4. This solution is blended on high just long enough to break open the cells, approximately 5 seconds. 5. This is then strained through 3 layers of cheese cloth into a beaker and kept cold in order to help prevent oxidation of the enzymes. 6. Each tube is filled up to the 1 cm mark with the solution matching the pH written on it. 7. Each test tube is then filled up to the 2 cm mark with potato extract and mixed. 8. Next, each tube is filled to the 3 cm mark with catechol and mixed well. 9. Any color changes are observed and documented using a plus and minus scale at 5 minute intervals for a total of 20 minutes. Raw Data:
Change in Color of Potato Extract with Different pH Values over Time Based on Color Intensity Scale
Tube 7 with the neutral pH and tube 3 with a pH of 3 did have an increase in color. However, after 10 minutes, their colors remained the same. Tube 6 containing a solution with a pH of 6 had the greatest steady increase in color throughout the 20 minutes. Therefore, 6 is the optimal pH for catechol oxidase.
This graph displays a visual for the result that tube 6 with a pH of 6 had the greatest color change signifying 6 as the optimal pH of this enzyme. Tube 7 with a pH of 7 is close behind it. Tube 3 had a slight increase in color, and tube 10 had absolutely no increase in color. This is because of the extremities of pH values. This caused the enzymes to denature. Post-Lab Questions
1. Based on your results, what was the optimum pH for catechol oxidase activity? pH of 6 2. What effect does a high or low pH have on enzyme activity? Why? When the pH of enzymes reaches too high or too low, the enzymes become inactive because the enzymes denature. 3. Based on your observations, why do cytoplasm and many body fluids have a pH measure near 7? 7 is the near the optimal pH for the potato extract. It works the best because it is the exact difference from both extremes on the pH scale that result in denaturation.
The hypothesis for Lab 6.A was that if temperature has an effect on enzyme activity, then there will be an increase in color change in the potato extract as temperature increases; however, when the catechol oxidase exceeds its optimum temperature, the reaction will stop abruptly due to denaturation. The data obtained from the lab supports this hypothesis. The greatest enzyme activity was apparent when the temperature was at 40ºC in tube 3 because it changed color the most and the quickest proving the most development of benzoquinone.
The two temperatures lower than this, 0ºC and room temperature (28ºC) in tubes 4 and 5, did have an increase in color, but it was not as defined as that of tube 3. Tube 1 containing the control, water, did not change color at all as expected. The solution in tube 2 at 100ºC did not have any color change either. This is due to the denaturation that occurred because of the extreme heat. It can be concluded that the optimal temperature for the enzyme catechol oxidase is somewhere between 40ºC and 100ºC.
Lab 6.Bs hypothesis was that if enzyme activity is impeded by inhibitors, then PTU will halt the oxidation process of the potato extract. This hypothesis was also supported by the data because PTU was the only potential inhibitor out of the three that did not result in a change of color. The addition of water and sodium phosphate result yielded a similar color change.
This suggests that they do not inhibit the enzyme catechol oxidase while PTU acts as a competitive inhibitor because there was no color change in the solution. PTU bonded to the active site of the enzyme catechol oxidase and did not allow the substrate catechol to enter. The hypothesis of lab 6.C stated that if enzyme activity is affected by the pH of a solution, then the enzymes will experience the greatest activity at a pH of 6. The data supports this hypothesis as well.
The test tube using the buffer solution with a pH of 6 had the greatest color change out of all of them. This suggests that there was the greatest amount of enzyme activity at this pH level because the greatest amount of benzoquinone was produced. Not much less color change than that is the tube with a pH of 7. The tube with a pH of 3 did have a change in color, but it was not very significant. Finally, the test tube with a 10 pH did not have any color change at all. It can be concluded from this that catechol oxidase denaturizes when it reaches the pH of 10, and the optimal pH is 6 where the enzymes experienced the greatest activity.