Experiment N0.

2 - Proteins and Amino Acids

I. Introduction:
Proteins are large, complex molecules that have critical roles in the body. Proteins work in cells and for
structure function and regulation of the tissues and organs in the body. The different levels of protein structure
are primary, secondary, tertiary, and quaternary structure. Examples of protein are actin, myosin, and
hemoglobin.
The building block or the basic unit of protein is the amino acid. Amino acids are composed of the amine
group, carboxyl group, and a variable side chain group. The 20 amino acids found within the proteins convey
a vast array of chemical versatility.

II. Objective:
1. To be able to test for the preserve of certain amino acids and characteristic structures in proteins like egg
albumin.
2. To be able to observe and describe the effects of denaturing agents such as heat, strong acids and bases
heavy metal ion solutions and alkaloidal reagents on protein solution.

III. Results/Discussion:
A. Color Reactions:
Test Observation +/- Specificity
Biuret (purple Biuret test is used for detecting compounds with
solution) Light purple color + peptide bonds. Histidine is the amino acid that
gives a positive result to the Biuret test.
Xanthoproteic Xanthoproteic test is used to detect amino acids
Pastel yellow color changed
(yellow to orange containing an aromatic nucleus in a protein
sol’n) into dark orange as some +
solution. Tyrosine/tryptophan and/or phenylalanine
reagent is added.
gives a positive result to Xanthoproteic test.
Millon’s (old rose or Old rose color changed into Millon’s test is based on the principle of nitrification
pink precipitate) dark rose mix with light + of the phenol group. Tyrosine is the amino acid that
brown shade. gives a positive result to the Millon’s test.
Hopkin’s Cole Hopkin’s Cole test is also known as the Glyoxylic
(violet or purple Small purple ring at the acid reaction. Tryptophan is the amino acid that
+
ring) junction of the liquids. gives a positive result in the Hopkin’s Cole test.

Pauly’s test (red Pauly’s test is used to examine proteins containing

coloration) Reddish in color. + tyrosine and histidine as the reagent. Histidine and
Tyrosine gives a positive result in the Pauly’s test.
Lead Acetate Lead Acetate Reaction is specific for Sulphur
Reaction (black containing amino acid. Only the sulfur-containing
sol’n/ppt) Black in color. + amino acid formulates the black precipitate. Either
S-S group in cysteine, or S-H group in cystine.
gives a positive result in this test.
Nitroprusside test Nitroprusside test is used to detect the presence of
(reddish-purple Only reddish in color. Not too ketoacids in the blood and the urine. Only the
sol’n) +
dark red color. Cysteine amino acid containing sulfhydryl group (-
SH). in the nitroprusside test is used for.
Sakaguchi Reaction The Sakaguchi Reaction is a colorimetric reaction
(Red orange sol’n) in identifying and quantitating of guanidino groups.
Standard Red orange color. +
Arginine is found and gives a positive result in the
said test.
Heller’s ring test Heller’s ring test is a test that shows that string
(white ring/ppt.) acids cause the denaturation of precipitated
White colored ring. +
proteins. Albumin, the protein gives a positive result
in the Heller’s ring test.

What do the results reflect about your protein sample (egg albumin)?
The result shows that the test gives a positive outcome based on the observation and the specificity of
the different tests of the color reactions. Through this, a good result presents the visibility of the expected color in
every analysis. Around eight amino acids are present in the protein sample egg albumin. Specifically, these are
histidine, tyrosine, tryptophan, phenylalanine, cysteine, arginine, and albumin.

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Precipitation Reactions/Denaturation:
1. Denaturation by Heat and Extreme pH
Observations
Test tube N0. After addition of reagent After pH has been adjusted to neutrality (ppt.
increased or decreased)
1. (protein + conc. HCl) Cloudy white Increase
2. (protein + conc. NaOH) Slightly clear gold and not cloudy Increase
3. (protein + water) Clear whitish and not cloudy Increase

Explain the effect of concentration Acid and base to protein sample.

Proteins are nearly neutral molecules. Some are neither acidic nor basic properties. Acidic carboxyl

groups of aspartic and glutamic acid are almost equivalent to the number of amino acids with elemental side

chains.

Acids and bases can substantially change the environmental pH of proteins, which derange the salt

bridges and hydrogen bonding formed between the side chains, take the lead to denaturation. A protein turns out

to be denatured when its regular shape gets distorted. Weak hydrogen bonds break when exposed to an acid.

3. Precipitation with Alcohol

Observation
Protein solution + ethanol White slimy jelly precipitation

Explain the result.

Ethanol or alcohol denatures proteins the same way as heat. It breaks bonds that hold parts of the protein
in a folded shape. Occasionally, alcohol molecules bond directly to some of the sections of the protein. It disrupts
the standard ways on how the protein would chain to itself.
Furthermore, ethanol denatures a protein by disrupting the intramolecular hydrogen bonding between the
side chains. It is vital in sustaining the tertiary protein structure. Alcohol interacts with the hydrophobic residues in
a protein, breaking its hydrophobic core and resulting in denaturation.

3. Precipitation with Heavy Metal Ions

Test tube No. Observations

After addition of reagents After addition of excess
reagents (inc. or dec. ppt.)
1. (protein + HgCl2)
White and cloudy Increase
2. (protein + AgNO3)
White and cloudy Increase
3. (protein + Pb(C2H3O2)2
White and cloudy Increase
4. (protein + CuSO4)
Milky white and cloudy Decrease

Explain the effect of heavy metal ions to protein sample.

Metals interfere with the biological activity of natural, folded proteins through various modes of interaction.

Metal ions may attach to free thiols or other functional groups in proteins. It may displace metal ions in

metalloproteins. Lastly, to catalyze the oxidation of amino acid side chains.

Heavy metals and metalloids show to inhibit refolding of chemically denatured proteins to disturb protein

folding and to cause the accumulation of emerging proteins in living cells.

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 4. Precipitation with Alkaloidal Reagents
Test tube No. Observations
After addition of reagents After addition of excess
reagents (inc. or dec. ppt.)
1. (protein + saturated picric acid Bright crystal yellow. Increase
solution
2. (protein + 10% tannic acid) Light brownish orange changed into Increase
darker orange
3. (protein + 5% potassium whitish Increase
ferrocyanide
4. (protein + 20% trichloroacetic Light clear white increase
acid (TCA)

Explain the use of strong tea solution as an emergency treatment for burns.
A burn is a wound or injury to the skin or any other organic tissue caused by heat or radiation, electricity,

friction, or contact with chemicals. (Davis, 2021)

The UCLA (the University of California at Los Angeles) Researchers found that the tannic acid in the tea

can smooth burns in as little as five minutes. The solid tea solution is an emergency treatment to cool and apply in

the burnt area to soothe irritation and redness. The tea has multiple ingredients which have anti-inflammatory and

anti-oxidative properties.

IV. Discuss:
What is the effect of denaturation on the biological function of proteins? Why?
Denaturation involves the breaking of linkage within a protein molecule. It is responsible for a highly

ordered structure of the protein in its natural state. In biology, it is the process of modifying the molecular structure

of a protein. For example, the egg white is irreversible. One of the frequent consequences of denaturation is the

loss of biological activity comparable to the loss of the catalytic of an enzyme.

V. Conclusion:

Proteins have a significant role in the body. Its building block is called an amino acid. There are 20 amino
acids found within the protein, which convey a vast array of chemical versatility.

In this laboratory experiment two, we can test for the preservation of assertive amino acids and
characteristic structures in proteins like egg albumin. Similarly, we can observe and describe the effects of
denaturing agents such as heat, strong acids, and bases heavy metal ion solutions and alkaloidal reagents on
protein solution.

In conclusion, there is a positive outcome based on the observation and the specificity of the different
tests of the color reactions because the amino acids are present in the egg albumin. Acids and bases can
substantially change the environmental pH of proteins, which takes the lead to denaturation. Denaturation means
to involve the breaking of linkage within a protein molecule. Additionally, ethanol or alcohol denatures proteins the
same way as heat. Ethanol breaks the hydrophobic core of a protein and resulting in denaturation. While in metal
ions, if added to the protein solution, it inhibits refolding of chemically denatured proteins to disturb protein folding.
It may catalyze the oxidation of amino acid side chains.

In regards for curing or treating burns at home, strong tea solution can be used as an emergency
treatment for burns because it contains tannic acid that can soothe burns round five minutes.

Protein changes its structure in the presence of certain chemicals, acids, or bases. Protein denaturation
plays a role in the biological processes. The way proteins act together with various simple molecules is crucial to
discovering new medicines.

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 Online References

Better Health Channel (2020). “Proteins”. Retrieved from

https://www.betterhealth.vic.gov.au/health/healthyliving/protein

L. F. Torres (2021). “Proteins and Amino Acids”. Retrieved from

https://www.youtube.com/watch?v=pCHpfAeU8SQ&feature=youtu.be

Love Food Love Science (2017). “Protein: Acid Denaturation”. Retrieved from
https://www.ifst.org/lovefoodlovescience/resources/protein-acid-denaturation

M. Abedin (2017). “How do heavy metals denature proteins to cause misfolding and aggregation?”
Retrieved from https://www.quora.com/How-do-heavy-metals-denature-proteins-to-cause-
misfolding-and-aggregation

Breaking Proteins. Retrieved from

https://askabiologist.asu.edu/sites/default/files/resources/activities/breaking_proteins/breaking-
proteins-activity-pdf-3.pdf

Charles Patrick Davis, MD, PhD (2021) “Medical Definition of Burn”. Retrieved from
https://www.medicinenet.com/burn/definition.htm

London Centre for Nanotechnology (2018). “Detecting protein denaturation and the interactions between
proteins and drugs at the nanoliter scale”
Retrieved from https://www.london-nano.com/research/detecting-protein-denaturation-and-
interactions-between-proteins-and-drugs-nanoliter-scale

SUBMITTED BY: QUINCY MARY M. CLARITO

COURSE AND YEAR: BSN 1- A
SUBMITTED TO: MAÁM ANN MARIE P. ALGUIDANO