Comparison of Catalysis Rates in the Decomposition of Hydrogen Peroxide using Apple, Lemon, and Avocado

Comparison of Catalysis Rates in the Decomposition of Hydrogen Peroxide using Apple, Lemon, and Avocado

Abstract: Peroxidase catalyzes oxidation of hydrogen peroxide. In denaturation in plants, enzymes like peroxidase are broken down under conditions of high heat, salinity, and high levels of pH. We hypothesized that in higher levels of acidity (lemons) the peroxidase would denature and therefore the rate of decomposition of hydrogen peroxide would be unaffected by the enzyme, and that the neutral pH of the avocado would be more effective at lowering the activation energy of the reaction. Our hypothesis was supported by our data, showing that the results of the acidic fruits were lower, as expected. The neutrality of the pH of the avocado proved the most effective.

Introduction: Peroxidase, an enzyme found mainly in plant cells,  catalyzes the oxidation (decomposition) of hydrogen peroxide. In plants, its main physiological functions include participation in late-stage lignin formation, and the protection and repair of tissues damaged by pathogenic microorganisms (Civello, et. all). Since peroxidase is an enzyme, its role in chemical reactions can be impaired by denaturation, the unraveling of the enzyme’s shape and subsequent loss of function. Denaturation occurs under conditions of extreme heat, salinity, or pH (Campbell). Hydrogen peroxide exists naturally in almost all organisms, and is produced as a by-product of cellular respiration. Therefore, enzymes (peroxidases) are needed in order to speed up the breakdown of hydrogen peroxide into water and oxygen (Pertucci). Our experiment examined the effects of the pH of different sources of peroxidase on the oxidation rate of hydrogen peroxide (H2O2) into liquid water (H2O) and oxygen gas (O2). Specifically, we examined three different fruits: lemons, apples, and avocados. All of these fruits contain peroxidases, but the relative acidity of the lemon and apple denature enzymes in the fruit and prevent them from catalysing the reaction as effectively as avocado, a relatively pH neutral fruit. In addition, the specific acid that is present in lemons and apples, ascorbic acid (Morton), and, has been shown to slow down the rate of oxidation reactions (Gonzales, et. all).

For the purposes of our experiment, we hypothesized that the lower pH levels in lemons and apples would cause the peroxidase to denature, subsequently reducing their catalysis rates in the decomposition of hydrogen peroxide, while the functional peroxidase enzymes in avocado would effectively catalyze and increase the rate of the reaction.

Materials

1 Eureka lemon

1 Honeycrisp apple

1 Hass avocado

84 mL of  Safeway brand 3% Hydrogen Peroxide

1 glass flask (volume of 37mL) and cap with tube inserted (1.98 mL)

1 plastic tube to attach to pressure sensor (7.54 mL)

1 thermometer

1 PASCO PasPort Absolute Pressure Sensor

1 DataStudio program or other data collecting method

1 USB connection cord

1 ruler

1 scalpel

1 Pair of forceps

1 Microwave (General Electric Co, model # JES1451WJ 02)

3 Microwave safe glass flasks

1 Graduated Cylinder

Procedure:

1. Assemble the data collecting system, by attaching the plastic tube into the cap of the reaction chamber and inserting the USB cable into a compute. Make sure DataStudio has recognized the device and it ready to record data.

2. Using the scalpel,  cut each fruit into two, 1 cm2 cubes.

3. Pour 14 mL of H2O2 into the flask.

4. Add the apple cube.

5. Close the system as tightly as is possible and start recording atm/s immediately using DataStudio. Make sure that the PASCO pressure sensor has been turned on.

6. Record the pressure (in atmospheres) every 5 seconds for 1:30.

7. After 1:30, stop recording and take the temperature of the system with the thermometer.

8. Using the forceps, dispose of the fruit piece and dispose of  the H2O2  

9. Repeat steps 3-8 twice more with the lemon and avocado, each time recording immediately.

10. Microwave the second piece of each fruit in the microwave safe glass containers on high power for one minute. These fruits will serve as the negative controls

11. Repeat steps 3-8 with the microwaved fruit.

12. Graph the data and find the average change in pressure in each test with respect to time.

13. Isolate the number of moles using the Ideal Gas Law to find the rate of the reaction.

Results:

Pictures:                          

Fig 1-Slicing the lemon into           Fig 2-The relatively inactive                   Fig 3- Measuring the temp.

cubes                                                 lemon reaction                                        of the reaction

                                     

Fig 4-Slicing the avocado               Fig 5- The highly active           Fig 6- The fruit after being

into cubes                                         avocado reaction                     microwaved

  

Fig 7-The microwave                      

Graphs: ** noted in our discussion, all our numbers are off by around a factor of 100 atmRate of Change of Pressure With Respect to Time

Trial	Pressure per second (atm/s)
trial 1 (apple)	.000111
trial 2 (lemon)	0
trial 3 (avocado)	.002666
trial 4 (dead apple)	.000111
trial 5 (dead lemon)	.000111
trial 6 (dead avocado)	0

(atm/s; found using (Pfinal-Pinitial)/(Tfinal-Tinitial))

Catalysis Rates:

V=42.04 mL

R= .082 (L*atm)/(K*mol)

Reaction Rate = (P/s) * V/(RT)

Trial #	Pressure/second (atm/s)	Temperature (K)	Reaction Rate (mol/s)
Apple	.000111	293.5	.000194
Lemon	0	293	0
Avocado	.002666	294	.004650
Dead Apple	.000111	292.5	.000195
Dead Lemon	.000111	293	.000194
Dead Avocado	0	293	0

Summary of Results

Taken as a whole, our data supports our hypothesis: The lemon and apple had slower reaction rates than the avocado. Trial 1 exhibited a quick jump of .01 atm to reach 1 atm at 5 seconds, and then remained at a constant pressure for the rest of the trial. Trial 1 had a reaction rate that was 183.9% that than the live avocado, Trial 3. The lemon in Trial 2 showed no change in pressure whatsoever. The Avocado in Trial 3 exhibited the fastest reaction rate of all the trials, a rate almost 184% faster than the next fastest trial, Trial 4 (.00465mol/second vs. .000195mol/second). Trial 4 also had the same initial jump in pressure from .99atm to 1atm, so its reaction rate also was .000195mol/sec. Trial 5 was nearly identical to Trial 4, with a reaction rate of .00194mol/sec. Finally, Trial 6 showed no changes in pressure at all, as with Trial 2.         

Discussion

Our experiment investigated the effect of various organic sources of peroxidase on the reaction rate of the decomposition of Hydrogen Peroxide. We hypothesized that the acidic nature of the lemon and the apple would inhibit the function of the peroxidase enzyme by denaturation, and not be as effective as the more pH neutral avocado in catalyzing the reaction rate. The data we collected from our trials supported our hypothesis.

Our results from the apple trials were as expected. Due to the high levels of ascorbic acid in the apples, the reaction rate of the system with apple cubes was close to zero. In fact, the levels were very similar to the rate of the reaction performed with the negative control apple cube. This suggests that the peroxidase in the apple was denatured by the high acidity of the apple and proved to be an ineffective catalyst in the reaction. The pH of an apple is between 3.3 and 3.9 (Food Safety). Similarly, the lemon, with a pH of 2.0-2.9, is much more acidic, and it shows in the results. The lemon trial resulted in a very low reaction rate, close to 0 moles per second. Once again, the lemon trial had a very similar outcome to the dead lemon trial, due to the fact that the acid in the lemon had denatured the peroxidase. Finally, the avocado has a pH of between 6.27 to 6.58. The drastic difference in the pH between the apple, lemon, and the avocado reflects the difference in the rate of the reaction. The reaction rate of the avocado trial was almost 183.9% faster than the apple trial, and .02666 mol/s faster than the lemon trial. Our experiment has clearly proven that peroxidase is more efficient in neutral conditions, because acidic conditions cause the protein to denature.

One source of human error in our experiment is the fact that we did not cut the fruits into precise and exact 1cm x 1cm cubes. Cutting them precisely would allow for near exact results. If the cubes are just slightly bigger, then the surface area would have increased, causing the reaction rate to increase because the hydrogen peroxide would be exposed to more peroxidase, and therefore yield inaccurate results. If the cubes were slightly smaller, then the surface area would have decreased along with the reaction rate and therefore also yield inaccurate results. Some of our cubes may have been slightly bigger or slightly smaller. To fix this we could use a more precise cutting tool or choose fruits that are easier to cut. A source of design error was the amount of time it takes to denature peroxidases in the cubes of fruit to yield a negative control. We heated each cube for a minute in the microwave. It may have not been long enough and therefore it may have not fully denatured the peroxidases in the fruits. This would allow them to partially react with the hydrogen peroxide and therefore would not serve as an effective negative control in a case like this. To fix this we would perform numerous trials in which we heat up the fruits for different amounts of time. This would allow us to confirm the amount of time it takes to denature a peroxidase. After confirming this amount of time we could then proceed to perform the experiment. The fact that we did not perform enough trials is also another source of design error. Ideally we would have done ten trials for the fruit cubes with peroxidases, and another ten for the cubes in which the peroxidases were denatured to yield more accurate results. Another source of design error was the fact that we did not use different fruits with a wider range of pH levels. Because we did not do this, our results were closer to zero than would have been optimal. To fix this we simply need to research more fruits and pick different ones to yield a wider variety of acidity in our experiment, and therefore yield a wider variety of data. Another source of design error was that we could have used a more accurate sensor, which would have resulted in  more accurate calculations and data. In addition to the lack of precision of our instruments, our pressure sensor clearly measured the pressure in all of our reactions to be in the range of 100 atms to 120 atms. Common sense would seem to say that this scenario is highly unlikely, but since we could determine no faults in our data collection, and because the probe clearly read 100 atms, we had no choice but to accept the readings of the probe as accurate. However, this inconsistency was corrected in our calculations, as we divided every calculation by 100 to compensate for the inaccurate measurement. A final source of design error would be the ripeness of the different fruits. Since ripeness is more of a subjective and qualitative observation that differs for each fruit, it would have been impossible to standardize a level of ripeness that could be replicated by another person doing the experiment (possibly at a different time of year, which could also affect the conditions of the fruit due to seasonal growing trends).

A follow up experiment can be performed. The procedure would be similar to ours but there would be fewer errors. In this experiment, cubes would ideally be cut into precise cubes (as close to perfect as possible) and the sensor would be of higher quality and more precise. Doing both would yield more accurate results. Also, the time it takes to denature the peroxidases in the fruits would be confirmed before performing the experiment to produce more accurate results. Also, there should be a wider range of pH levels in this follow-up experiment to produce a wider range of results. And of course, more trials would be performed to yield more data. Having more data would result in higher accuracy.

Peroxidase enzymes have great potential for use in the world today: not only do they have the potential to assist in the manufacturing process of adhesives, computer chips, and car parts (Tuhela, et. all), but they also are crucial in the breakdown of potentially harmful hydrogen peroxide into harmless water and oxygen gas. Since hydrogen peroxide is used extensively in everyday life (as a bleach, a disinfectant for inanimate objects, in toothpaste, and in acne treatments), a peroxidase could be used to accelerate the rate of the reaction in one of these applications, perhaps when too great a dose of hydrogen peroxide has been used and needs to be quickly converted into water and oxygen gas. Therefore, it is important to know the organic sources of functional peroxidases (and non-functional, denatured peroxidases) for a potential application in everday situations.

References

Campbell, Neil A., and Jane B. Reece. Biology Seventh Edition. San Francisco: Pearson Education, 2005. Print.

Morton, Julia F. "Lemon." Lemon. N.p., n.d. Web. 21 Oct. 2013.

"Peroxidase from Strawberry Fruit (Fragaria Ananassa Duch.): Partial Purification and Determination of Some Properties." ACS Publications. N.p., 1995. Web. 22 Oct. 2013.

Petrucci, Ralph H., and William S. Harwood. General Chemistry Principles & Modern Applications. New York: Macmillan, 1993. Print.

"PH Values of Common Foods and Ingredients." Food Safety. N.p., n.d. Web. 21 Oct. 2013. <http://www.foodsafety.wisc.edu/business_food/files/Approximate_pH.pdf>.