CHEM 5 - BIOCHEMISTRY

EXPERIMENT NO. 6
PROTEINS

I. OBJECTIVES
1. To be able to make visual observations on the effects of some physical and chemical agents on
proteins.
2. To be able to perform some color tests on proteins and amino acids.
II. INTRODUCTION
Proteins are most abundant molecules in the cells after water account for about 15% of a cell’s
overall mass. It is a naturally-occurring, unbranched polymer in which the monomer units are amino
acids. Amino acid is an organic compound that contains both an amino (-NH2 ) and carboxyl (-COOH)
groups attached to same carbon atom.
Denaturation refers to the unfolding and rearrangement of the secondary and tertiary structures
of a protein without breaking the peptide bonds. Some denaturation are reversible, while others
permanently damage the protein, irreversible. The agents which cause denaturation are heat causes
reversible denaturation of protein but vigorous heating denatures protein irreversibly by disrupting
several types of bonds., alcohol coagulates all types of proteins by forming H-bonds that compete with
naturally occurring H-bonds in process thus increasing precipitation but the process is irreversible, salt
and heavy metals denature protein by disrupting salt bridges and disulfide bonds present in the protein
thereby decreasing precipitation when added in excess, alkaloidal reagents denatures protein
irreversibly by disrupting salt bridges and hydrogen bonds, radiation denature proteins irreversibly by
disrupting the H-bonds and the hydrophobic bonds present in the protein, pH changes disrupts H-bond
and salt bridges causing irreversible denaturation, salting out separates a protein from a mixture of
other substances particularly insoluble salts, and oxidizing agents denature protein irreversibly by
disrupting disulfide bonds.
What is being tested when the following tests are done on proteins?
1. Biuret test
A Biuret test is a chemical test used to determine the presence of a peptide bond in a
substance. It is based on the biuret reaction in which a peptide structure containing at least two peptide
links produces a violet color when treated with alkaline copper sulfate. In presence of an alkaline
solution, blue-colored copper II ion can form a complex with the peptide bonds since the peptide has
unshared electron pairs in nitrogen and oxygen of water.
2. Millon’s test
Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which is the
only amino acid containing the phenol group. Millon’s test is a specific test for tyrosine, but it is not a
specific test for protein as it also detects the phenolic group present in other compounds as well.

3. Xanthoproteic test
Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine,
tryptophan and phenylalanine) in a protein solution which gives yellow color nitro derivatives on heating
with conc. HNO3.

4. Hopkin’s-Cole test
This test is specific test for detecting tryptophan. The indole moiety of tryptophan reacts with
glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product.
III. RESULTS AND DISCUSSION
Part 1: DENATURATION
A. Heat Denaturation
Observations:

 After boiling: Formation of white precipitate

 After adding acetic acid observe the effect: It further increased the volume of precipitation
B. Denaturation using Concentrated Mineral Acids
Observations:

 Color of the precipitate formed: White precipitate

 After addition of excess acid observe the effect (note whether the precipitate that forms will
dissolve in excess acid): the precipitate disappeared, it became clear and the precipitate that
formed dissolved in the excess acid.
C. Action of Heavy Metals

Observation after
addition of excess
Observation after
reagent (observe
Test tube No. Reagent added addition of a few drops
whether the precipitate
of reagent
will increase or
decrease in amount)
1 1% HgCl2 White precipitate The precipitate
decreased in amount
2 1% AgNO3 White precipitate The precipitate
decreased in amount
3 1% Pb(C2H3O2)2 White precipitate The precipitate
decreased in amount

D. Denaturation using Alkaloidal Reagents

Observation after
addition of excess
Observation after
reagent (observe
Test tube No. Reagent added addition of a few drops
whether the precipitate
of reagent
will increase or
decrease in amount)
1 Satd. picric acid Yellow precipitate The precipitate
increased in amount
2 20% TCA White precipitate The precipitate
increased in amount
3 5% K4Fe(CN)6 Yellowish white The precipitate
precipitate increased in amount

Discussions:
1. Explain the use of strong tea solution as an emergency treatment for burns.

An infusion of tea contains the necessary tannic acid. Tannic acid is strongly astringent and is
mildly anesthetic and slightly antiseptic in it it’s action. It coagulates the damaged proteins. The
scab which forms on the surface restrains excessive granulations and protects the ingrowing
epidermis resulting in a stipple scar.
2. What is the effect of denaturation on the biological functions of proteins? Why?
Denaturation is a structural change in a protein that results in the loss (usually permanent) of its
biological properties because the way a protein folds determines its function, any change or
abrogation of the tertiary structure will alter its activity, it usually is caused by temperature and
pH. High levels of thermal energy may disrupt the hydrogen bonds that hold the protein
together. As these bonds are broken, the protein will begin to unfold and lose its capacity to
function as intended. Amino acids are zwitterions, neutral molecules possessing both
negatively (COO–) and positively (NH3+) charged regions. Changing the pH will alter the
charge of the protein, which in turn will alter protein solubility and overall shape.
 E. Salting Out
Observations:

 After addition of (NH4)2SO4, is there a precipitate formed? Describe the ppt.: yes, a white
precipitate formed
 After addition of water, what happened to the ppt.?: The white precipitate turned to a clear
solution
Discuss:
1. Does the salting out cause protein denaturation? Why?
Salts strip off the essential layer of water molecules from the protein surface
eventually denaturing the protein. Acids and bases alter the pH of the solution as well as disrupt
the salt bridges which are primarily stabilizing ionic interactions between opposite charged
amino acid residues on protein surface because salting out is an effective means for initial
molecule purification.
Part 2: COLOR REACTIONS
F. Color Reactions
Protein sample: egg albumin

TEST Observations Result (+/-)

Biuret Purple coloration +

Xanthoproteic White precipitate at first but +

after heating it slowly turned
yellow then after cooling, the
color changed to orange.
Yellow to orange coloration
Millon’s White precipitate at first but +
after heating, cooling, adding
the reagent, and the water
bath, old rose precipitate
Hopkin’s-Cole Violet ring appears at the +
junction of the two layers

Discuss:
1. What do the results reflect about your protein sample?

The result reflects that the tests are positive since those tests were specifically used to measure
the amount of proteins present in the samples.

2. Are your results correct? Justify your answer.

Yes, my results are correct based on the positive results of the tests since peptide bonds are
responsible for the purple coloration in biuret test. The aromatic compounds are responsible for
the coloration in xanthoproteic test since it is used to detect the amino acids. Millon’s test is
used to detect the tyrosine in phenol group which the phenolic groups are the one’s responsible
for the old rose precipitate as well as with Hopkin’s-Cole test which tryptophan is the product
responsible for the violet ring that appears at the junction of two layers. The result changes in
their shapes and affect their functions like in the denaturation of proteins.

IV.CONCLUSION
The physical agents are the heat, high pressure, ultraviolet, surface action. In the chemical agents, the
acids and alkalis attack directly the hydrogen bonds in the secondary and tertiary structure of proteins. The
denatured protein is greatly decreased in solubility at its isoelectric point. The chemical groups are exposed to
chemical reactions and more readily detected as a result of the unfolding process in denaturation. Enzymatic
or hormonal activity is usually destroyed by denaturation. If the denaturation is severe, the protein molecules
become insoluble and precipitation results as well as the changes in the properties of the proteins are
permanent and “irreversible”. In case of mild denaturation, there is “reversible denaturation” leading to the
slight changes in the properties of the protein which can be restored to the native state after suitable treatment.
Protein reacts with a variety of reagents to form colored products. These tests, known as color reactions of
proteins, are of importance in qualitative detection and quantitative estimation of proteins, and of their
constituent amino acids in body fluids and other biological materials. They are Biuret reaction to detect peptide
bonds which gave a purple coloration, Xanthoproteic reaction to know the presence of aromatic amino acids
which gave an orange coloration, Millon’s test to know the presence of tyrosine which gave an old rose
precipitate, Hopkins-Cole’s Test for indole nucleus to detect the presence of tryptophan which gave a violet
ring at the junction of two layers. Similarly, when egg albumin is heated till it is coagulated, the denaturation is
irreversible and the secondary and tertiary structure of the proteins are completely lost resulting in a mixture of
randomly arranged polypeptide chains.

V. REFERENCES

Anupama Sapkota, (2020), Millon’s Test- Definition, Principle, Procedure, Result, Uses, retrieved last
December 1, 2020 from, https://microbenotes.com/millons-test/

A. Taneja, denaturation of proteins (with denaturing agents), retrieved last from December 2, 2020 from,
https://www.biologydiscussion.com/proteins/denaturation-of-proteins-with-denaturing-agents/41902

Bio-OER, (2019), Proteins, retrieved last December 2, 2020 from,

https://bio.libretexts.org/Bookshelves/Biotechnology/Bio-
OER_(CUNY)/02%3A_Chemistry/2.09%3A_Proteins

H. Stephen Stoker, (2009), General, Organic, and Biological Chemistry, 5th edition,

M.V.P. Peiris, (1937), the use of tea in the treatment of burns, retrieved last December 2, 2020 from,
https://pdfs.semanticscholar.org/ef83/69cbdac2c7c428a7ab265bf3eea157c342d6.pdf

Sagar Aryal, (2020), biuret test for protein, retrieved last December 1, 2020 from,
https://microbenotes.com/biuret-test-for-protein/