Analysis of Peroxidase Content in Apples and Bananas
S. Anderson, N. Downey, G. Soso, M. Van de Brooke
Miramonte High School, Orinda, CA
Introduction:
Peroxidases consist of a family of isozymes that catalyze a variety of reactions in the presence of peroxides (Hamid 2009). A peroxide is any organic compound having two oxygen atoms joined together {-O-O-} (Canada Center 2009). The role peroxidases play in the living plants is not completely understood, although they have been associated with cell wall biosynthesis, response to injury, disease resistance, and wound repair. The properties of peroxidase and its physiological role in pre/post harvest fruits and vegetables has been studied more extensively. It has been known to be involved in deteriorative changes in flavor, texture and color in raw and processed fruits and vegetables (Neves 2000). It is still under investigation, the magnitude of correlation there is between peroxidase activity and fruit ripening (Neves 2000). Different fruits are hypothesized to have different amounts of peroxisomes depending on the fruit, and the stage of ripening of said fruit.
The purpose of our experiment was to test the different levels of peroxidases between an apple and a banana by placing each in a 3% H2O2 solution and recording the pressure change as more or less O2 gas was produced. The difference between O2 gas produced showed which fruit contained more peroxidases.
Abstract:
If we measure the change in pressure during a reaction between Honeycrisp Apple/Chiquita Banana and 3% H2O2 solution, then we will find that bananas yield a greater pressure change because they have a greater peroxidase content, the chemical responsible for reacting with the hydrogen peroxide to produce O2 gas. To test this, we prepared 1cm3 blocks of banana and apple, placed them in 10 mL of solution, capped off the test containers, and used a PASCO Chemistry Sensor to gauge changes in pressure. Then we used the equation PV=nRTto find the mols of O2 gas produced. We also had a negative control test group in which we measured the pressure exerted by apples and bananas that had been microwaved for 3 (three) minutes. The heating should have denatured the peroxidase, rendering it inert. For further reference, we also measured the change in pressure when nothing was placed in a 3% H2O2 solution. With our data, we were able to conclude that bananas are a greater source of Peroxidase than apples.
Materials:
1 DOMEX superfresh growers Organics Honeycrisp Apple
1 Chiquita Banana
1 Carolina SS Pakistan Scalpel
2 Eye-droppers
1 Microwave (1100 Watt, GE Sensor Microwave Oven)
5 (10ml amounts) of Safeway brand 3% Hydrogen Peroxide solution
1 PASCO Chemistry Sensor w/ Pressure Attachments and USB link
Clear Tubing (61 CM)
1 System Container (Glass) with inverted dropper top (See equipment photos)
2 10 mL plastic graduated cylinder
1 100 mL Mallinckrodt graduated cylinder
2 plastic micropipette
1 Computer with DataStudio installed
3% Hydrogen Peroxide Solution Scalpel and Apple Pasco Chemistry Sensor
Chiquita Banana System Container Lab during experimentation
Procedure:
Step 1- Using a ruler, measure and cut 1 cm3 blocks of both banana and apple.
Step 2 - Fill a graduated cylinder halfway up with water, and record the initial volume. Next, drop in a cube of either banana or apple and record the final volume. Remove the cube from the water and dry it off. Calculate the displacement to verify the 1 mL volume of the cube.
Step 3 - Prepare the System Container by attaching the inverted eye-dropper top to the top of the glass container (dropper tube facing up and out of the bottle), then attach the 61 CM clear plastic tubing to the eyedropper tube. Fill both the system container and tube with water and pour into a graduated cylinder. Record volume. Disassemble and dry out the system container and tube.
Step 4 - Reassemble system container, leaving the cap off, and attach the PASCO Chemistry Sensor w/ Pressure Attachment to the free end of the plastic tube. Connect the USB link to the computer, and open DataStudio. Start a new experiment and register the sensor. Calibrate it for pressure tests using atmospheres (atm).
Step 5 - Pour 10 mL of 3% H2O2 solution into a graduated cylinder. Verify 10 mL volume, then quickly pour it into the system container.
Step 6a - For the experimental control Do not place anything into the system container other than the H2O2.
Step 6b - For experiments using uncooked banana or apple Place a single 1 cm3 cube of either banana or apple into the open system container, then immediately close the container.
Step 6c - For experiments using cooked banana or apple Place a cube of either banana or apple into a 1100 Watt microwave and heat it on high for 3 minutes prior to measuring out the 10 mL of H2O2 (this is to prevent any O2 gas from escaping by natural decomposition). Shortly after cooking, measure out 10 mL of H2O2, and pour it into the system container. Place the heated cube into the system container and close the container.
Step 7 - Start gathering data and let it sit for 90 seconds.
Step 8 - After 90 seconds have elapsed, stop the experiment.
Repeat until data for both cooked and uncooked apple and banana have been recorded.
Results:
Graph 1
Chart 1
Peroxidase Source
|
Control
|
Uncooked
Banana
|
Cooked Banana
|
Cooked Apple
|
Uncooked Apple
|
Pressureinitial (atm)
|
.974
|
.974
|
.974
|
.974
|
.974
|
Pressurefinal (atm)
|
.978
|
1.038
|
.981
|
.985
|
.987
|
Pressure (atm)
|
.004
|
.064
|
.007
|
.011
|
.013
|
Mols of O2
|
4.99 × 10-6
|
7.98 × 10-5
|
8.73 × 10-6
|
1.37 × 10-5
|
1.62 × 10-5
|
We began gathering data for all tests once the lids to the system containers were secured, each with an initial pressure of .974 atm. For the next 90 seconds, reactions between H2O2 and peroxidase increased pressure within the container with the production of O2 gas. Our pure hydrogen peroxide control resulted in a positive .004 atm pressure increase; this was the lowest pressure change. Next lowest was cooked banana with a total pressure change of .007 atm, a 75% increase from control. After that was cooked apple with a pressure difference of .011 atm, a 175% increase from control. Next was uncooked apple with a .013 atm pressure change, a 225% increase from control. Lastly, uncooked banana had the greatest pressure change with a net pressure difference of .064, a 1600% increase from control. This data goes along with our hypothesis, banana having given the greatest pressure change thus showing its greater peroxidase content. In addition, our pure hydrogen peroxide solution had the lowest pressure change with the two cooked tests following as their peroxidase enzymes have been denatured and rendered inert. It is worth noting that the cooked apple only had a .02 atm pressure change difference with its uncooked counterpart while the cooked banana had a .057 atm pressure change difference from its uncooked counterpart.
Equations
Pressurefinal(after the 90 second period)-Pressureinitial=Pressure
(Pressure)(Volume)=(Number of Moles)(0.0821)(Temperature(Kelvin))
Discussion and Conclusions:
Our goal was to determine which, between Chiquita Bananas and Honeycrisp Apples, is a better source of peroxidase. We were able to figure this out by placing equally sized chunks of each into a 3% H2O2 solution and measuring the pressure exerted by the O2 gas produced in the reaction.
Our results show that bananas are in fact a greater source of peroxidase. This conclusion is supported when a 1cm3 block of banana in peroxide results in a net pressure change of roughly .06 atm while an apple chunk of equal dimensions yields a net pressure change of about .01 atm. Such a great difference in change signifies bananas’ greater peroxidase content. The banana’s rate of reaction was also much faster. 30 seconds into their 90 second trials, our un-microwaved banana test had caused a pressure change of .034 atm while apple had only caused a pressure change of .008 atm. Based on this data, the banana, only one third of the time in had already released half of the overall gas, while the apple had released less than a third.
There were many sources of error in this experiment. Firstly we failed to control the temperature for the entire experiment. The external environment (the temperature of the room) was changing throughout the course of the experiment and could have easily messed up our data. We could have avoided this if we were in a temperature controlled environment as opposed to a drafty lab room of fluctuating temperature. Also there were a number of problems involved with the microwaving of the two sources of peroxidase. When we microwaved the apple, 3 minutes seemed to be quite effective and the apple cube retained most of his shape and volume. However when we microwaved the banana for the same amount of time, the banana was reduced to a mushy banana powder. This may be a result of the fact that microwaving boils the water within the cube, disrupting its structure, whereas heating within an oven would have been a more natural and stable approach to denaturing the peroxidase. Next, while gathering data, we had to hold the straw connected to the sensor’s tubing upright to keep airflow. This also caused slight fluctuations in pressure within the container, skewing our results. To fix this, we could have arranged for the straw to remain rigid somehow or remain incredibly still for the duration of the experiment. Moreover, it is more than likely that there was a small amount of air leakage where the tube connected to the sensor and straw. This would have lost us mols of oxygen and decreased our change in pressure. Had we taken extra steps to seal all possible leakage sites, this problem could have been fixed or minimized significantly. In addition, we used a ruler to measure the dimensions of our fruit cubes. This is not a very accurate form of measurement and definitely created variation in the sizes of the chunks. To minimize variation in chunk size, it may have been helpful to create some type of 1cm3 mold that we could have used to sort of cookie cut the chunks equally. Furthermore, we placed the peroxidase source in the container and then screwed on the cap, so there was a period of time where the peroxidase was reacting but the pressure was unaffected due to the lack of an appropriate seal. This could have been prevented by placing the peroxidase in the cap and then sealing the capsule. What’s more is that we didn’t take any steps to try and use peroxidase sources of equal freshness and ripeness. The difference in freshness or stage of development may mean a different quantity of peroxidase. If we were able to use fruit of equal quality and development, then it would be even more fair to compare their peroxidase contents. And we wouldn’t have a complete error analysis without mentioning that the number of significant figures we used in finding pressure and other variables limited the accuracy of our data. As always, increasing the number of significant figures would have made our data even closer to its true value.
The next step in peroxidase research of this nature would be to identify and test more sources of effective fruit based peroxidases. This should be followed up by isolating the peroxidase from the specific fruit using centrifuges. Next, synthesization of the peroxidase should be attempted. This would provide a better understanding of peroxidases in nature, their structure, and natural occurrence. Another experiment to try would be to test how the amount of peroxidase within a fruit affects its deterioration of flavor, texture, or color.
There are countless directions to take peroxidase research in. It is relevant to fields of biology and chemistry making it a legitimate subject to learn more about. In the future we will achieve a greater understanding of this enzyme, its role in nature, and practical applications for things like medicine or crop cultivation.
References:
Neves, Valdir Augusto. "Ionically Bound Peroxidase from Peach Fruit."
Brazilian Archives of Biology and Technology. N.p., 31 Oct. 2000. Web.
18 Oct. 2013. <http://www.scielo.br/
scielo.php?pid=S1516-89132002000100002&script=sci_arttext>.
Préstamo, G., and P. Manzano. "Peroxidases of Selected Fruits and Vegetables and
the Possible Use of Ascorbic Acid as an Antioxidant." Hort Science.
American Society for Horticultural Science, 6 Jan. 1993. Web. 20 Oct. 2013.
"What are organic peroxides?" Canadian Center for Occupational Health and
Safety. Canada, 1 Mar. 2009. Web. 19 Oct. 2013. <http://www.ccohs.ca/
oshanswers/chemicals/organic/organic_peroxide.html>.
Hamid, Mohsina, and Khalil -ur- Rehman. "Potential Applications of Peroxidases."
Science Direct. N.p., 15 Aug. 2009. Web. 19 Oct. 2013.
<http://www.sciencedirect.com/science/article/pii/S0308814609002234>.
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