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Running head: ANION AMYLASE STARCH DIGESTION

Effect of Different Anions on Amylase Digestion of Starch


Daniel Black
Dalhousie University

March 20, 2014


P. Briggs
BIOC 2610.03

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Introduction

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Methods

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Results

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Discussion

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References
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ANION AMYLASE STARCH DIGESTION

Abstract
Amylase breaks down the glycosidic bonds in starch molecules producing glucose and maltoses.
It is an enzyme and may be influenced by its substrate, especially with anion concentrations.
Depending on the type and amount, anions can act as activators and increase the enzyme activity
and overall rate, or they can act as inhibitors and decrease the enzyme activity and rate. Starch
digestion can be timed by using an iodine solution to keep track of the total digestion in terms of
the undigested starch forming a complex with the iodine producing a dark colour. As the colour
fades, the starch content decreases, until it reaches the achromatic point when no more starch is
being digested. The achromatic point should be reached quicker than the control without any
ions if the anion is an activator. If the anion is an inhibitor the achromatic point will be reached
after the control, or not at all. Of all the anions that were tested, the NaCl and NaNO3 acted as
activators and significantly improved the rate. The other anions were not as efficient are were
only slight activators or inhibitors of amylase. The activators turned out to be monovalent anions,
maybe due to their common occurrence where amylase is needed.
Introduction
Amylase in the enzyme found in saliva that is responsible for the breakdown of starch
molecules. It is an -amylase which means it hydrolyzes -(1-4) glycosidic bonds which are
between the one and four carbon positions of neighboring glucose molecules. Starch consists of
amylose straight chains, and amylopectin branched chains. Digestion of the amylose with
amylase produces glucose and maltoses, which are two glucose residues linked by am -(1-4)
bond. Digestion of the amylopectin yields glucose, maltose, -(1-6) isomaltose, and dextrins.
Dextrins are polysaccharides smaller than amylopectin but consist of the same type of bond
(Briggs, B, 2014).

ANION AMYLASE STARCH DIGESTION

Amylases enzyme activity may be increased or decreased by anionic activators or


deactivators. The type of change each anions will produce depends on mutations in amylase
between individuals. This can be determined experimentally by finding if each anion inhibits or
benefits amylase and by how much it changes from a normal amylase enzyme. A way of
determine the rate of digestion, you may use the Iodine Test to measure the time it takes to digest
a set amount of starch solution. High starch content is dark blue in colour where as a lack of
starch is a fading of the colour back to the colour of iodine solution (Briggs, A, 2014).
Performing the iodine test with amylase and several anion solutions allow you to
determine whether each anion is an activator or deactivator, using amylase without an anion as a
time control. If a solution of amylase and an anion results in a lower starch digestion time than
the water control, the anion is an activator and sped up digestion time. If the anion results in a
longer digestion time, it slowed down the digestion of starch, meaning that the anion is a
deactivator and inhibited amylase (Briggs, B, 2014).
Materials and Methods
The materials and methods followed were as per the BIOC2610 Introductory
Biochemistry Lab Manual (Briggs, A, 2014). First a dialysis was executed using 5mL of saliva
solution in a sealed dialysis sac with a 12000kdal M.W. cutoff. It was placed in 500mL of
distilled water for 45 minutes, changing the distillate every 15 minutes. While this was carried
out, an initial assay was conducted to determine an appropriate dilution of amylase. 500L of
water and 4.5mL of starch were incubated at 37oC and the wells of the spot plate were spotted
with one drop of iodine solution (0.01M of I2 and 0.12M of KI). 1mL of undialyzed-saliva was
diluted with 9mL of water for a 1/10 dilution. While timing, 1.0mL of the diluted saliva was
added to the incubated starch. At 30-second intervals, a drop of this mixture was added to a well

ANION AMYLASE STARCH DIGESTION

with the iodine solution. This is completed until the achromatic point was reached, when there is
no visible change in colour. This process was repeated using different dilution factors until the
achromatic point was reached at around four minutes.
After the appropriate dilution factor was determined and the dialysis is complete, collect
the dialyzed material. Dilute this collected dialyzed amylase according to your determined
dilution factor making about 10mL total. Set up five reaction tubes all with 4.5mL of the 0.5%
starch solution and 0.5mL of the corresponding additive. Water was added to tube 1, 0.1M NaCl
was added to tube 2, 0.1M NaNO3 was added to tube 3, 0.1M Na2SO4 was added to tube 4, and
0.1M Na2HPO4 was added to tube 5. The tubes were incubated for 5 minutes. While preparing to
time and still incubating, 1.0mL of the diluted, dialyzed amylase was added Tube 1. At one
minute intervals, one drop of the tube was added to a prepared spot plate with iodine solution.
This was continued until the achromatic point was reached, or until after twenty minutes has
passed. In the case of the later, there is no observed reaction. This was repeated for all five tubes.
Results
We completed one preliminary experiment with the undialyzed saliva. We used 1mL of
amylase diluted with 9mL of water resulting in a dilution factor of ten times. The achromatic
point was achieved in four minutes. This gives an enzyme amount of 1U for the diluted amylase
and an enzyme amount of 10U in the stock and dialyzed solutions as calculated in Figure 1. As
seen in Table 1, the no salt added control with the dialysed amylase took about twenty minutes to
reach the achromatic point. The NaCl and NaNO3 additions both showed significant time
decreases so they are activators of amylase activity. The Na2SO4 was about one minute faster
than the water control so it activated the amylase slightly. The Na2HPO4 showed no colour
change after twenty minutes so it is an inhibitor of amylase.

ANION AMYLASE STARCH DIGESTION

Discussion
We determined the ideal dilution of the amylase to be a 1:10 dilution, however when
proceeding with part two the achromatic point of the water control was not reached at the twenty
minute mark, however a few more minutes would have shown the point. This may be due to our
dilution determination being inaccurate due to inadequate mixing of the solution or by the
removal of ions with the dialysis causing slight inactivation of the enzyme slowing it down. The
NaCl and NaNO3 salt additions were definitely activators, and NaCl worked slightly better at
increasing the rate. Na2SO4 reached achromatic point just before the twenty minute mark making
it a slight activator of the enzyme and definitely not an inhibitor. The Na2HPO4 showed no
change, so it was definitely slower than the water control making it an inhibitor of the amylase
enzyme.
The hypothesis of the maximal rate of an enzyme being in the presence of monovalent
anions held true for the experiment. The two monovalent anions we tested were Cl- and NO3which both increased the rate of starch digestion when compared to the anions that were not
monovalent. This is probably because of the lack of the more complex molecules being in the
system of a living body. The enzyme is more likely to encounter Cl- and NO3 when it has to
dissolve starch since it is found in saliva and these anions can be found in food. This will ensure
that the enzyme is most active when it is needed, when it is in contact with starch which is found
in food.

ANION AMYLASE STARCH DIGESTION

References
A) Briggs, P. (2014). BIOC2610 Introductory Biochemistry Lab Manual 2014. Halifax, Canada:
Dalhousie University. amylase.1-amylase.6
B) Briggs, P. (2014). Electrophoresis of Amino Acids [Powerpoint]. Retrieved from
http://www3.biochem.dal.ca/2610/

ANION AMYLASE STARCH DIGESTION

Table Descriptors
Table 1 The Results of the 1/10 Dilution Dialysed Amylase Digestion with Different
Salts Added Producing Anions that Act as Inhibitors or Activators with No Salt Added as a
Control Determined as the Time in Minutes it Takes to Reach the Achromatic Point of the Iodine
Complex of Starch Digested

ANION AMYLASE STARCH DIGESTION

Table 1
The Results of the 1/10 Dilution Dialysed Amylase Digestion with Different Salts Added
Producing Anions that Act as Inhibitors or Activators with No Salt Added as a Control
Determined as the Time in Minutes it Takes to Reach the Achromatic Point of the Iodine
Complex of Starch Digested
Salt Added

Achromatic Point

Activator/Inhibitor

(min)
No Salt Control

20

Control

NaCl

5.0

Activator

NaNO3

7.0

Activator

Na2SO4

19

Slight Activator

Na2HPO4

No Reaction

Inhibitor

ANION AMYLASE STARCH DIGESTION

Figure Descriptors
Figure 1 Shows the Calculation of the Number of U per mL of Amylase in the Stock
Solution of Saliva using the Definition 1U is the Enzyme Required to Digest 4.5mL of 0.5%
Starch Solution at 37oC to the Achromatic Point in 4 Minutes.

ANION AMYLASE STARCH DIGESTION

10

1:10 dilution for 4 minutes to achromatic point.


1U in 1:10 dilution, 1U x 10 = 10U in stock solution per mL
Figure 1 Shows the Calculation of the Number of U per mL of Amylase in the Stock Solution of
Saliva using the Definition 1U is the Enzyme Required to Digest 4.5mL of 0.5% Starch Solution
at 37oC to the Achromatic Point in 4 Minutes.