GIMNASIO DEL NORTE

LAB REPORT
IB BIOLOGY

Name of the student: Maria Jose Rodriguez

Date: February 5th, 2018
Name of the practice: Enzymatic activity

The objective of the practice is to evaluate the enzymatic activity of amylase when it reacts
with starch in acidic and alkaline pH’s.

An enzyme is built from proteins folded in complicated shapes and they are present
throughout the body, the chemical reactions that develop inside of our bodies rely on the
enzymes. Functions such as metabolism and respiration depend on enzymes, this proteins
help speed up chemical reactions that without them could take a very long time, this
process is called catalysis. The way catalysis occurs involves an enzyme, an active site and a
substrate. A substrate is a molecule that acts with an enzyme. Enzymes catalyze chemical
reactions involving the substrates. The substrate bonds with the enzyme active site, and
that creates an enzyme-substrate complex. The active a site is a specific region of the
enzyme to which the substrate binds, this is an element of the protein that determines
whether the protein is functional when having a reaction with an enzyme. After the
reactions the substrate are converted into products and these products leave the active
site, allowing other substrates to bind with the active site and produce more reactions.

In the first step the polar regions of amino acids attract the substrate and the active site of
the enzyme. The active site is the surface of an enzyme to which substrates bind and that
catalyze the reaction. Once the substrate has been bound, the reaction is catalyzed. The
products are released, and the enzyme is used again.

Figure 1 (Substrate-enzyme complex)

A substrate molecule can only bind to the
active site if it moves very close to it. The
getting together of a substrate molecule and
an active site is known as collision. With most
reactions the substrates are dissolved in
water around the enzyme. Because of water
being a liquid, the particles dissolved in it are
in contact with each other and are in
constant motion. Both substrates and
enzymes are able to move, either way as substrates are typically known to be smaller than
enzymes they tend to move faster.
Collisions between substrates and active sites occur because of random movement of the
substrate and the enzyme. The successful collisions are those in which the substrate and
active site are in site, allowing the binding to occur.

Figure 2 (Denaturation of enzymes)

Enzymes, like other proteins can have their
structure altered by certain conditions. This
process called denaturation can be caused by
high temperatures and either high or low pH.
When an enzyme is denatured, the active site is
altered, so the substrate can no longer bind,
and if it binds the reaction probably won't
occur. In many cases denaturation causes
enzymes that were dissolved in water to
become insoluble and form a precipitate.

Most enzymes have an optimum pH in which their activity is in Figure 3 (pH scale)
It’s highest peak. If the pH is increased or decreased from
that optimum, enzyme activity decreases and then stops
altogether, when this happens the structure of the enzyme is
altered, including its active site. Beyond a specific pH the
structure is irreversible altered, this is another example of
denaturation.
Not all enzymes share the same optimum pH, actually the
range is pretty large. This reflects the wide range of pH
environment in which enzymes work.
The image by the right side shows the wide range of optimum
pH in different places where enzymes function.

It is important to add that pH is involved with the concentration of hydrogen ions, acidic
solutions have a high concentration of hydrogen ions, which is higher than the one in pure
water. Basic solutions have a low concentration of hydrogen ions, lower than the one in
pure water. Solutions are marked as alkaline or acid based on their concentration of
hydrogen ions in relation of pure water

Alpha-amylase is a common among living organisms. In the digestive systems of humans

and many other animals, an alpha-amylase called ptyalin gets produced by the salivary
glands. Ptyalin is mixed with food on the mouth, and it acts with starch. Even if the food
remains in the mouth for only a short time, the action of ptyalin continues for long in the
stomach, until the food is mixed with the stomach solutions, then the high acidity that
inactivates ptyalin. When food passes to the small intestine, the molecules left of starch are
catalyzed to maltose by pancreatic amylase. The products of amylase hydrolysis are broken
down by other enzymes into molecules of glucose, that are absorbed really fast in the
intestine wall.
Starch is the most abundant biomolecule on earth after cellulose and the major
carbohydrate. It is found as a powder which contains smaller amylose molecules. By far the
biggest source of starch is corn, some other sources are wheat, potato, and rice.

Starch when mixed with Iodine (a chemical indicator) turns a deep shade of blue because
starch contains amylose. Amylose is a soluble component of starch, in addition to other
carbohydrates.

Amylose molecules are made up of single strands of glucose that are shaped like springs.
When iodine is added to a starch, it adheres to amylose molecules because of their
solubility. The starch pushes the iodine into a line in the middle of the amylose spirals and
creates a transfer of charge between the iodine and starch. This causes a change in the
order of electrons and energy level spaces. The new spaces absorb light differently and
create the deep blue color that is visible to the human eye.

In the first hand, for the purpose of the laboratory a hypothesis was planned and developed,
which was If pH changes from acid levels (4 and 5) to alkaline levels (9, and 10) instead of
conserving amylase optimum pH (7) , then enzymatic activity will decrease, because 𝛼
amylase has an optimum pH, and if that level is altered the enzyme undergoes the process
of denaturation, in which the protein changes shape, as well as it’s active site, resulting in
unsuccessful collisions between the substrate (starch) and the previously named enzyme.

In the second hand, variables were essential for the experiment. The independent variable
is the pH level, which will be managed by using different pH buffers with levels of 4, 5, 9
and 10, then, this variable will be measured using a pH sensor. The dependent variable is
the monosaccharide concentration after 10 minutes, which will be measured using a scale
of monosaccharide concentration indicated with lugol iodine solution.

For this practice there are two controlled variables that are substrate concentration and
temperature, in order to have liable results these variables need to be kept in the same
conditions throughout the whole experiment. Substrate concentration will be constant in
10ml of 1% starch solution and temperature will be kept in 37 C in a stove with a
thermometer. Moreover, the control variable will be a solution in a pH level of 7, with 10ml
of 1% starch solution, and it will be kept in 37 C, this variable contains all the factors in
optimum conditions for enzymatic activity of amylase.

This experiment required the use of the following materials, one graduated cylinder, two
200ml beakers, two 100ml beakers, one stove, one ceramic square, one pipette, one
thermometer, two measuring boats, one pH sensor (Lab Quest), one dropper, one tong and
one ring stand. In addition, for the three repetitions it will be needed to have 15gr of 
amylase, 38 gr of starch, 110ml of distilled water, 30ml of iodine solution, 15ml of each pH
buffer (4, 5, 7, 9 and 10), and 1.275ml of water.
Before the practice a measuring method was developed in order to correctly obtain
quantitative results for the trials, in order to do so a monosaccharide concentration scale
was created before the experiment, the values used were the following, 1%, 3%, 5%, 7%,
10-% and 12%, for doing this 100 ml of water were filled in 3 beakers and 3 Erlenmeyers,
then in one beaker 1 gr of starch was added, in another one 3gr and in the last one 5gr, then
in one Erlenmeyer 7gr, in another one 10gr and in the last one 12 gr. In each beaker and
Erlenmeyer 2ml of iodine solution was added in order to expose the color of the solutions
depending on the concentration of the monosaccharides.

The method required for each repetition was the following. First , boil 100ml of distilled
water in a 200ml beaker using the stove in “high” level, while doing so, mix 10ml of distilled
water in and 1gr of starch ,when the boiling water starts to evaporate, add the 10ml of
distilled water and starch in the boiling water and leave it in the stove for one minute, then
use the tongs to put the mixture in a ceramic square and leave it cooling. Second, mix 45ml
of water, 5ml of pH buffer level 7 (measured with the pipette) and 1 gr of  amylase in a
100ml beaker. Third, use the graduated cylinder to measure 10ml of the 1% starch solution
made before into the  amylase solution with pH of 7, heat the mixture in a stove and with
a thermometer placed in the ring stand wait until the mixture is in 37 C, then using the
tongs put it in a ceramic square, then with a chronometer, count 10 minutes. Third, check
with pH sensor that the pH is in its expected level. Fourth, with the pipette add 2ml of
iodine solution and take pictures of the results. Lastly, repeat from the second step changing
the pH levels (4, 5, 9 and 10).

Due to the liability of the results, the practice will be repeated 3 times,
this way the results can be analyzed more precisely, having in mind the
results that will be obtained after each repetition.

The picture in the left shows the control variable, which have the optimum
conditions for enzymatic activity of amylase (1% substrate concentration,
pH 7 and 37 C

The monosaccharide concentration scale can be seen down below.

MONOSACCHARIDE CONCENTRATION
Chemical indicator: Lugol iodine

Low concentration High concentration

Results
These results were taken in relation with the monosaccharide scale*

Table 1 Pictures #1
Monosaccharide concentration Solutions in the first trial
in the first trial
pH Monosaccharide
concentration
4 1%
5 3%
7 12%
9 5%
10 5%

Table 2 Pictures #2
Monosaccharide concentration Solutions in the second trial
in the second trial

pH Monosaccharide
concentration
4 3%
5 5%
7 12%
9 7%
10 3%

Table 3 Pictures #3
Monosaccharide concentration Solutions in the third trial
in the third trial
pH Enzymatic
activity
4 1%
5 3%
7 12%
9 7% pH 4 pH 5 pH 9 pH 10
10 5%
What can be seen in tables 1,2 and 3 is that enzymatic activity decreases when the pH of
the solution isn’t in its optimum peak, the highest concentration in every repetition
occurred when the pH was in level 7, this means that reactions happened faster, and
successful collisions were more probable when the solution was in optimum conditions.

Moreover, the lowest concentrations in every repetition were visible when the reaction
occurred in acidic and alkaline pH levels, the reason for those results were related to the
denaturation of the enzymes, which happens slowly and causes the rate of reaction to be
slower as well, which is also a reason for the low concentration in the 10 minutes in which
the solutions were left to react. Equally important, even if the amylase enzyme works
between pH levels of 3 to 11 the reaction didn’t happen as successfully in acidic and alkaline
levels as it did in the neutral pH in which it is supposed to work. In addition, results that
were more prominent for the acidic pH during the experiment where the ones in which the
monosaccharide concentration was around 1% to 3%, which means that low pH levels are
more susceptible to having few reactions.

When the iodine solution enters the spiral in the amylose it colors a dark blue, which is also
close to brown-like color. Equally relevant, starch is a monosaccharide that contains
amylose, which is the reason why iodine stains starch blue. Furthermore, the most acidic
pH (4) was the solution that didn’t stain as dark as the other ones in the three repetitions,
this means that when the enzyme-substrate complex was going through the reaction in
strong acidic levels collisions weren’t successful and the rate of reaction could potentially
be slow as a consequence, causing the iodine to stain lightly in the solution. It is also
important to add that the pH levels that were closer to 7, which were five and nine were
the ones who had a higher monosaccharide concentration than the other ones, this
indicating that strong bases and acids alter the reaction in a bigger way.

The results obtained indicate that amylase-starch solution is not a buffer solution, as a
buffer solution is able to endure the shock of changing pH levels because it has a weak acid
and a base, this explains why enzymatic activity was in its lowest when the pH level included
a strong acid. pH levels are indicated by the amount of hydrogen ions that they contain, it
is important to remember that hydrogen has a molecular mass of one, which means that it
is able to move easily and rapidly, creating drastic changes in the solution that it acts upon,
this is relevant to understand why levels closer to the optimum pH didn’t have a large
difference in concentration when compared to the control variable, as hydrogen didn’t
move in big proportions from the optimum point, enzymatic activity didn’t decrease as
much as it did when exposed to the strong acid and base.

Amylase-Starch complex, as said before works efficiently in a pH level of 7, in this level the
solution contains a necessary and leveled amount of hydrogen ions, when this pH drops
into acidic levels hydrogen ions are released, which cause a decreasing action in the
catalysis of the reaction, reflected in monosaccharide concentration. On the contrary, when
the pH increases to alkaline levels it receives hydrogen ions, that as well as with the acidic
levels cause enzymatic activity to decrease.
Additionally, the hypothesis was valid, as enzymatic activity did decrease when the enzyme-
substrate complex was reacting in pH levels different to its optimum peak. Besides, the
objective of the practice was completed throughout the practice and in the analysis of the
quantitative results obtained using the scale created for the purpose.

Lastly, the practice had strong factors that helped the recollection of results, such as the
responsible use of materials in the laboratory and the precision in choosing the tools that
were used in each trial. However, the recommendations for this practice are valuable and
necessary, for instance, the monosaccharide scale, even if it was effective could include
more values, in order to have more liable and precise results. In addition, it is essential to
calibrate the pH sensor, so that it is confirmed that the solutions are in the pH level that
they are expected to be.

BIBLIOGRAPHY

1. Why Does Iodine Turn Starch Blue? Reference* (2015)

https://www.reference.com/science/iodine-turn-starch-blue-70931cf7c358f2e2
2. Is pH the Measurement of Hydrogen Ion Concentration or Ion Activity? YSI (2015)
https://www.ysi.com/ysi-blog/water-blogged-blog/2015/01/is-ph-the-
measurement-of-hydrogen-ion-concentration-or-ion-activity
3. Amylase Britannica (2018) https://www.britannica.com/science/amylase
4. Starch Water structure and science (2017)
http://www1.lsbu.ac.uk/water/starch.html
5. Dissociation of water Biology Corner (2010)
https://www.biologycorner.com/worksheets/acids_bases_coloring.html
6. 2.5 Enzymes Bioknowledgy https://www.bioknowledgy.info/25-enzymes.html