syringe. About 25 mL of catalase stock solution, a 1:4 solution of potato juice to the 7.2 pH citrate buffer, was made.
The first experiment that was performed was the optimal catalase concentration experiment. This was done by preparing 4 flasks with varying catalase concentrations by adding different volumes of solutions to the flasks. 1.5 mL of catalase solution and 3.5 mL of 7.2 pH citrate buffer was added to the 25% catalase concentration flask. The 33% catalase flask had 2 mL catalase solution and 3 mL of buffer. The 50% catalase flask had 3 mL catalase solution and 2 mL of buffer. The 75% catalase flask had 4.5 mL catalase solution and 0.5 mL of buffer. After making up the flasks, 1 mL of H2O2 was added to one flask through the stopper and data was collected every 15 seconds for two minutes by reading the meniscus, with this process repeated for each flask, including setup of the eudiometer for each trial.
After each experiment, the leftover solution in the flasks was disposed of in a correct manner due to the potential of having leftover H2O2 and glassware was cleaned. After this first experiment, a 33% catalase solution was used throughout the rest of the lab due to determining that it was the most efficient enzyme concentration. It was considered most efficient because the most amount of oxygen gas was produced without the eudiometer emptying completely.
Moving forward, we conducted a varying temperature experiment. An additional 40 mL of catalase solution was made for the temperature experiment. 5 flasks were prepared by adding 4.5 mL catalase solution and 0.5 mL buffer to each flask. Each flask was then subjected to one of the temperatures of 5°C, 20°C, 25°C, 30°C, 40°C and then introduced to 1 mL of H2O2. Each flasks was maintained at its designated temperature for at least 5 minutes before beginning the reactions with H2O2 and recording was done as previously every 15 seconds for 2 minutes.
The following experiment tested how variation in hydrogen peroxide concentration impacted reaction velocity. An additional 50 mL of the 1:4 juice to buffer mixture had to be made. 6 flasks were prepared by adding 5 mL of the 33% catalase solution to each and then diluting each flask to the proper dilution with a volume of 6 mL. 6 different microcentrifuge tubes were prepared to make the proper dilutions to be added the flasks. Table 1 shows the mixtures that were prepared in the microcentrifuge tubes and added to the corresponding flask. 1 mL of H2O2 was injected into each flask and again data was recorded every 15 seconds for 2 minutes.
The final experiment that we performed retested how variation in hydrogen peroxide concentration impacted reaction velocity but with a sodium cyanide inhibitor added to the mixture for this experiment. 6 additional flasks were made with the proper volume of 33% catalase stock solution. In this experiment, sodium cyanide replaced the volume of some of the buffer to make the 33% concentration solution. To make the 33% stock solution, 2 mL of catalase solution was mixed with 2.5 mL of buffer and 0.5 mL of the 1.5 mM sodium cyanide inhibitor. Microcentrifuge tubes were once again made as described in Table 1 above. After properly creating the 6 mixtures, the flasks incubated for at least 5 minutes in proper temperatures before adding the H2O2 and recording data.
Our data collection consisted of us recording the meniscus value every 15 seconds over 2 minute time periods, with the meniscus value corresponding to the amount of Oxygen gas created. Multiple graphs were then created in order to more easily analyze our data and share our results. Volume of Oxygen gas evolved (mL) versus time (seconds) graphs were created for each of the reactions above. By locating a linear portion of each graphed line, tangents lines were graphed to represent the velocity of each reaction. Other graph, such as a velocity versus temperature graph and Lineweaver-Burk plots were also generated and the Lineweaver-Burk plots were utilized to identifying the substrate concentration molarities of the graphs.
The first experiment that we performed was used as a control to determine the most efficient enzyme concentration in which to use for the remainder of the experiments. For this reason, the graph is not included within the report as it does not help us experimentally but rather procedurally. The data that we recorded showed that the 25%, 50%, and 75% trials quickly reached saturation of Oxygen gas, whereas the 33% gradually increased in the amount of Oxygen gas produced before the eudiometer ran out of water and overall produced the most Oxygen gas. For all of the experiments, the meniscus reading of the eudiometer was recorded and in order to determine the volume of gas produced, the difference of the meniscus reading and the volume after the initial bubble were calculated.
In the temperature experiment there were to be 6 flasks at 5°C, 20°C, 25°C, 30°C, 40°C and also at 60°C. We did not test for 60°C because of how poorly 40°C performed. We concluded that almost nothing would happen at 60°C because almost nothing happened for 40°C. For the temperature experiment, the data was graphed in a normal line plot and the tangent lines were calculated and graphed along with the points. Reaction velocity could then be derived from the tangent lines and those values are graphed in Figure 1 for the 5 different temperature treatments that we tested. The reaction temperatures that performed the poorest by producing the least amount of oxygen gas were at 5°C and 40°C, the highest and lowest temperatures that we tested. The highest amount of oxygen gas was produced between the temperatures of 20°C and 30°C. The temperature that produced the most oxygen gas was 25°C and so it is at 25°C that the velocity of the reaction is the most rapid of the five temperatures.
Data for the next experiment was collected using the same technique of reading the meniscus but in this experiment and the last experiment, the data reflected substrate concentration rather than temperature. The third experiment performed was variations of hydrogen peroxide concentrations and how that might impact catalase reaction velocity. A normal line plot was graphed yet again, along with its corresponding tangent lines which tell us the initial velocity of the reactions. Figure 2 depicts a Michaelis-Menten plot which represents the relationship between the molar concentration of hydrogen peroxide compared to the initial reaction velocities of each reaction. Viewing the Michaelis-Menten plot for without inhibitor, it can be concluded that the 30% concentration at 1.5 M exhibited the greatest velocity of the reactions.The graph shows that there is a positive linear correlation between H2O2 concentration and velocity. Additionally, Lineweaver-Burk plots were created for the substrate concentration with and without inhibitor experiments. The substrate concentration at which the reaction velocity is half of Vmax (Km), the reaction velocity that the enzyme attains when enzyme is fully saturated with substrate (Vmax), and the rate which allows determination of how fast the reaction from substrate to product is proceeding (Km/Vmax) can all be obtained from the Lineweaver-Burk plots. Figure 3 is a Lineweaver-Burk plot and from it we can calculate the Km and Vmax of the substrate concentration experiment with no inhibitor, which are approximately 3.14 and 4.82.
The last experiment that we performed was still varying substrate concentration but in the presence of a sodium cyanide inhibitor. The procedure remained the same for data collection except that this time there was an inhibitor present in the reaction mixture. Figure 2 and Figure 3 also relate to this experiment, with the relevant data being labeled “vary hydrogen peroxide concentration with sodium cyanide”. The data from the Michaelis-Menten plot (Figure 2) indicates that the 22.5% H2O2 concentration at 1.1 M with sodium cyanide exhibited the greatest velocity of the reactions. The Km and Vmax calculate from Figure 3 for sodium cyanide present were 0.99 and 1.25.
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