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Iodine Test Reveals CaCl2 Up to 0.0676M
Enhance αöAmylase Digestion of Solanum tuberosum
Starch
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By:
Jon Engstrom
Amanda Gnau
Nathan
Johnson
Hong-Phuc
Nguyen
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Abstract Salivary αöamylase is a key component in the initial chemical processing
of food. Chloride was discovered to be an allosteric effector of αöamylase for activation. A
structural Ca2+ ion is also necessary. Because calcium and
chloride ions are vital for αöamylase function, CaCl2 may be a rate limiting
factor of the enzyme. We examined the digestion of starch in potato
when high (0.090M) and low (0.045M) concentrations of CaCl2 were
added to saliva. The samples were subjected to several assays. In
Benedictâs test, the samples all produced an orange/red precipitate,
indicating the presence of reducing sugars. The timed Barfoedâs test
generated all negative results; no monosaccharide reducing sugars were
detected.Ê The protein assay confirmed
that the amounts of protein present in the saliva samples were not
significantly different. The absorption spectrum of the three leaves
samples were similar, with two peaks of absorption around 400 and 680
nm. For the qualitative Iodine test, the potato-saliva sample was
darkest, the potato-saliva-0.045M CaCl2 sample was of intermediate
color, and the potato-saliva-0.090M CaCl2 was the lightest color
at 3 and 6 minutes after contact with saliva.Ê That suggests the least amount of starch was found in the
latter sample. In the quantitative Iodine test, we measured the
absorbance for samples of α-amylase,
potato starch, and either 0M, 0.025M, 0.045M, 0.0676M, or 0.090M CaCl2 at 0 and 5 minutes.Ê Higher concentrations of CaCl2 yielded lower
absorbance, correlating with a lower amount of starch.Ê However, the sample with 0.090M CaCl2 did not follow that trend. We conclude
that higher concentrations of CaCl2 enhance αöamylase action, but there is an optimal
concentration (0.0676M) above which it is no longer beneficial for enzymic
activity. |
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Figure 8. Quantitative Iodine test.
The solutions measured contained amylase, 1% starch, IKI, and one of the five
different concentrations of CaCl2. Their absorbance values were measured with a spectrophotometer
immediately after mixing the solutions. As the concentration of CaCl2 increased, the absorbance
decreased. This trend continued until the 0.090 M concentration of CaCl2, which showed an increase of
absorbance. |
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Discussion
We observed the effect of CaCl2 on the productivity of salivary
amylase through various tests.Ê CaCl2 is a cofactor to salivary amylase, which denotes that its
addition to the saliva and potato samples would increase the rate of the
chloride binding, which activates salivary amylase (Numao et al., 2002). We
predicted that the CaCl2 will enhance the efficiency and
overall productivity of salivary amylase.Ê
According to the tests performed, our hypothesis that the addition of CaCl2 increased the rate at which salivary amylase broke down starch
was supported.Ê Benedictâs test determines the presence of
reducing sugars such as ketones and aldehydes.Ê The Benedictâs test came up positive for reducing sugars in the
saliva/potato solution as well as the potato-saliva- CaCl2 solutions (Figure 1). The Benedictâs positive results may be due
to the presence of maltose, which is a disaccharide reducing sugar (Wang,
unknown). The results show that the addition of the cofactor had no effect on
the presence of reducing sugars in the amylase/starch solutions. ÊÊÊÊÊÊÊÊÊÊÊ Barfoedâs test determines the presence of
monosaccharide reducing sugars.Ê No
monosaccharides were detected for any of the samples except the positive
control which was fructose (Figure 2).Ê
After performing the conventional Barfoedâs test which produced no
positive results, our group tried a new approach to the test.Ê The original test added all components
together, including the Barfoedâs reagent, and placed them into the hot water
bath for the determined timed intervals.Ê
In the new approach, our group added the saliva to the potato and
potato- CaCl2 solutions and let them sit for periods of three, six and ten
minutes.Ê The Barfoedâs reagent was
then added and the samples were placed in the hot water bath. We were still
not able to detect the presence of monosaccharides (Figure 2).Ê The data suggests that little to no
monosaccharides present in our starch/saliva solutions.Ê Our predictions that the CaCl2 would affect the presence of monosaccharides in varying
concentrations were not supported because monosaccharides were not
detected.Ê This contradiction from our
expectations may be due to the saliva not having time to break down the
starch to glucose.Ê The saliva used might
not have contained an adequate amount of amylase or the potato solution may
have been too diluted in order to produce positive results.Ê One other possible explanation is that the
alpha amylase could only break down the starch up to a certain point
(McCloskey, 2004).Ê A factor that
could have affected the results was the concentration of the potato solution
which was 10% by weight / volume.Ê The
solution may have been too dilute. The Iodine test
determines the presence of starch in a solution.Ê Our group predicted that the addition of the cofactor would
reduce the amount of starch found using the Iodine test.Ê The Iodine test came up with data that was
inconclusive the first time through using 1 minute intervals and
photographing (Figure 3A & B).ÊÊ
Our group then incubated the amylase with potato samples for intervals
of three minutes and six minutes before pipetting the IKI. The data for the
new approach was conclusive (Figure 4A & B).Ê The surrounding temperature of the samples was a variable to
our experiment, so we immersed the samples in a 37 ¡C water bath to attempt
to keep the temperature consistent and bring the temperature to the
approximate temperature of the mouth.Ê
The heat served to optimize the conditions for salivary amylase to
function (Zaremba, 2004).Ê Solutions
with the higher concentrations of CaCl2 were lighter in color, and the
solutions were more translucent.Ê
Therefore, the empirical evidence could suggest that the more CaCl2 placed in a solution, the more starch was broken down.Ê Since there was still starch present in
the solution containing CaCl2, the concentration of CaCl2 might not have been high enough to cause complete hydrolysis of
the starch.Ê Bradfordâs assay was performed to determine that the amount of protein in our
saliva samples was consistent (Figure 6).Ê
After applying the tested solutions (saliva and high/low
concentrations of CaCl2) to the standard curve (Figure 5),
the data showed that there was no significant difference between the
concentrations of protein in the samples (Table 1).Ê If we had more saliva/enzyme in our samples, it would result in
increased and faster hydrolysis of starch (Team Absolute, 2003), thereby
skewing our results.Ê The data shows
that it is indeed the addition of the cofactor that increases the rate of
starch breakdown.Ê While the test
doesnât directly test for alpha amylase, the results suggest the protein
content among the saliva samples was consistent.Ê The results were similar to the predictions that our group had
made about the test.
Our group tested the effects of saliva and
its cofactor on photosynthesis.Ê The
absorption spectrum test determines which wavelength(s) has the highest
absorbance for each of the samples.Ê
We predicted that the absorbance spectrum would not change
considerably for all three of the samples.Ê
The most absorbed wavelengths were expected to be the red and blue
parts of the visible light spectrum, approximately 700 and 400 nanometers
respectively (Freeman, 138-139).Ê The
least absorbed wavelength was expected to be the color green at approximately
550 nanometers.Ê Our actual results
show a linear plot of data that decreases in absorption starting at 400nm and
ending at 700nm.Ê The results showed
that as the concentration of CaCl2 was increased, the absorbance of
the samples increased.Ê The results
suggest that little to no digestion occurred in the amylase/leaf solution
(Figure 7).Ê No digestion occurred and
no components in the amylase/leaf solution were broken down because the
absorption levels did not drop.Ê If
there is no digestion occurring, then the addition of a cofactor would serve
no purpose.Ê Furthermore, since the
absorbance increased as the concentration of CaCl2
increased, the results
could suggest that the addition of unused CaCl2
was responsible for
the higher absorption.Ê In our independent lab, we quantified the
amount of starch across five treatments of varying CaCl2 levels.Ê Our earlier
results suggest that the addition of CaCl2 enhances the activity of
amylase.Ê For the quantitative Iodine
test, five different mixtures containing increasing amounts of CaCl2 concentrations were measured by the spectrometer for absorbance.
The first measured solution contained no CaCl2, and the other four had increasing
amounts of: 0.0225 M, 0.0454 M, 0.0676 M, and 0.090 M concentrations of CaCl2. All mixtures contained 1% starch solution, amylase solution,
IKI, and one of the five concentrations of CaCl2. It was found that the sample with
no CaCl2 had the highest absorbance at the initial and five minute
reading. As concentration of CaCl2 increased in the samples, the
absorbance value decreased at both measured time readings. This trend
continued, except for the 0.090 M concentration of CaCl2, which showed a higher reading of absorbance in both cases
(Fig. 8,9).Ê It was expected that as
the concentration of pure starch was increased, the absorption value could
increase according to the best fit line (McCloskey, 2004).Ê The figures suggest that higher
concentrations of cofactor used in the solutions results in a lower
absorbance reading.Ê The results
suggest that the 0.0225M CaCl2 solution is the closest to the most
optimal concentration of cofactor for salivary amylase.Ê The rise in the absorbance of the higher
concentrations of CaCl2 solutions could be due to an excess
of CaCl2 that is not being utilized.Ê
The results suggest CaCl2 has a positive affect on amylase productivity.Ê There were a few flaws or complications
with the structure and procedure of the experiments performed.Ê Prior knowledge from our TAs confirmed
that the pure potato solutions would have extremely high starch
concentrations and might make our data gathering more difficult.Ê We decided on 1 gram of potato solution in
100 ml of water, but we found the solution to be too diluted and changed the
solution to 10 grams of potato solution in 100ml of water. The concentrations
of the solutions we used throughout our tests were based on what we thought
was best, and might not necessarily be the optimum concentrations for the
tests performed.Ê Since different
potatoes were used each week, and the freshness of the potatoes was not the
same for every experiment, there may have been an error in the data due to
these conditions. Our experiment required a large volume of
saliva in order to perform all of the tests.Ê
Since acquiring the entire supply from one person would have taken
longer than desired, all group members contributed to the saliva stock and
the entire solution was mixed to keep consistency.Ê In order to achieve the high volume of saliva, Quench gum was
used to ãforceä salivation.Ê Since the
effects of the gum on amylase are unknown, the gum could have potentially
altered the data. ÊÊÊÊÊÊÊÊÊÊ When it came time to perform our original independent
lab, our group could not get the potato solutions to produce a transmittance
of higher than ten percent.Ê We were
having difficulty finding the right concentrations for both the potato
solutions and the cofactor solutions.Ê
Our lab assistant suggested an alternative setup using pure potato
starch and pure amylase based on the format of an already accomplished
experiment.Ê Our group followed the
new experiment and altered the solutions to fit our needs.Ê Using the pure starch solution and pure
amylase solutions were very beneficial to our experiment because they
isolated exactly what our experiment was meant to test.Ê The only components in the experiment were
CaCl2, pure amylase, and pure potato starch.Ê There were no impurities or any other
variables effecting what we set out to test.Ê
The pure potato solution was acceptable to use because the starch came
from potato, just as all of our other starch solutions did so there were no
discrepancies with the source of the starch. A way to further examine our experiment would be to investigate the effects of the Quench gum on amylase and saliva. We knew that the gum increases the amount of saliva produced in the mouth, but the chemical makeup and the effects of the gum on salivary amylase are still unsure. |
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