Breaking Down Starch Lab Report
Introduction: Everyday thousands of chemical reactions happen in our bodies. These reactions produce the energy for our bodies to use. During these chemical reactions there are enzymes, biological catalysts, which help speed up metabolic reactions by lowering the energy barriers without being used up or altered in the reaction. (Campball, 2008) Every enzyme has an optimum pH at which it is most active. An increase or decrease in the pH of the solution will cause the enzyme to have a change in its three dimensional shape. If an enzyme is placed in an environment that is to basic or acidic the reaction will take longer to digest the starch because the enzyme shape has changed and the substrate will have a harder time connecting to the active
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site. A substrate is the material which the enzyme reacts with. An example of pH is water, which is neutral with a pH close to 7.0. Water is in the exact middle of the pH scale so any solution with a pH less than 7 is called an acidic environment and solutions with a pH higher than 7 are considered basic. pH measurements on food or medicine are extremely important to know for human health. For example if we were working at a coca cola plant and the machine added more sorbic acid into the batch than needed it would change the drink’s pH to an unsafe level for humans to ingest. Another important example of pH is that of the blood. Its nominal value of pH = 7.4 is regulated very accurately by the body. If the pH of the blood gets outside the range 7.35 to 7.45 the results can be serious and even fatal. pH is measured by the concentration of hydrogen ions in the solution. The letters in pH stand for “power of hydrogen” and the numerical value given to the solutions is the negative power of 10 of the molar concentration of H+ ions. (Ebbing, 1990) A low pH indicates a high concentration of hydronium ions which makes the solution acidic and a low concentration means the solution is basic.
Amylase is found in the saliva of humans and is responsible for the first step in breaking down starch. Starch is a polysaccharide composed of a large number glucose monomers joined together. Amylase breaks down starch by separating the glucose molecules into maltose, which is a two glucose-unit compound. (Morgan, 2008) During this experiment we investigated the influence of pH on the activity of the enzyme Amylase. We are able to use the fact that when I2KI is placed in a starch solution the solution turns a dark purple which shows that the starch has not been digested. When solution remains a yellow amber color this means that all the starch has been digested. Amylase was placed in five separate test tubes with each test tube having a different pH level. Using a test plate we placed a drop of the amylase solution on top of a drop of I2KI every 10-seconds to test the time it takes amylase to digest the starch. The optimal pH for amylase is determined by the shortest time of starch digestion. The pH of the saliva in the mouth ranges from 5.76- 7.96 depending on the person. (Larsen, 1998) If saliva pH ranges from 5.76 to 7.96 we believe that amylase activity will be most active between 6 to 7 pH, so the tests tubes with a pH level of 6 and 7 should have the
lowest reaction time.
Materials and Methods: Five separate test tubes were placed into a rack and labeled with a wax pencil to indicate pH value of the solution ranging from four to eight. Each test tube was givin 5-mL of the appropriate buffer by using a graduated pipette. The pipette was rinsed with distilled water after putting in each buffer. Using a new 5-mL graduated pipette 1.5mL of amylase solution was distributed to all five test tubes and mixed by rolling the test tube in our hands. Then using a test plate each compartment was administered one drop I2KI. Using only one of the test tubes at a time 2.5 mL of the 1% starch solution was placed into the first test tube with the pH of 4 using a clean 5-mL pipette. The test tube was then mixed again by rolling it in our hands. As soon as the starch hits the solution the reaction has started and this is time 0. Using a disposable pasteur pipette a drop of the reaction mixture is placed in the compartment of the test plate which already has a drop of I2KI. Add a drop of the reaction mixture to a different compartment in the test place every 10 seconds until the blue color is no longer produced by the of I2KI and remains an yellow-amber indicating the amylase has finished digesting all the starch. The test plate and pasteur pipette were rinsed after reaction time recorded on Table 1. The test plate was then set up again with one drop of I2KI in each compartment and 2.5 mL of the 1% starch solution was placed into next test tube until all pH solutions were tested and reaction time recorded. (Morgan, 2008)
Results: In this experiment testing the effect of pH on the activity of Amylase we found that the test tube with the pH of 6 had the fastest time of starch disappearance. Any increases or decreases in the pH caused amylase to digest the starch at a much slower rate.
Test Tube pH Time of Starch Disappearance
(in seconds)
1
4
90
2
5
60
3
6
0
4
7
60
5
8
140
Table1:
Graph 1:
Discussion: The pH at which amylase was most active was in the solution with the pH of 6. We were correct in our predictions about the optimum pH at which amylase would be most active but gave to wide of a range. We said that amylase would be most active between the pH of 6 and 7. Looking at Table 1 you can see that at pH 6 that the amylase digested the starch almost instant and as the pH gets farther away from pH 6 the amylase activity slows down tremendously. If we were to do this experiment again we would find out where the amylase was from because the pH level differs for each species. For example another experiment shows the optimum pH, for amylase activity in bile and liver of crow, corresponds with the luminal pH of the duodenum which is 4.9. (Bhattacharya, 1970) The experiment was accurate when compared to other experiments testing human amylase activity. The amylases in the salivary glands of Lygus hesperus and L. lineolaris were isolated and purified by ion exchange chromatography, and by isoelectric focusing, respectively. The amylase from L. hesperus had an isoelectric point (pI) of 6.25, and a pH optimum of 6.5. The amylase from L. lineolaris had a pI of 6.54, and a pH optimum of 6.5. (Zeng, 2000) Overall the experiment was accurate providing the optimum pH at which amylase is most active. If we were to retest this experiment again we would try different starch extracts to see if there would be a change amylase activity. Also we would use new pipettes every time instead of rinsing after each use to ensure no residue was left from the other pH solution.
Literature Cited:
1. Bhattacharya S., Ghose K.C. “Occurrence of biliary amylase in vertebrates: Influence of NaCl and pH.” Comparative Biochemistry and Physiology, 1970, Volume 37, Issue 4, Pages 581-587.
2. Campbell N. Reece J., Urry L., Cain M., Wasserman S., Minorsky P., Jackson R. Biology, 8th edition, San Francisco, CA: Pearson, 2008.
3. Ebbing D. General Chemistry, 3rd edition, Boston, MA: Houghton Mifflin, 1990.
4. Larsen M.J, Jensen A.F, Madsen D.M, Pearce E.I.F. ”Individual variations of pH, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva.” Archives of Oral Biology, 1999, Volume 44, Issue 2, Pages 111-117. 5. Morgan J., Brown-Carter M.E. Investigating Biology, 6th edition, San Francisco, CA: Pearson, 2008.
6. Zeng F., Cohen A.C. ”Partial characterization of α-amylase in the salivary glands of Lygus hesperus and L. lineolaris.” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2000, Volume 126, Issue 1, Pages 9-16.
site. A substrate is the material which the enzyme reacts with. An example of pH is water, which is neutral with a pH close to 7.0. Water is in the exact middle of the pH scale so any solution with a pH less than 7 is called an acidic environment and solutions with a pH higher than 7 are considered basic. pH measurements on food or medicine are extremely important to know for human health. For example if we were working at a coca cola plant and the machine added more sorbic acid into the batch than needed it would change the drink’s pH to an unsafe level for humans to ingest. Another important example of pH is that of the blood. Its nominal value of pH = 7.4 is regulated very accurately by the body. If the pH of the blood gets outside the range 7.35 to 7.45 the results can be serious and even fatal. pH is measured by the concentration of hydrogen ions in the solution. The letters in pH stand for “power of hydrogen” and the numerical value given to the solutions is the negative power of 10 of the molar concentration of H+ ions. (Ebbing, 1990) A low pH indicates a high concentration of hydronium ions which makes the solution acidic and a low concentration means the solution is basic.
Amylase is found in the saliva of humans and is responsible for the first step in breaking down starch. Starch is a polysaccharide composed of a large number glucose monomers joined together. Amylase breaks down starch by separating the glucose molecules into maltose, which is a two glucose-unit compound. (Morgan, 2008) During this experiment we investigated the influence of pH on the activity of the enzyme Amylase. We are able to use the fact that when I2KI is placed in a starch solution the solution turns a dark purple which shows that the starch has not been digested. When solution remains a yellow amber color this means that all the starch has been digested. Amylase was placed in five separate test tubes with each test tube having a different pH level. Using a test plate we placed a drop of the amylase solution on top of a drop of I2KI every 10-seconds to test the time it takes amylase to digest the starch. The optimal pH for amylase is determined by the shortest time of starch digestion. The pH of the saliva in the mouth ranges from 5.76- 7.96 depending on the person. (Larsen, 1998) If saliva pH ranges from 5.76 to 7.96 we believe that amylase activity will be most active between 6 to 7 pH, so the tests tubes with a pH level of 6 and 7 should have the
lowest reaction time.
Materials and Methods: Five separate test tubes were placed into a rack and labeled with a wax pencil to indicate pH value of the solution ranging from four to eight. Each test tube was givin 5-mL of the appropriate buffer by using a graduated pipette. The pipette was rinsed with distilled water after putting in each buffer. Using a new 5-mL graduated pipette 1.5mL of amylase solution was distributed to all five test tubes and mixed by rolling the test tube in our hands. Then using a test plate each compartment was administered one drop I2KI. Using only one of the test tubes at a time 2.5 mL of the 1% starch solution was placed into the first test tube with the pH of 4 using a clean 5-mL pipette. The test tube was then mixed again by rolling it in our hands. As soon as the starch hits the solution the reaction has started and this is time 0. Using a disposable pasteur pipette a drop of the reaction mixture is placed in the compartment of the test plate which already has a drop of I2KI. Add a drop of the reaction mixture to a different compartment in the test place every 10 seconds until the blue color is no longer produced by the of I2KI and remains an yellow-amber indicating the amylase has finished digesting all the starch. The test plate and pasteur pipette were rinsed after reaction time recorded on Table 1. The test plate was then set up again with one drop of I2KI in each compartment and 2.5 mL of the 1% starch solution was placed into next test tube until all pH solutions were tested and reaction time recorded. (Morgan, 2008)
Results: In this experiment testing the effect of pH on the activity of Amylase we found that the test tube with the pH of 6 had the fastest time of starch disappearance. Any increases or decreases in the pH caused amylase to digest the starch at a much slower rate.
Test Tube pH Time of Starch Disappearance
(in seconds)
1
4
90
2
5
60
3
6
0
4
7
60
5
8
140
Table1:
Graph 1:
Discussion: The pH at which amylase was most active was in the solution with the pH of 6. We were correct in our predictions about the optimum pH at which amylase would be most active but gave to wide of a range. We said that amylase would be most active between the pH of 6 and 7. Looking at Table 1 you can see that at pH 6 that the amylase digested the starch almost instant and as the pH gets farther away from pH 6 the amylase activity slows down tremendously. If we were to do this experiment again we would find out where the amylase was from because the pH level differs for each species. For example another experiment shows the optimum pH, for amylase activity in bile and liver of crow, corresponds with the luminal pH of the duodenum which is 4.9. (Bhattacharya, 1970) The experiment was accurate when compared to other experiments testing human amylase activity. The amylases in the salivary glands of Lygus hesperus and L. lineolaris were isolated and purified by ion exchange chromatography, and by isoelectric focusing, respectively. The amylase from L. hesperus had an isoelectric point (pI) of 6.25, and a pH optimum of 6.5. The amylase from L. lineolaris had a pI of 6.54, and a pH optimum of 6.5. (Zeng, 2000) Overall the experiment was accurate providing the optimum pH at which amylase is most active. If we were to retest this experiment again we would try different starch extracts to see if there would be a change amylase activity. Also we would use new pipettes every time instead of rinsing after each use to ensure no residue was left from the other pH solution.
Literature Cited:
1. Bhattacharya S., Ghose K.C. “Occurrence of biliary amylase in vertebrates: Influence of NaCl and pH.” Comparative Biochemistry and Physiology, 1970, Volume 37, Issue 4, Pages 581-587.
2. Campbell N. Reece J., Urry L., Cain M., Wasserman S., Minorsky P., Jackson R. Biology, 8th edition, San Francisco, CA: Pearson, 2008.
3. Ebbing D. General Chemistry, 3rd edition, Boston, MA: Houghton Mifflin, 1990.
4. Larsen M.J, Jensen A.F, Madsen D.M, Pearce E.I.F. ”Individual variations of pH, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva.” Archives of Oral Biology, 1999, Volume 44, Issue 2, Pages 111-117. 5. Morgan J., Brown-Carter M.E. Investigating Biology, 6th edition, San Francisco, CA: Pearson, 2008.
6. Zeng F., Cohen A.C. ”Partial characterization of α-amylase in the salivary glands of Lygus hesperus and L. lineolaris.” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2000, Volume 126, Issue 1, Pages 9-16.