The table above shows the qualitative results from each iodine test trial at varying enzyme concentration. It also shows the intervals (in seconds) in which the results were recorded.
Table 3. Results for the Benedict’s solution test for test tubes 16-20
This table shows the qualitative results from the Benedict’s test for each of the test tubes (16-20) after the end-point was reached for the solutions.
Graph 1. Time required to reach end-point VS salivary amylase concentration graph
The graph above shows the relationship between the different salivary amylase concentrations and the time that it takes to reach the end-point.
Table 4. Results of the iodine test for test tubes 1-7
The table above shows the qualitative results from the iodine test for each test tube. Every iodine test was done at 3 minute intervals, for approximately 20 minutes. In test tube #4 and #7, instead of fresh phosphorylase, boiled phosphorylase was used.
Discussion
In the first part of the experiment, the hydrolysis of starch through the use of salivary amylase, the theoretical expectations stacked up with the actual results of the lab. In table 1, the iodine and the Benedict’s test was conducted on test tubes 1 through 5. In the iodine test results in table 1, which tests for starch/glycogen, test tubes 1 (10% salivary amylase solution), 2 (5% salivary amylase solution), 3 (2% salivary amylase solution), and 4 (1% salivary amylase solution) were all yellow, which indicates a negative result in the detection of starch. This is to be expected as amylase is an enzyme and not a starch. Test tube 5 (1% starch) however, tested positive in the iodine test in table 1, for it has a small amount of starch in the solution. In the Benedict’s solution test, test tube 1 (10% amylase solution), and 2 (5% amylase solution) produced a green precipitate, which indicates a positive result for a reducing sugar. The amylase solution produced a positive result for it contains small amounts of lactose within the solution. Test tubes 3 (2% amylase solution), 4 (1% amylase solution), 5 (1% starch suspension) remained a blue solution after being placed in the boiling bath, thus the test tubes are negative for reducing sugars. In these first tests, the starch solution was the positive control for iodine. These first two tests are done so that comparisons can be made after the enzymes have been added (Department of Biology, 2009).
In table 2, starch is added to the enzyme solutions and the reaction was allowed to take place. Test tube 14 (1% amylase solution + 1% starch), was recorded at 60 second intervals and it took, 8 iodine tests before it produced a negative result, thus it required 480 seconds for test to reach the end-point (the point at which the iodine test first become negative). Test tube 13 (2% amylase solution + 1% starch), was recorded at 30 second intervals and the time it took to reach the end-point was 270 seconds. Test tube 12 (5% amylase solution + 1% starch), was recorded at 15 seconds intervals and it reached the end-point at 75 seconds. Test tube 11 (10% amylase solution + 1% starch), recorded at 5 second intervals, took 25 seconds to reach end-point. Test tube 15 (water + 1% starch), was the positive control of this part of the lab, and because this test tube had no enzymes, the reaction could not take place, thus, the test tube continued to test positive throughout the tests. What is shown in the table 2 and in
graph 1 indicates that the relationship between the concentrations of enzymes is indirectly proportional to the amount of time for the end-point to be reached. Therefore, if the concentration of the substrate remains the same, as the concentration of enzymes increase, the amount of time to reach the end-point decreases.
In the next part of this experiment, a small amount of the solutions from the previous part that underwent reaction was removed and tested with the Benedict’s test. Table 3 shows that test tubes 16 (containing 2mL of solution from test tube 11), test tube 17 (containing 2mL of solution from test tube 12), 18 (containing 2mL of solution from test tube 13), and 19 (containing 2mL of solution from test tube 14) produced an orange precipitate on the top layer. Therefore, these test tubes (16, 17, 18, and 19) are positive for reducing sugars. This is expected as starch is made up of glucose monomers, and as the reaction took place, the starch molecules were broken up into singular glucose monomers, thus producing a positive test in the Benedict’s test. Test tube 20, which contained the water and the 1% starch suspension, again acts as a negative control. The starch was not broken down in test tube 20, therefore a negative result in the Benedict’s test.
In the phosphorylase section of the lab, the purpose was to investigate the effects of an enzyme on a reaction at equilibrium. The reaction goes as follows:
(Glucose)n+HPO42 ←→ (Glucose)n-1 + Glucose-1-phosphate
Because of the fact that enzymes do not change the direction of reactions but merely the rate of which the reactions reach equilibrium, they are able to work in both directions (Department of Biology, 2009). Enzymes can only accelerate the rate at which a favourable chemical reaction proceeds (Karp, 2008).
In table 4, test tube 1 (1.5mL of 0.01M glucose, and 1 drop of 0.2% starch suspension) tested negative for the first 5, 3 minute intervals, but for the last two intervals (at 18 minutes and 21 minutes), the test was positive. This was not an expected result as phosphorylase does not work on just glucose on its own (Department of Biology, 2009). The expected results were it to be negative in all the intervals. For test tube 2 (1.5mL of 0.01M glucose-1-phosphate, and 1 drop of 0.2% starch suspension), the iodine tested negative for the first 6 minutes, and at 9 minutes on, the iodine tested positive for starch. This result was expected because in the beginning, there was too little starch to detect, but as the synthesis continued, more glucose was attached to the starch primer molecule and eventually starch became long enough to be detected by the iodine test. Test tube 3 (containing 1.5mL of 0.01M glucose-1-phosphate) tested negative for all of the intervals. This was expected as the enzyme did not have a pre-existing starch primer to attach the glucose from glucose-1-phosphate to. In test tube 4 (1.5mL of 0.01M glucose-1-phosphate, and 1 drop of 0.2% starch suspension) boiled phosphorylase was used, the iodine tests in all of the intervals resulted in negative result. Although the right substances are present in the test tube for the reaction to take place, the enzyme could not function. The reason for this is because, since the enzyme is boiled, it is denatured thus destroying its secondary and higher structures. Therefore, the enzyme loses its 3-dimensional form and the substrate can no longer bind to the active site (Karp, 2008). Test tube 5 (containing 1.5mL of 0.01M glucose-1-phosphate, 0.5mL of 0.2M potassium phosphate, and 1 drop of 0.2% starch suspension) is negative for all of the intervals. This also expected, on one side of the reaction, starch reacts with the phosphate ion, thus breaking up the starch and in turn creating glucose-1-phosphate. On the other side of the reaction, the glucose from glucose-1-phosphate elongates the starch primer. These reactions keep going on, as starch is being broken down in phosphorolysis, it is also being synthesized; therefore the results will be negative. For test tube 6 (containing 0.5mL of 0.2M potassium phosphate, and 1.5mL of 0.2% starch), the results were all positive in all of the intervals. However, the color was lightening up at 15 minutes, and continued to lighten up, this matched up with the theoretical predictions. The reason for this is because the reaction is going through phosphorolysis with starch and the phosphate ion in potassium phosphate, thus effectively breaking apart the starch into glucose-1-phosphate (Green & Stumpf, 1942). Therefore at each interval there was less starch, therefore the colour of the iodine test lightened at the end of the interval. For test tube 7 (containing 0.5mL of 0.2M potassium phosphate and 1.5mL of 0.2% starch) boiled phosphorylase was used, the results were positive through all of the intervals, and no lightening of colour. Again, the denaturing of the enzyme destroys the enzyme’s function by effectively changing the shape of the enzyme. Thus, the substrate cannot bind to the enzyme and the starch cannot be broke down, and therefore throughout the intervals, the results for the iodine test will be positive (Department of Biology, 2009).
During the experiment there were many sources of error that could have altered the results of the test. One being contamination of the test tubes through the handling or initial condition of the equipment. Another source of error is from the techniques employed during the lab, for example the moving the solution from one test tube to another, or handling the pipettes.
In conclusion, the concentration of the enzyme, the reaction time, and the concentration of the substrate are all related to each other. In the first section of the lab, using amylase in a series of tests has determined that the concentration of enzyme is indirectly proportional to the reaction time. In the second section of the lab concerning phosphorylase, it is clear that enzymes do not change the direction of the reaction, but just accelerating it by lowering the activation energy required for the reaction to take place (Karp, 2008).
References
Department of Biology 2009 cell Biology Laboratory Manual. University of Waterloo, Waterloo. pp. 38-43.
Green, D. E. & Stumpf P. K. 1942. Starch Phosphorylase of Potato. Journal of Biological Chemistry.
Karp, G. 2008. Cell and Molecular Biology: Concepts and Experiments, 5th edition. John Wiley and Sons, Inc. pp.94-105;114-116.