The Characterization of Biological Macromolecules

Angelo Phillip A. Bautista, Ales Mariya Clarion, Zyrelle John A. Precilla, Jana Raiza F. Pati ño, and Rheiz Janae P.
Montecillo

Philippine Science High School – Central Visayas Campus, Department of Science and Technology, Talaytay,
Argao, Cebu, 6021, Philippines

Corresponding Author Email: aabautista@cvisc.pshs.edu.ph, aclarion@cvisc.pshs.edu.ph,

zaprecilla@cvisc.pshs.edu.ph, jfpatino@cvisc.pshs.edu.ph, rpmontecillo@cvisc.pshs.edu.ph

Abstract

Biological macromolecules, or biomacromolecules, are the basic substances produced by cells. Protoplasm,
a key component in the living part of cells, comprises several types of these biomacromolecules. In this study, five
experiments were performed for the detection of these molecules, namely, the starch, sugar, coagulation, biuret, and
grease spot tests. The experiments aimed to detect these macromolecules by observing the reactions of biomolecule
samples to the different reagents/acids/bases, such as IKI, NaOH, HCl, etc. The starch test was to detect the starch
Amylase enzyme in the samples, which resulted in only the starch sample reacting when compared to glucose and
water. The sugar test was to detect reducing sugars in the samples, with sucrose showing 0.1% - 0.5% reducing
sugar content, glucose having 1.0% - 1.5%, and fructose having 1.5% - 2.0%. The coagulation test was to observe
the change in protein structure of the egg albumen (protein sample) in response to heat or a change in pH. Based on
the reactions, it can be concluded that the albumen was a coagulable protein. The biuret test was to detect a presence
of protein, and it resulted positively as the albumen sample showed that it contained protein. Lastly, the grease spot
test was to test the lipid contents of a substance, which resulted positively as the vegetable oil showed a higher
presence of fats and oils when compared to water. In conclusion, this study was successful in developing more
background knowledge about biomacromolecules, and discovering how they react to different environmental and
chemical stresses.

Introduction Methodology

Biological macromolecules, or Starch Test

biomacromolecules, are large organic chemical
structures that are created by living organisms, more
specifically, the cells. They are made up of covalent
bonds of simpler chemical structures called
monomers. Biomacromolecules are generally
classified into 4 groups: carbohydrates, proteins,
lipids, and nucleic acid. They take part in various
different functions, including structural support, a
source of energy and genetic information, etc. The
protoplasm, the viscous fluid that acts as the living
part of a cell, is a mixture of several kinds of these
biomacromolecules. This study aims to determine
what kind of environmental or chemical stresses can
form biochemical reactions with the biomolecule
compounds, to attain a deeper knowledge on the
characteristics and functions of the stated
compounds. This study includes the detection of
starch amylase, reducing sugars, coagulability,
proteins, and lipids. Due to the lack of necessary
apparatuses, no experiments were performed under
the topic of nucleic acids.
3 test tubes were numbered respectively from 1-3.
1 ml of starch solution was added to test tube #1, 1
ml of glucose solution to test tube #2, and 1 ml of
water to test tube #3. 2 drops of iodine potassium
iodide (IKI) solution were added to each tube.
Changes in appearance were recorded.
Sugar Test
Test tubes were labeled from numbers 1-4, each
with 1 ml of glucose solution, fructose solution,
sucrose solution, and water, respectively. 2 ml of
Benedict's solution was added into each test tube
before being placed over the alcohol lamp to boil.
The tubes were shaken in a back-and-forth motion
before being lined up. Any noticeable changes in
appearance were recorded.

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Coagulation Test
Four 10mL test tubes were each added with 2 ml
of a dilute egg albumen solution, with distilled water
as the solvent. Initial observations of the solution
were recorded. 10 drops (1 ml) of ethanol (C2H5OH)
were added to the first test tube. 10 drops of
concentrated hydrochloric acid (HCl) were added to
the second test tube. 10 drops of sodium hydroxide
(NaOH) were added to the third test tube. The fourth
test tube was placed on an alcohol lamp to heat until
it boiled. All the test tubes were given around 6
minutes to settle and were shaken well. Changes in
the substances’ appearance were observed and
recorded.
Biuret Test
3 ml of egg albumen solution was placed in a test
tube and 20 drops of concentrated sodium hydroxide
(NaOH) was slowly added. Initial observations of the
solutions were recorded. After that, 20 drops of a
dilute solution of copper sulfate (CuSO4)
were added to the solution. Final changes and
observations were recorded.
Grease Spot Test
2 similar-sized circles were drawn on a sheet of
paper. A drop of water was placed in one circle and a
drop of vegetable oil was added to the other. Both
were left to dry for at least 3 minutes. A light was
then shone under the paper to see which liquid has
more fat and oil. This can be determined by checking
which one of the 2 dried liquids leaves a spot.
Results
Starch Test
From clear, the starch in the test tube turned a
very dark brown with hints of orange after adding 2
drops of IKI solution. From a clear and colorless
substance, the glucose in the test tube turned bright
orange after adding 2 drops of IKI solution. From
clear, the water in the test tube turned to a similar
color to the results of the second procedure, bright
orange, however, it appears darker and redder
compared to the glucose solution. The three test tubes
are as shown in Fig. 1.
Figure 1
Starch Test - Test Tubes For (1) Starch, (2) Glucose,
And (3) Water
Sugar Test
After the addition of 2 ml of Benedict’s solution,
the results included changes in color. The glucose
solution changed from a clear to a sky-blue color. The
fructose solution went from clear to sky blue to a
cloudy sea green or blue color. The sucrose solution
went from a blurry light yellow to a greenish blue.
The water went from a clear to a blue color.
However, after heating over an alcohol lamp, the
color changed again. The glucose solution changed
from sky blue to orange and then finally settled with
a bright orange color. The fructose solution, when
heated, bubbled from orange to bright orange to
red-orange and finally, a brown color. The sucrose
solution changed from its last seen color, a greenish
blue, to green before coming to a boil, turning into a
swampy green color. After settling, however, it
turned yellow. Finally, the water, upon boiling, did
not react nor change from when Benedict’s solution
was added. See Fig. 2 for a more accurate depiction
of the colors.
Figure 2
Sugar Test - Test Tubes For (1) Glucose, (2)
Fructose, (3) Sucrose, And (4) Water
Coagulation Test
For the test tube with ethyl alcohol (C2H5OH),
the results showed precipitation and a bit of
cloudiness but mostly precipitation on top of the
albumen. For the second test tube with concentrated
hydrochloric acid (HCl), there is precipitation at the
bottom, causing the solution to be a little cloudy. For
the third test tube with concentrated sodium
hydroxide (NaOH), the solution did not have
precipitation and it retained the same color it had
before the concentrated sodium hydroxide was added
only slightly cloudier. For the final test tube which
2
only contained the diluted egg albumen solution, the
solution was boiled for too long and was nearly
cooked. It was fully white but still moved when the
test tube was shaken. The final test tubes are as
shown in Fig. 3.
Figure 3
Coagulation Test - Test Tubes For (1) Ethanol, (2)
HCl, (3) NaOH, And (4) Diluted Albumen
Biuret Test
Adding 20 drops of concentrated sodium hydroxide
(NaOH) to the Albumen solution resulted in a cloudy
white mixture. Upon adding 20 drops of a dilute
solution of copper sulfate (CuSO4), the mixture’s
color first turned blue, then purple, then green, and
finally, dark blue, as shown in Fig. 4.
Figure 4
Biuret Test - Test Tube Sample
Grease Spot Test
As shown in Fig. 5, when held up to the light, the
circle with vegetable oil left a more visible and wider
spot than the drop of water, which appeared to have
already dried.
Figure 5
Grease Spot Test - (1) Water Spot and (2) Vegetable
Oil Spot
Discussion
All tests came up with different results per
different combinations of compounds and chemicals.
Results included observational elements, specifically
in terms of color.
In the starch test, with the three solutions starch,
glucose, and water, all three ended up with different
results. This is due to the Amylose interacting with
the starch, resulting in the change in color. If starch
amylose is present, the color will turn a blue-black
color while staying orange if it is not present
(Libretexts, 2022).
The results for the starch test showed that only
the starch solution showed a different observational
result in terms of color. Though what we have
observed was the color being a dark brown color, it
could have been a misinterpretation of a blue-black
color due to the color being dark in value. This
indicates that only the starch solution showed signs of
starch compared to both the glucose solution and
water
In the sugar test, the solutions glucose, fructose,
sucrose, and water were all clear in color before the
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addition of the bright blue Benedict's solution. All
turned into a different color after they were heated on
the oil lamp.
The solutions that are Benedict's solution are
added to changes in color depending on the
concentration of the reducing sugar present (Loqman,
n.d.). Based on the result interpretations in a research
by Aryal (2022), we can determine the percent of
reducing sugars present.
The glucose solution contains about 1% to 1.5%
reducing sugars present because of its bright orange
color. The fructose solution contains about 1.5% to
more than 2% reducing sugars due to it being red in
color before dying down to a brown color. The
sucrose solution contains about 0.1% to 0.5%
reducing sugars due to its green color. Finally, the
water has no trace of reducing sugars because it didn't
change in color.
In the coagulation test, it is a test to see the
change in the structure of proteins as a result of heat
and change in pH (Sapkota, 2022). Also according to
the research of Sapkota (2022), in the case of
coagulable proteins, when these proteins are heated at
their isoelectric pH, the polypeptide chains become
uncoiled and matt with each other to form an
insoluble mass. The mass formed doesn’t dissolve
back to the liquid. Based on our results, we can
conclude that the Albumen is a coagulable protein.
In the biuret test, from the Albumen from the
coagulation test, the results were yet again a change
in color. According to Vedantu (2022), in the
presence of protein, it changes its color from blue to
violet and that the color intensifies as the number of
peptide bonds increase in protein. With this, after the
addition of the 2 ml of HCl, we can conclude that the
change in color to a darker blue of the Albumen
solution was the result of the presence in protein.
Since it was of a darker color, plenty of peptide bonds
were increasing in protein.
Finally in the grease spot test, according to the
Ministry of Education, a translucent spot or stain on
fabrics indicates the presence of lipids. The results of
our test conclude that the vegetable oil has more
lipids therefore more fats and oils compared to water
when dried and shown with light.
Summary and Conclusion
The majority of our findings were in line
with what was anticipated based on a number of other
studies and reference materials. We were able to get
the results we anticipated from the starch test, which
was designed to test for the presence of starch.
Several articles have said that when there is starch
present in the solution, the iodine changes from
brown to blue-black or purple (Do It Yourself:
Causes of Color., n.d) The same is true for the other
tests (biuret, coagulation, and sugar test); we were
able to obtain the color changes predicted by the
reference articles (Sourcing Sugars: Benedicts., n.d)
Although there were no color changes with the grease
spot test, once more, our findings were consistent
with those of other academic studies. (Tests of oils
and fats, 2023.) Therefore, it is recommended to
understand these biomolecules along with their
importance, so that healthy diets are easier to
maintain.
Acknowledgment
The authors would like to express their gratitude
to Mr. Paul Isaac Dizon from the Biology Lab for
assisting them with their experiments and providing
them with the materials and tips to successfully
complete their experiments. Credits should also be
given to Mr. Eleazar Guia, their research adviser,
Biology teacher and supervisor in the performance of
their experiment.
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