The second enzyme phosphorylase also acts on starch however it breaks down glucose molecules using phosphoric acid. This process is called phosphorolysis, when the chemical reaction uses phosphoric acid to break down molecules. The difference between salivary amylase and phosphorylation is the water concentration doesn't affect the direction of the enzyme activity. During phosphorlysis, the energy from the breaking the bonds between glucose-glucose is used to create glucose-1-phosphate. Therefore, the direction of the enzyme reaction is determined by the concentrations of reactants and products. (Fesht, A., 1977) In the experiment, glucose-1-phosphate and lguose were used to indicate the presence of starch when performing the iodine test.
Materials
The materials is to be followed as described in the Biology 130L lab manual, fall 2009 (Department of Biology, 2009). Pp. 28-45.
Method
The method is to be followed as described in the Biology 130L lab manual, fall 2009 (Department of Biology, 2009). Pp. 28-45. However, start with part two of the experiment, Phosphorylase.
Results
Phosphorylase
Table 1. Test Tube Solutions
The following test tubes were filled with the following solutions. Test tubes #1-7 were tested with for starch with the iodine test, and test tube #8 was boiled in the water bath for 10 minutes.
Table 2. Initial Iodine Test
These are the results of the first iodine test before phosphorylase was added to the solutions.
Table 3. Test Tube Solutions for Iodine Test
These are the final solutions that will be in the test tubes for the iodine test. Test tube #1-3, 5,and 6 had fresh phosphorylase added and test tube #4 and 7 had boiled phosphorylase added (test tube #8).
Table 4. Iodine Test
These are the results of the iodine test after the addition of phosphorylase to the solutions.
Amylase
Table 5. Test Tube Solutions
Test tubes #1-5 were filled with these solutions and then tested with iodine and Benedict test.
Table 6. Initial Iodine Test
After the iodine test, the following results were obtained.
Table 7. Initial Benedict Test
After the Benedict test, the following results were obtained.
Table 8. Test Tube Solutions for Water Bath
The following solutions were added to test tubes #6-15. These test tubes were immersed into a water bath at 37oC for 5 minutes. This is the optimal temperature for salivary amylase activity.
Table 9. Test Tube Solutions for Iodine Test
After the water bath, one by one the test tubes were mixed and were put through the iodine test under time intervals.
Table 10. Iodine Test for Test Tube #11
The following results were obtained after completing the iodine test at 5 second time intervals
Table 11. Iodine Test for Test Tube #12
The following results were obtained after completing the iodine test at 15 second time intervals
Table 12. Iodine Test for Test Tube #13
The following results were obtained after completing the iodine test at 30 second time intervals
Table 13. Iodine Test for Test Tube #14
The following results were obtained after completing the iodine test at 60 second time intervals
Table 14. Iodine Test for Test Tube #15
The following results were obtained after completing the iodine test at 30 second time intervals
Table 15. Benedict's Test
The following results were obtained after the test tubes were in a boiling bath for 5 minutes.
Figure 1. Graph of salivary amylase enzyme vs. time
This graphs shows the effect of concentration salivary amylase on the time it takes for the enzyme reaction to reach the end point.
Discussion
Overall, many of the obtained results were as expected. The phosphorylase experiment consisted of two iodine tests. During the first iodine test, refer to Table 2. Initial Iodine Test, test tube 1-5 were yellow therefore a negative. This is as expected because these there is any detectable starch present in the test tubes. Test tubes 6 and 7 were dark blue black thus they were positive. This result is also as expected since the solutions that these test tubes contained had the presence of starch. After the addition of either fresh or boiled phosphorylase, the iodine test was performed once again. (Refer to Table 3. Test Tube Solutions for Iodine Test for which phosphorylase was added to each test tube).
This time, all the solutions of the test tube were iodine tested based on a 3 second interval. The results of this test is on table 4. As a result, test tube 1,3, and 4 were negative. As expected, this shows that there was no detectable starch in these test tubes. Test tubes 2, 5, 6, and 7 were positive during the second round of iodine test. During round one of the iodine test, test tubes 2 and 5 were tested negative, now they are positive. This means that primer starch was present in the tube, and that the phosphorylase enzyme made the primer starch molecules longer. As expected, the starch was detected during the second round of iodine test. Test tubes 6 and 7 were positive since round one of iodine test, this means that starch molecules were always present in the solutions.
The salivary amylase experiment consisted of both iodine test for starches and Benedict test for reducing sugars. Test tubes #1-5 were put through both of these tests. During the iodine test, test tubes 1,2,3,4, resulted in a negative reaction. This result is as expected because these test tubes only contained salivary amylase solution, therefore no starch molecules should be present. Test tube 5 was the only one who tested positive in the iodine test. This is as expected because this test tube contained 1% starch solution. During the Benedict test, test tubes 3, 4, and 5 were negative. This means that there wasn't any reducing sugars present in the solutions. Salivary amylase enzyme optimal temperature is at 37oC, and this is why we water bath test tubes 5-16 for 5 minutes.
Refering to table Table 9. Test Tube Solutions for Salivary Amylase Iodine Test, there were 5 different iodine test. The iodine test for test tube 11, which was mixed with #6, reach the end point at 45 seconds. The concentration of salivary enzyme is 10%, the highest, in this test tube, therefore it should take the shortest amount of time for the enzyme to break the starch molecules. Referring to Figure 1. Graph of concentration of salivary amylase enzyme vs. time, this result is true. The iodine test for test tube 12, which was mixed with #7, reach the end point at 135 seconds. This test tube contained 5% of salivary amylase solution, therefore the rate of enzyme activity was the second fastest compared to the next two concentrations. The iodine test for test tube 13, which was mixed with #8, reached the end point at 270 seconds. This test tube contained 2% of salivary amylase. The iodine test for test tube 14, which was mixed with #9, reach the end point at 480 seconds. This test tube contained 1% of salivary solution. Test tube 15, which was mixed with #10, reached the end point the same time as test tube 13. As one can see, as the concentration of salivary solution decreases, the rate of enzyme activity takes longer. This is because there is less enzymes working on breaking down the maltose off the ends of the starch chains.
In conclusion, the results of the lab were as expected. When the concentration of substrate was increased, the rate of enzyme activity also increased accordingly. When the rate of enzyme decreased, the rate of enzyme activity also decreased. A possible source of error is while timing the intervals for iodine testing, human reaction is always delayed. Although the instructions say to test it for example every 5 seconds, the time the iodine test performed might actually be approximately 7-9 seconds. This small delay may cause errors in the results.
References
Department of Biology Introductory Cell Biology Laboratory Manual. University of Waterloo, Waterloo. Pp. 38-45
Fesht, A. (1977) Enzyme structure and mechanism. New York: W.H. Freeman and Co. Pg. 288-291
Hasselberger, F.X. (1978). Uses of Enzymes and Immobilized Enzymes. Chicago: Nelson-Hall. Pg. 1-8
Karp, G. (2008). Cell and molecular biology. 5th edition. USA: John Wiley & Sons, Inc. Pp. 94-104
Kuby, S.A. (1991). A study of enzymes. Volume 2. Mechanism of enzyme action. Florida: Library of Congress Catalogingin-in-Publication Data. Pp. 383-388
Shadan, S. (2007). Evolutionary genetics: You are what you ate. Nature, 449, 155.