Running head: LAB 5: INVESTIGATING AN ENZYME-CATALYZED REACTION

Lab 5: Investigating an Enzyme-Catalyzed Reaction

September 24, 2014
Principles of Biology 120.601
Mrs. Annemarie Duncan

Abstract: (Burmania)
This experiment was performed in order to examine ways in which a potato catalase enzyme reacts to various assays with differing variables. To do so a baseline assay (undiluted extract and room temperature H2O2) was used within the experiment with only one other variable changed in the other assays. These variables included a boiled, frozen and then thawed, and frozen potato extract and dH2O instead of the potato extract. It was noted that the temperature and or way the potato extract was prepared effects how the enzyme with the potato will react. Therefore the results of each assay varied, suggesting there is more than one way that a catalyzed reaction can occur.

Introduction: (Burmania)

The main purpose for this experiment was to explore how an enzyme catalase caused a reaction to catalyze through doing various assays using potato extracts. Enzymes are catalysts that are crucial in helping to speed up reactions, and catalase is a common enzyme found in almost every living organism that is exposed to oxygen. For this experiment, potatoes were used because they were previously known to have significant catalase activity. The potato catalase was mixed with hydrogen peroxide because the catalase helps to breakdown the hydrogen peroxide which helps determine how long a paper disk takes to float to the surface, which in return provides the time of the complete reaction. Since various assays were mostly used in combination with the potato extract, the extract should aid in speeding up the reactions with the assays that it is used. With the following information, the various ways that the assays were prepared within the realm of temperature differences, it was hypothesized that there would be a significant rate of reaction. If the reactions were to be similar, it would suggest a null hypothesis. Safety goggles were worn whenever handling the hydrogen peroxide.

Materials and Methods: (Allen)
Before this experiment begins, potato extract will be used as a source of catalase enzyme. In order to prepare the extract, 300 grams of potato are blended with 300 ml of ice and water. It than will be filtered through cheesecloth and the filtrate adjusted to a final volume of 600 ml. The extract should be kept on ice throughout this whole experiment. There will also be 3 other extracts experiments to do, one test tube full of the extract that will be thawed for 30 minutes before anyone is able to use it. The question is does different temperatures of the extract affect the outcome of the soaked paper disk. The materials needed for this experiment will be 0.3% of Hydrogen Peroxide, a 10ml of pipettes, pipetters, P1000 micropipette, blue tips, test tubes, racks, filter paper disks, graduated cylinder, flasks, beakers, forceps, timer, tape, markers. (Should be wearing goggles at all times during this experiment.) Also, the experiment will need some additional supplies, ice bucket, hot plate, water baths, and refrigerator. Experimental Procedure: (Allen)
In this experiment, before anything else clean all glassware that is being used in this experiment to get better results, than obtain about 100 mL of 0.3% H2O2 in a clean flask. After that obtain about 50 mL of potatoes extract and keep it on ice. Pipette 5 ml of 0.3% H2O2 into a clean test tube. Use tweezers to take a single piece of filter paper (do not touch with hands) and soak the paper disk in potato extract for 5 seconds or until it is completely covered in the solution.) Than place the disk into the test tube of H2O2 and wait for it to sink to the bottom and then start the timer. Once the disk rises to the top stop the timer and record the time. Do this 4 more times and record each result. Once that one is done do the Boiled assay two times. (Stop after 10 minutes), the frozen extract one time (Stop the trail after 10 minutes), the Frozen thawed one times (stop trial after 10 minutes) and the dH2O one time as well (stop the trail after 5 Minutes). Record all the data.
Results: (Allen) * Raw Data:
Table 1: Took other students results and compared them. This table shows all of the results from each result the whole class got. Down below in the red is the average for each of the assays.

Table #2: This is the Anova of the assays including all of the other student’s results except for the average of all.

Table 3: The averages of each assay only no other data in involved with this one. | ANOVA | | | | | | | | Source of Variation | SS | df | MS | F | P-value | F crit | | Rows | 107983.3 | 4 | 26995.83 | 1.172483 | 0.371082 | 3.259167 | | Columns | 464015.6 | 3 | 154671.9 | 6.71771 | 0.006532 | 3.490295 |

Figure 1: This is a graph of the averages of each assay including the other student’s results as well.

Important Results: (Allen)
The Average of the Boiled and dH20 assay were the only two that did not rise to the top at all. The averages just leveled out. Boiled was 600 seconds average and dH2O was 300 seconds, but the baseline assay has the disks float to the top in a matter of seconds.
Calculations: (Allen)
There was only one calculation that was able to be used for this experiment and it was to convert everything from minutes into seconds. For example, for 10:00 minutes, do 10.00 x 60 = 600 seconds. Once converted into second, it was able to be converted into a graph. That was the only calculation that was used during this experiment.

Discussion: (Oury)
A catalyst is a substance within all living things that speeds up a chemical reaction. An enzyme is a molecule that allows the chemical reaction to take place. In this experiment, we used a potato extract enzyme (catalase) in a reaction with hydrogen peroxide to observe decomposition, or the breakdown of substances. The catalase works to speed up the decomposition of hydrogen peroxide into oxygen and water. This reaction can be seen by the bubbling of hydrogen peroxide once the filter paper discs are submerged in the liquid. This experiment tested how different temperatures of catalase will affect the decomposition process.
Referring back to Table 1 in the results, you can clearly see that the boiled catalase produced little to no reaction at all. This is because when you boil a potato, you are destroying the catalase enzyme making it unable to decompose the hydrogen peroxide. The frozen and thawed catalase produced a slow reaction, with less bubbles, showing that it was able to decompose hydrogen peroxide, but at a slow rate. Room temperature catalase seemed to produce the most obvious and quickest reaction, allowing us to understand that catalase works best at room temperature.
Our group tried to use a different type of paper to replicate this experiment. Changing this variable, we did not expect to see any significant change in our results. The first time we conducted this experiment, we used filter paper. Filter paper is highly absorbent allowing liquid substances to attach to it. When we dipped the filter paper in the potato extract, the catalase enzyme was absorbed into the filter paper allowing the reactions with hydrogen peroxide to take place. On the second day, we used colored printer paper with a smooth finish purchased from office depot. When repeating the experiment with this paper, we noted that the paper discs would not sink, and if they did sink, they immediately rose to the surface of the hydrogen peroxide. No bubbling occurred which alerted us that no reaction had taken place. We suspect that the smooth finish of the paper did not allow the catalase to absorb into the paper. This would be why the paper discs did not sink at all. When the paper discs did sink, but immediately rose to the surface of the hydrogen peroxide, we suspected that a small amount of catalase may have attached to the surface of the paper (adsorption).
By conducting this experiment, we are now better able to understand the effects of a catalyst on a reaction. If all living things contain catalysts, such as catalase, that means that our bodies also contain catalysts. By understanding that a catalyst is used to speed up a reaction, we can also understand that our bodies require temperature control to be able to perform different reaction efficiently.

Conclusion: (Puccini) In conclusion, we have seen how a catalase enzyme can cause a reaction by breaking down or decomposing hydrogen peroxide. Through performing the experiment (soaking a paper Disc in potato extract and submerging it in hydrogen peroxide) and observing the data that was collected (how long it took the disc to rise to the top of the hydrogen peroxide) we have seen and proved our hypothesis that temperature differences or changes will affect the reaction of a catalase enzyme. We have also seen how the preparation of the potato extract will affect how the enzyme with the potato will react.

References: (Oury)
Lab 5 & 6 Instructions provided by WCC http://www.education.com/science-fair/article/activator/ http://www.officedepot.com/a/products/170719/Neenah-Astrobrights-Bright-Color-Paper-8/;jsessionid=0000rGCdAB2-lbq8dlgKOHOe_ui:17h4h7c4g#firstTab