The pH; even a fairly noticeable change in the pH can change the reaction heavily. The optimum pH is found where the reaction works best. When the pH is then still rising the enzyme will not be broken down, (most of the times), the reaction will start decreasing again.(refer to graph #1)
For starch the optimum pH is supposed to be around 7 which is neutral.
Therefore I predict that the reaction will work best at a pH of 7. It will be lowest at around 1, at two I expect it to slightly increase, so with 4. because of the fact that the effect of pH is irreversible, at 9 I expect it to start decreasing again.
Temperature, substrate- and enzyme concentration will have to be at a constant level otherwise the results will be affected.
Planning b:
Materials:
6 boiling tubes
6 test tubes
2 spotting tiles
marker pen
2 dropping pipettes
stopwatch
iodine solution
100 cm³ of buffer solution with 5cm³ graduated pipette
100 cm³ 2% starch solution with 5 cm³ graduated pipette
Method:
First of all 6 boiling tubes have to be labelled with the different pHs from 1 to 9.afterwards 2.5cm³ of 2% starch solution will be added to each tube. Meanwhile two or three spotting tiles will be filled with samples of iodine solution. Another 5 test tubes will be filled with 5 cm³ of amylase solution. The reaction starts when the amylase is added from a test tube to another test tube containing the starch and pH8buffer) mixture, and mixed well by shaking.
Immediately a sample will be taken and placed into a tile. This will be continued every 30 seconds and for minutes. After 5 minutes samples will be taken only every minute.
Supposedly twp tubes will be run at the same time.
The process will be continued for twenty minutes if necessary. Meanwhile the experiment has to be started with other tubes, otherwise there will not be enough time.
It is important to rinse the pipettes after each experiment. If the different pHs would mix the pH concentration would change it this would severely affect the results.
After each experiment the time it took the reaction to end will be noted. The reaction will have stopped when any blue/ black colour disappears, this will signify that the starch has broken down completely.
Data processing and presentation:
Data analysis:
When referring to graph #2 and checking the results for pH 1 the hypothesis seem to be correct. The time for the blue/black color to disappear was 170 seconds, means the starch was broken down completely after this time.
The time for the pH 2 was 80 seconds, the result for the pH of 4 was 120sec. The time in pH 4 is a bit of an anomaly because the reaction is supposed to go faster with increasing pH. Still, the reaction rate was not a s slow as in the experiment with pH 1.
I expected the reaction to be at its maximum (fastest) with a pH of 7, but this did not occur. The time for starch to brake down completely was 140 sec. Almost as slow as with the pH 1.what could have been the reason for this anomalous result?
First of all, as I said in my method it could be that the pH of 7 was mixed by using the same pipette for two different experiments. This could have changed the pH concentration easily.
Another reason could be the measuring of the time. We did not use a stopwatch, instead we used a normal wall clock. Even though I tried to measure the time accurately, some times I just forgot to take a sample after 30 seconds and I did took after 40/ 50 seconds.
At the pH concentration of 9 the reaction rate increased further, which is another anomalous result. Supposedly the reaction should take even more time because once the optimum pH is reached the reaction does not speed up anymore. However the enzyme is not denaturated because the pH effect is reversible (in most of the times), but the reaction decreases.
When referring to graph #2 it can also be seen that the reaction rate decreases at 4 and 7, therefore something must have gone wrong.
Overall the pH concentration where the enzyme worked best was at the pH around 2. if we would have analysing a protein digesting enzyme in our stomach this result would have been reasonable because there reaction rates are supposed to work very good at very acid pH concentrations.
Conclusion and evaluation:
Overall the experiment was conducted as described in the method.
But I cannot really accept my hypothesis, because the was nor a constant increase in the reaction rate nor was the reaction working best at the neutral pH of 7.
As I said before we did not use a stopwatch to measure the time, instead a normal wall clock. I believe that this did affect the results in some way. We tried to measure the time accurately but sometimes we forgot the time were we had started or during the 30 seconds we were filling tiles with iodine solution instead of paying attention to the time. To improve the results time should measured accurately with a stop watch. In addition enough tiles should be filled with iodine solution.
Another factor which probably influenced the result heavily was that the dropping pipettes were either not cleaned properly or not rinsed at all.
Also it could be that the reaction rate had stopped before or between the 30 seconds, so it is hard to get the right time.
Due to the fact that we got such unexpected results for the pH concentration of 7, we did the experiment for this concentration again, next time to approve or improve the results the whole procedure should be repeated.
Bibliography
-Written notes from class
-“Chemistry in action “
Michael freemantle
London, 1987
- “Advanced Biology, principles and applications”
CJ Clegg with DG mackean