Peroxidase Enzyme Lab Report

Jacob Sweet

10/23/14
Period 5

Peroxidase Enzyme Lab Report

Does mixing enzymes mixed with different pH levels change their rate of reaction?

Introduction:

     In our experiment, we will find out how an enzyme's rate of reaction changes when mixed with different pH levels. To do so, we will get the enzyme hydrogen peroxidase from a bindweed plant and mix it with varying pH levels. After this, the solution will be mixed with hydrogen peroxide to see how large and how fast the reaction occurred. The bindweed enzyme that we are extracting from it corresponds to hydrogen peroxide so it should produce noticeable results. When the enzyme acts as a catalyst to hydrogen peroxide, the hydrogen peroxide enters as 2 H2O2 and is broken into 2 H2O and O2. The amount of O2 produced as a result of the enzymatic reaction will be recorded in terms of the height of bubbles produced.

Purpose:
     The purpose of this lab is to test how much a specific enzyme can benefit from being mixed with different pHs and therefore seeing if the bindweed plant can benefit from being watered with different pH levels since that would help their enzymes work faster.

Hypothesis:
     If the pH levels of enzyme solutions influence their rates of reaction, then the lower the pH level, the faster the reactions will occur.

Justification:
     This was chosen as a hypothesis because the enzymes were taken from plants which naturally live off of rainwater which has a pH level that is lower than neutral (7). Since this is how they are kept naturally they should benefit from that experience and a further extreme of it.

Independent Variable:
     The pH level of the enzyme solution

Dependent Variable:
     The amount of oxygen (bubbles) produced as a result of the mixture

Controlled Variables:

• Temperature of all enzyme solutions/hydrogen peroxide
• Amount of enzyme solution/hydrogen peroxide combined
• The area the bindweed was taken from
• How much water was put into the enzyme solution to help mix it

Materials:

• 21 mL of distilled water
• 10 grams (about 1 handful) of fresh bindweed vines and leaves
• Mortar and pestle
• 1 paper towel (for filtration)
• 3 100-liter glass/plastic beakers
• 1 mL syringe
• 5 mL syringe
• 1 glass test tube
• Test tube rack or holder
• Small plastic ruler (at least 8 cm)
• 1 mL of acid solution with pH between 2 and 3
• 1 mL of acid solution with pH between 3 and 4
• 1 mL of base solution with pH of 10
• 1 mL of base solution with pH of 12
• 10 mL of hydrogen peroxide
• Safety glasses
• Tape (about 4 mm total)
• Timer/stopwatch

Hydrogen peroxide

Bindweed

1 mL syringe

Procedure:

Overview-

     For this lab, crushed bindweed will be mixed with different pH levels and then combined with hydrogen peroxide. The height of the bubbles produced as a result of enzymatic reaction will be measured every two seconds. This will be done with each pH and once that is finished, charts and graphs will be created to show the data.

Steps-

     1. Obtain all materials

     2. Fill one beaker with 21 mL of water and one with 10 mL of hydrogen peroxide

     3. Mix a handful of bindweed with 20 mL of water with a mortar and pestle until most of the bindweed is mushed into a sloshy solution

Mixing bindweed with water

Final solution

     
    
     4. Place a paper towel over the empty beaker
     5. Pour the bindweed solution on top of the paper towel
     6. Pick up the paper towel and squeeze out only the liquids and throw out the rest

Extracting the liquid from the enzyme solution

     7. Tape a plastic ruler to the test tube

Test tube with ruler attached

     8. Pour 1 mL of enzyme extract into the test tube

     9. Add 1 mL of the pH level being tested (2-3, 3-4, 7, 10, or 12) to the enzyme extract

   10. With a timer ready, pour 2 mL of hydrogen peroxide into the test tube and every two seconds record the height of the bubbles in mm to the nearest whole number

   11. Clean out the test tube once the bubbles stop forming or decrease in height

   12. Repeat from step 8 with a different pH level until all of them are tested

   13. Analyze the data and create graphs to represent them

Observations and Data:

Data-

Time and Bubble Height Data Chart

Time in Seconds	pH 2-3 Height in mm	pH 3-4 Height in mm	pH 7 Height in mm	pH 10 Height in mm	pH 12 Height in mm
0	0	0	0	0	0
2	0	0	0	0	0
4	0	0	0	0	0
6	0	0	0	0	0
8	0	0	0	1	0
10	0	0	1	2	1
12	0	1	3	3	3
14	0	3	5	4	4
16	0	4	6	5	6
18	0	5	7	6	6
20	0	5	8	6	7
22	1	6	8	6	8
24	1	6	8	6	10
26	1	6	9	7	12
28	1	7	9	7	12
30	1	7	9	7	13
32	2	8	9	7	13
34	2	8	9	7	14
36	3	8	9	7	14
38	3	9	9	7	15
40	3	9	9	7	15
42	3	9	9	7	15
44	3	9	9	7	15
46	3	9	9	7	15
48	3	9	9	7	15
50	3	9	9	7	15
52	3	9	9	7	15
54	4	9	9	7	15

*Note - The last measurement on this chart was the highest point of the bubbles

This chart was created through Google Spreadsheet to show the exact measurements of bubble height for each recorded time so that all of the data is easily accessible.

Observations - There was not any noticed data that was not shared between all of the test groups. For example, all of the test groups had bubbles form at the bottom and once they stopped forming, some of them stuck to the sides making it look like only the center went down. In addition, the bubbles were relatively small but joined together to create a frothy appearance. Finally, all of the groups retained the same dark lime green color.

Control test at about 10 to 12 seconds

Data Analysis:
     

This graph was also created through Google Spreadsheet to represent the rate of reaction for each pH group tested.

By using this graph it is clear that the pH 12 group had the highest rate of reaction. However, the pH 2-3 test had by far the lowest. The pH 3-4 and 10 had about the same rate with 3-4 being a bit faster overall. Finally, the control group (pH 7) was only a little quicker than the pH 3-4 group but still not close to the pH 12 group.

Discussion:

     With our results, our hypothesis that using a lower pH enzyme solution would help its enzymatic reaction be quicker was proven false. The control group had a faster rate of reaction than the other acidic groups. In addition, the highest pH level surprisingly had much more success creating a faster rate of reaction, contrary to my hypothesis. This should mean that the bindweed plant will benefit from being watered with a higher pH level. I believe this happened due to the bindweed naturally growing in rainwater which has a pH level of about 5.6, meaning its enzymes should also naturally be closer to that pH level. That would happen because the plant is mostly made out of that rainwater. In addition, the hydrogen peroxide is also slightly acidic. Since when opposite pH levels combine they react, that additional reaction of combining a base with acids could have caused a larger reaction, resulting in more bubbles. In conclusion, it is extremely evident that mixing enzymes with different pH levels changes their rate of reaction somewhat drastically.

There was unfortunately some factors that flawed our test data. First of all, the heights were only recorded in whole numbers causing our information to be quite less accurate than it could have been. In addition, the test tubes were tilted when the bubble measurements were being taken which may have caused the heights to appear higher than they actually were. Finally, we had small test groups because we only tested each pH level once which may have caused our data to be far off from the true average.
While our experiment was successful and answered our question and hypothesis, there could be much better versions of it by fixing the previously mentioned issues. Based on our results, we can create a future experiment that tests how high the rate of reaction could go by testing higher and higher pH levels. Likewise, we could also create an experiment that tests how slow the reaction can go by testing lower and lower pH levels until no reaction occurs at all. In addition to more specifically enzyme tests, we can test if watering bindweed with a high pH level increases its growth since the high pH helped its enzymes work faster. In conclusion, nothing went majorly wrong in our experiment so the same strategies and procedures can be applied to similar tests.