Turnip Peroxidase Lab

In all living organisms enzymes function as catalysts for chemical reactions. Enzymes are a type of protein which fold in such a way to orient the reaction's reagents into a spot that makes the bond physically easier to be performed. Like all proteins, they are affected by temperature and pH. In this lab, we're technically going to determine the optimal pH of peroxidase from turnips by measuring one of the products of that reaction (O₂), at different pH levels with all other conditions fixed. The educational purposes of this lab are to teach us how to take data in video format and analyze it later.

I hypothesize that the catalytic ability of the peroxidase will be most optimal at a slightly acidic pH, perhaps around 6.0. The reason for this is because in biology, most reactions are performed under acidic conditions.

You measured the color change at different times. Which time will you use for your later assays? Why? (The time/color change that you select will serve as your baseline for additional investigations.)

We will compare the test tubes to determine enzymatic efficiency at around one minute because at one minute the enzymatic rate is still quite linear and does not yet taper off as a result of running out of hydrogen peroxide. For later timestamps, there will be little hydrogen peroxide left in any of the pH levels that worked, because by then the peroxidase will have decomposed all of it.

When you use this assay to assess factors that change enzyme activity, which components of the assay will you change? Which will you keep constant?

We will keep constant the volumes between test tubes, the temperature, and the concentration of the peroxidase enzyme. The only thing that we're going to change is the pH of the liquid water that we add to the test tubes. By varying this factor only, we will be able to determine the optimal pH for which peroxidase operates most optimally.

1.  First, we assembled our test tube racks and set two test tubes in one of the racks.

2. Next using a 10-mL graduated cylinder we measured 7.3 mL of distilled water and added it to one test tube allocated for the peroxidase.

3. Using the same graduated cylinder, we measured 7.0 mL of distilled water and added it to the other test tube allocated for the guaiacol and hydrogen peroxide.

4. Using a clean 10-mL graduated pipette with a pipette pump, we added .2 mL of 3% guaiacol to the test tube allocated for guaiacol and hydrogen peroxide, being sure to shake the guaiacol to be sure it was fully mixed.

5. After that, we used another clean 10-mL graduated pipette to add .3 mL of 0.1% hydrogen peroxide to the same test tube.

6. Next, we used another clean 10-mL graduated pipette to add .2 mL of the 8x peroxidase solution into the test tube allocated for peroxidase.

7. Now, while recording for 5 minutes, we poured one test tube into the other, and back again to be sure they were fully mixed.

8. After our first run, we repeated steps 2-7 only replacing the 7.0 mL of distilled water with 7.0 mL of one of the 6 buffer solutions that we picked (pH 3.0, 4.0, 5.0, 6.0, 8.0, 9.0), and replacing 7.3 mL of distilled water with 1.3 mL of distilled water and 6 mL of the same buffer solution for that run.

The attachment below is a video that we recorded of each test run at different pH levels with all other conditions constant. It's organized from a pH of 4 to a pH of 10, going left to right respectively. We did not make a pH buffer of 7, as that is the pH of distilled water.

Below is the graph constructed by looking at the relative colors of each test tube with the y-axis representing the relative enzymatic activity (as depicted by the color of the test tube,) at one minute, and the x-axis representing the pH that we used for that test tube.

From the data that you collected from your independent investigation, graph the results. Based on the graph and your observations, compare the effects of biotic and abiotic environmental factors on the rate(s) of enzymatic reactions and explain any differences.

If the pH of the solution that the enzymes were in was off, it was clear by our experiment that that effected the enzymes' ability to catabolize hydrogen peroxide into oxygen and water was reduced. Specifically that these enzymes were denatured by the inappropriate pH level, which caused them to function less effectively.

Overall in this lab we learned about conditions affecting enzymatic activity, as well as recording, graphing, and comparing data taken from a lab. We hypothesized that the enzymes would operate most optimally at a slightly acidic environment with a pH of 6.0, however, we partially reject this hypothesis because we found that the optimal pH, although still acidic, was at a pH of 5.0.

If this value is incorrect, it may be due to some of the following experimental or human errors; for one, measuring relative color is not a great standard of measurement for this experiment, and it would've been much more accurate and standard among everyone if we used a spectrophotometer to determine the amount of oxygen produced, or if instead of using guaiacol, we directly measured the oxygen volume over a pneumatic trough. Another potential experimental error has to do with the fact that guaiacol and hydrogen peroxide decompose when exposed to UV radiation, which could've partially impacted our results.

Overall I felt that this lab was a relatively simple one. Enzymes are a concept that we learned much earlier in advanced biology, and I feel like we could've done something a little more rigorous with our time away from the AP curriculum. However, it's still cool to measure enzymatic activity, as this lab is not just about the optimal pH of peroxidase, but the optimal pH of most all enzymes, as most enzymes do operate in acidic environments. There are of course exceptions to this, for example your blood pH is slightly alkaline at 7.4, but it's still good to know that in general, biology tends to prefer acidic over basic.

Ryan Kornas' Lab Report

Keegan Hull's Lab Report