Porcine Pancreatic Alpha-Amylase
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Affect of pH on Porcine Pancreatic Alpha-Amylase Activity
Introduction
Proteins function in a variety of different ways, and one of their fundamental tasks is to act as enzymes. Enzymes are extremely important in controlling reaction speed (by initiating and regulating biological activity), cell communication, and growth. One particularly significant enzyme is amylase, which catalyzes the hydrolysis of alpha glycosidic linkages of amylose, starch components, and other oligosaccharides (Qian, et al., 1994).
Porcine pancreatic alpha-amylase can be found in pancreatic secretions, and works most efficiently at pH 6.9 for the majority of substrates. This ideal pH, however, has shown to shift to as low as 5.2 for the hydrolysis of some …show more content…
low molecular substrates (Ishikawa, et al., 1991).
Amylase is activated by chloride and calcium ions, and the active site contains five sub-sites for binding glucose units (catalytic attack occurs between sub-sites three and four) (Qian, et al.
1994). Analysis shows the shift of the ideal pH down to 5.2 occurs only when the fifth sub-site is being occupied by the glucosyl residue of the specific substrate (Sivaramakrishnan, et al., 2006).
This indicates the way a substrate binds to the enzyme, directly affects its catalytic power as well as pH.
In this experiment, the six tubes which contained the same amount of enzyme to substrate were tested for reaction rate with pHs ranging from 4 to 9 as the only difference, in order to determine what the optimum pH was. According to previous experiments, protenation of the nucleophile limits catalysis at low pH, and deprotenation of the hydrogen donor limits it at high pH (Nielsen, Vriend & Borchert, 2001). In addition, a review paper states alpha-amylase …show more content…
1
from porcine pancreas’ experimental optimum pH ranges between 6.5 and 7 (Sivaramakrishnan, et al., 2006). Due to the substrate used in this experiment, it is hypothesized the optimal pH will be 6.8.
Methods
In order to test the hypothesis, six test tubes were labeled 1-6, and tube 1 was labeled with pH 4, tube 2 with pH 5, and so on until tube 6 was labeled with pH 9. 5 mL of the buffer solution which corresponded to what was labeled on each respective tube was added. 1.5 mL of porcine pancreatic alpha-amylase solution was then added to each tube and mixed by rolling the tube between our hands. One or two drops of iodine potassium iodine solution was put into the wells of each compartment on the test plate. Beginning with test tube one only, we added 2.5 mL of the 1% starch solution, mixed it by rolling between out hands, started the time at zero, and immediately took a drop and placed it on the test plate. This continued for 10-second intervals until the solution was no longer blue, but instead an amber color. The blue color means starch is present, and the amber color indicates it is not. If the color did not change after 7-minutes, the experiment was terminated for that reaction mixture. These steps were repeated for each test tube, and reaction times were recorded.
Results
Figure 1: Test plates showing effect of pH on the effectiveness of porcine pancreatic alphaamylase. Each plate represents a pH solution from pH 9 to pH 4 (left to right respectively).
2
As seen in Figure 1, the test plates which correspond to pH 9, 8, 4, and 5 see no reaction after 7 minutes. The plate corresponding to pH 7 had the fastest reaction time, followed by pH6.
Amongst all the groups, the solutions with pH 9, 8 and 4 also had no reaction. Only one group received a reaction time for pH 5, which I am omitting from the data, due to the fact the standard
Reac4on%Time%per%Tiral%for%Porcine%Pancrea4c%
Alpha9Amylase%in%1%%Starch%
Average and Standard deviation of
Time for pH 6 and 7 alpha-amylase solution in 1% starch
250"
Time%(seconds)%
200"
Average
Time (sec)
Standard
Dev.
pH 6
148
45.64
pH 7
185
28.87
150" pH"6" 100"
pH"7"
50"
0"
1"
2"
3"
4"
Group%Trial%
Figure 2: Time taken per group for reaction with porcine pancreatic alpha-amylase in pH 6 and 7. Faster rates on average
Chart 1: Shows the average and standard deviation for the reaction time of porcine pancreatic alpha-amylase in 1% starch solution in a pH 6 and 7 buffer solution.
deviation is extremely large; therefore, pH 5 also had no reaction. It can be see in figure 2 that the overall group times showed pH 6 as more optimal than pH 7. Chart 1 shows the average time for pH 6 of 148 seconds with standard deviation 45.64, and pH 7 has an average time of 185 seconds with a standard deviation of 28.87.
Discussion
Unlike the hypothesis, the optimal pH obtained from this experiment was at 6, according to the average reaction time, which disproves the hypothesis.
One could argue the standard deviation is too large for the pH 6 to have a credible mean. This could be due to error in terms of how time was taken, also in when it was decided the solution no longer had starch. In terms of time, some groups may not have started the time as soon as the enzyme and substrate were together, while others may have not taken consistent ten-second intervals. As for the iodine test
3
for starch, knowing when the starch is gone is relative to the person conducting the experiment.
The initial color change is subjective, because there could still be starch in a solution if the iodine is an amber color but has specks of dark blue in the well which could cause some to state the reaction time as earlier than it actually is. There are several other human errors that could have resulted in a lower optimal pH, one of the biggest being contamination. If one did not use a different pipette, solutions will become contaminated with other pH levels.
A great majority of journals have experimentally found the optimal pH for pancreatic alpha-amylase to be between 6.7 and 6.9 (Nielsen, et al., 2001). While the ideal pH has
been experimentally seen to go as low as 5.2, this is only for molecules such as maltotroise. Starch is an extremely simple polymer which does not affix a glucosyl residue to the fifth binding site, thereby not altering the optimal pH level (Sivaramakrishnan, et al., 2006). In addition, acid treatment of starch particles have been found to prevent the adsorption of amylase. This is because the acid degrades the amorphous areas of the starch, and only leaves it with highly ordered material to which amylase binds poorly (Slaughter, Ellis & Butterworth, 2001). With the plethora of experiments contradicting our results, human error is the most plausible cause of having such a low pH as ideal conditions for the enzyme. The experiment would have been much more successful if time was being kept properly, and if the results from the iodine test were read correctly. Also, ensuring contamination was at a minimum by discarding used pipettes, and properly cleaning test plates and test tubes before each use, would have resulted in more reliable data. This enzyme is important in not only the digestion of carbohydrates, but also that of dead white blood cells (Iwata, et al., 2010). Amylase is also extremely significant in forensic
4
investigations of body fluids, and can be a reliable indicator of pancreas-related complications.
Due to its specific functionality, the artificial synthesis of this enzyme is very attractive. It is vital to understand the conditions in which it works in order best recreate a successful protein. The artificial enzyme could help those with diabetes, or those who cannot digest fat and so instead intake an amount of sugar that causes amylase deficiency. Carbohydrate breakdown is of great economic importance in the food industry, and knowing what this enzyme’s optimal conditions are reduce cost as well as time. By further understanding the complexities of this protein, more medical and manufacturing advancement can be made.
References
Ishikawa, K., Matsui, I., Honda, K., Kobayashi, S., & Nakatani, H. (1991). The ph dependence of the action pattern in porcine pancreatic wamylase-catalyzed reaction for maltooligosaccharide substrates. Archives of Biochemistry and Biophysics, 289(1), 124-129.
Iwata, N., Kodera, Y., Eguchi, T., Ohashi, N., Nakayama, G., Koike, M., Fujiwara, M., & Nakao,
A. (2010). Amylase concentration of the drainage fluid as a risk factor for intra-abdominal abscess following gastrectomy for gastric cancer. World Journal of Surgery, 34(7), 1534-1539.
Nielsen, J., Vriend, G., & Borchert, T. (2001). The determinants of α-amylase ph–activity profiles. Protein Engineering, 14(7), 505-512. doi: 10.1093/protein/14.7.505
Qian, M., Haser, R., Buisson, G., Duee, E., & Payan, F. (1994). The active center of a mammalian .alpha.-amylase. structure of the complex of a pancreatic .alpha.-amylase with a carbohydrate inhibitor refined to 2.2-.ang. resolution. Biochemistry, 33(20), 6284–6294. doi:
10.1021/bi00186a031
5
Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K., Soccol, C., & Pandey, A. (2006). aamylases from microbial sources – an overview on recent developments. Food Technology &
Biotechnology, 44(2), 173–184.
Slaughter, S., Ellis, P., & Butterworth, P. (2001). An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches. Biochimica et Biophysica Acta (BBA) General Subjects, 1525(1-2), 29-36.
6
Affect of pH on Porcine Pancreatic Alpha-Amylase Activity
Introduction
Proteins function in a variety of different ways, and one of their fundamental tasks is to act as enzymes. Enzymes are extremely important in controlling reaction speed (by initiating and regulating biological activity), cell communication, and growth. One particularly significant enzyme is amylase, which catalyzes the hydrolysis of alpha glycosidic linkages of amylose, starch components, and other oligosaccharides (Qian, et al., 1994).
Porcine pancreatic alpha-amylase can be found in pancreatic secretions, and works most efficiently at pH 6.9 for the majority of substrates. This ideal pH, however, has shown to shift to as low as 5.2 for the hydrolysis of some …show more content…
low molecular substrates (Ishikawa, et al., 1991).
Amylase is activated by chloride and calcium ions, and the active site contains five sub-sites for binding glucose units (catalytic attack occurs between sub-sites three and four) (Qian, et al.
1994). Analysis shows the shift of the ideal pH down to 5.2 occurs only when the fifth sub-site is being occupied by the glucosyl residue of the specific substrate (Sivaramakrishnan, et al., 2006).
This indicates the way a substrate binds to the enzyme, directly affects its catalytic power as well as pH.
In this experiment, the six tubes which contained the same amount of enzyme to substrate were tested for reaction rate with pHs ranging from 4 to 9 as the only difference, in order to determine what the optimum pH was. According to previous experiments, protenation of the nucleophile limits catalysis at low pH, and deprotenation of the hydrogen donor limits it at high pH (Nielsen, Vriend & Borchert, 2001). In addition, a review paper states alpha-amylase …show more content…
1
from porcine pancreas’ experimental optimum pH ranges between 6.5 and 7 (Sivaramakrishnan, et al., 2006). Due to the substrate used in this experiment, it is hypothesized the optimal pH will be 6.8.
Methods
In order to test the hypothesis, six test tubes were labeled 1-6, and tube 1 was labeled with pH 4, tube 2 with pH 5, and so on until tube 6 was labeled with pH 9. 5 mL of the buffer solution which corresponded to what was labeled on each respective tube was added. 1.5 mL of porcine pancreatic alpha-amylase solution was then added to each tube and mixed by rolling the tube between our hands. One or two drops of iodine potassium iodine solution was put into the wells of each compartment on the test plate. Beginning with test tube one only, we added 2.5 mL of the 1% starch solution, mixed it by rolling between out hands, started the time at zero, and immediately took a drop and placed it on the test plate. This continued for 10-second intervals until the solution was no longer blue, but instead an amber color. The blue color means starch is present, and the amber color indicates it is not. If the color did not change after 7-minutes, the experiment was terminated for that reaction mixture. These steps were repeated for each test tube, and reaction times were recorded.
Results
Figure 1: Test plates showing effect of pH on the effectiveness of porcine pancreatic alphaamylase. Each plate represents a pH solution from pH 9 to pH 4 (left to right respectively).
2
As seen in Figure 1, the test plates which correspond to pH 9, 8, 4, and 5 see no reaction after 7 minutes. The plate corresponding to pH 7 had the fastest reaction time, followed by pH6.
Amongst all the groups, the solutions with pH 9, 8 and 4 also had no reaction. Only one group received a reaction time for pH 5, which I am omitting from the data, due to the fact the standard
Reac4on%Time%per%Tiral%for%Porcine%Pancrea4c%
Alpha9Amylase%in%1%%Starch%
Average and Standard deviation of
Time for pH 6 and 7 alpha-amylase solution in 1% starch
250"
Time%(seconds)%
200"
Average
Time (sec)
Standard
Dev.
pH 6
148
45.64
pH 7
185
28.87
150" pH"6" 100"
pH"7"
50"
0"
1"
2"
3"
4"
Group%Trial%
Figure 2: Time taken per group for reaction with porcine pancreatic alpha-amylase in pH 6 and 7. Faster rates on average
Chart 1: Shows the average and standard deviation for the reaction time of porcine pancreatic alpha-amylase in 1% starch solution in a pH 6 and 7 buffer solution.
deviation is extremely large; therefore, pH 5 also had no reaction. It can be see in figure 2 that the overall group times showed pH 6 as more optimal than pH 7. Chart 1 shows the average time for pH 6 of 148 seconds with standard deviation 45.64, and pH 7 has an average time of 185 seconds with a standard deviation of 28.87.
Discussion
Unlike the hypothesis, the optimal pH obtained from this experiment was at 6, according to the average reaction time, which disproves the hypothesis.
One could argue the standard deviation is too large for the pH 6 to have a credible mean. This could be due to error in terms of how time was taken, also in when it was decided the solution no longer had starch. In terms of time, some groups may not have started the time as soon as the enzyme and substrate were together, while others may have not taken consistent ten-second intervals. As for the iodine test
3
for starch, knowing when the starch is gone is relative to the person conducting the experiment.
The initial color change is subjective, because there could still be starch in a solution if the iodine is an amber color but has specks of dark blue in the well which could cause some to state the reaction time as earlier than it actually is. There are several other human errors that could have resulted in a lower optimal pH, one of the biggest being contamination. If one did not use a different pipette, solutions will become contaminated with other pH levels.
A great majority of journals have experimentally found the optimal pH for pancreatic alpha-amylase to be between 6.7 and 6.9 (Nielsen, et al., 2001). While the ideal pH has
been experimentally seen to go as low as 5.2, this is only for molecules such as maltotroise. Starch is an extremely simple polymer which does not affix a glucosyl residue to the fifth binding site, thereby not altering the optimal pH level (Sivaramakrishnan, et al., 2006). In addition, acid treatment of starch particles have been found to prevent the adsorption of amylase. This is because the acid degrades the amorphous areas of the starch, and only leaves it with highly ordered material to which amylase binds poorly (Slaughter, Ellis & Butterworth, 2001). With the plethora of experiments contradicting our results, human error is the most plausible cause of having such a low pH as ideal conditions for the enzyme. The experiment would have been much more successful if time was being kept properly, and if the results from the iodine test were read correctly. Also, ensuring contamination was at a minimum by discarding used pipettes, and properly cleaning test plates and test tubes before each use, would have resulted in more reliable data. This enzyme is important in not only the digestion of carbohydrates, but also that of dead white blood cells (Iwata, et al., 2010). Amylase is also extremely significant in forensic
4
investigations of body fluids, and can be a reliable indicator of pancreas-related complications.
Due to its specific functionality, the artificial synthesis of this enzyme is very attractive. It is vital to understand the conditions in which it works in order best recreate a successful protein. The artificial enzyme could help those with diabetes, or those who cannot digest fat and so instead intake an amount of sugar that causes amylase deficiency. Carbohydrate breakdown is of great economic importance in the food industry, and knowing what this enzyme’s optimal conditions are reduce cost as well as time. By further understanding the complexities of this protein, more medical and manufacturing advancement can be made.
References
Ishikawa, K., Matsui, I., Honda, K., Kobayashi, S., & Nakatani, H. (1991). The ph dependence of the action pattern in porcine pancreatic wamylase-catalyzed reaction for maltooligosaccharide substrates. Archives of Biochemistry and Biophysics, 289(1), 124-129.
Iwata, N., Kodera, Y., Eguchi, T., Ohashi, N., Nakayama, G., Koike, M., Fujiwara, M., & Nakao,
A. (2010). Amylase concentration of the drainage fluid as a risk factor for intra-abdominal abscess following gastrectomy for gastric cancer. World Journal of Surgery, 34(7), 1534-1539.
Nielsen, J., Vriend, G., & Borchert, T. (2001). The determinants of α-amylase ph–activity profiles. Protein Engineering, 14(7), 505-512. doi: 10.1093/protein/14.7.505
Qian, M., Haser, R., Buisson, G., Duee, E., & Payan, F. (1994). The active center of a mammalian .alpha.-amylase. structure of the complex of a pancreatic .alpha.-amylase with a carbohydrate inhibitor refined to 2.2-.ang. resolution. Biochemistry, 33(20), 6284–6294. doi:
10.1021/bi00186a031
5
Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K., Soccol, C., & Pandey, A. (2006). aamylases from microbial sources – an overview on recent developments. Food Technology &
Biotechnology, 44(2), 173–184.
Slaughter, S., Ellis, P., & Butterworth, P. (2001). An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches. Biochimica et Biophysica Acta (BBA) General Subjects, 1525(1-2), 29-36.
6