Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University

Date Performed: June 20, 2017 Score: ________

Date Submitted: June 29, 2017 Prepared by:BS Chem-2

Experiment No. 6
Proteins

Objectives:
 Identify the structural patterns of proteins.
 Use the isoelectric point of casein in milk to isolate the protein.
 Use chemical tests to identify amino acids and protein.
 Observe the denaturation of proteins.

Results:
A. Separation of a Protein (Casein) from Milk
Table 1. Masses of the Materials
Part Materials Mass (grams)
Erlenmeyer Flask (250-mL) 116.14
A-1 Erlenmeyer flask (250-mL) with milk 163.63
Milk 47.49
A-2 Casein Product 6.58

Percent Casein in milk: 14%

Tests for Amino Acids and Proteins

B. Biuret Test
Table 2. Test for the presence of Peptide bonds (2 or more).
Compound Color Conclusion
Lysine Blue Negative for the presence of 2 or more peptide bonds.
Alanine Blue Negative for the presence of 2 or more peptide bonds.
Aspartame Blue Negative for the presence of 2 or more peptide bonds.
Gelatin Violet Positive for the presence of 2 or more peptide bonds.
Albumin Violet Positive for the presence of 2 or more peptide bonds.
Casein Violet Positive for the presence of 2 or more peptide bonds.
C. Ninhydrin Test
Table 3. Test for the presence of Amino Acids & Proteins.
Compound Color Conclusion
Lysine Red violet An amino acid.
Alanine Blue An amino acid.
Aspartame Blue Positive for the presence of protein and amino acids.
Gelatin Violet Positive for the presence of protein and amino acids.
Albumin Violet Positive for the presence of protein and amino acids.
Casein Blue Positive for the presence of protein and amino acids.

D. Xanthoproteic Test
Table 4. Test for the Presence of Aromatic Ring(s).
Compound Color Conclusion
Tryptophan Yellow Aromatic ring is present in the molecule.
Tyrosine Yellow Aromatic ring is present in the molecule.
No aromatic ring present in any molecule of the
Aspartame White
compound.
Gelatin Yellow Aromatic ring is present in the molecule.
No aromatic ring present in any molecule of the
Albumin White
compound.
No aromatic ring present in any molecule of the
Casein Colorless
compound.

E. Sulfur Test
Table 5. Test for the presence of Sulfur.
Compound Color Conclusion
Cysteine Yellow-orange Sulfur is present in the molecule.
Alanine Colorless No sulfur is present in the molecule.
Aspartame Not performed Not performed
Gelatin Yellow Sulfur is present.
Albumin Yellow Sulfur is present.
Casein Dark red Sulfur is present.

F. Denaturation Test
Table 6. Test for the Denaturation of Protein.
Denaturing Agent Observation
Heat Turbid solution
Heavy AgNO3 White precipitate & small amount of violet compound were formed
Metals PbAc2 Turbid solution
6M HNO3 White precipitate was formed
Ethanol Cloudy solution
Discussion
Upon subtracting the mass of Erlenmeyer flask with milk by the mass of Erlenmeyer flask, the mass
of milk was obtained, which was equal to 47.49 g. After heating the milk the casein was therefore,
separated and was dried afterwards. The dried casein weigh 6.58 g. Then, the percent of casein in milk
was obtained by dividing the mass of casein by the mass of milk and then multiplied by 100, and this
resulted to 14 percent.
From the six samples that were obtained, three gave blue solutions and the other three
produced violet solutions upon the addition of 10% NaOH and 0.5% CuSO4. Those samples, lysine,
alanine, & aspartame, which produced blue solutions indicate the absence of polypeptide since lysine
and alanine are individual amino acids and aspartame is the combination of phenylalanine and
aspartic acid, so, no peptide bond and only one peptide bond was present, while those samples,
gelatin, albumin, & casein, which produced violet solutions indicate the presence of polypeptide since
these compounds are composed of substances called protein, which is made up chains of more than
two amino acids, thus, have several peptide bonds.
In ninhydrin test, all the samples, lysine, alanine, aspartame, gelatin, and albumin, produced
blue (alanine & aspartame) and violet (gelatin, lysine, and albumin) solutions. The blue and violet
solutions confirmed the presence of amino acid(s). The casein also produced blue solution indicating
the presence of protein and amino acids in the compound. Ninhydrin is a test for amino acids and
proteins. Therefore, the samples were positive in the said test since they are amino acids (lysine &
alanine) and are made up of proteins (gelatin, albumin, casein, & aspartame).
With concentrated nitric acid, HNO3, amino acids with aromatic rings in their molecule reacted
with the strong acid to produce nitro-substituted benzene rings which appeared yellow. Since
tryptophan and tyrosine have aromatic rings in their molecules, they produced the yellow solution after
reacting with nitric acid. Gelatin has also produced yellow solution when added with nitric acid,
therefore, gelatin contains amino acid(s) with aromatic ring(s). For aspartame, albumin, and casein,
they produced yellow and white solutions, which indicate the absence of aromatic rings in the
molecules that composed the said compounds.
Between cysteine and alanine, it was the cysteine that produced yellow-orange solution
indicating the presence of sulfur in its molecule. By looking back to the molecular structure of cysteine
in the previous experiment (experiment no. 5: amino acids), it has sulfur atom attached to the carbon
atom that is attached to the alpha carbon. Thus, this amino acid reacts with the lead (II) acetate
producing a lead sulfide, PbS, precipitate. Gelatin and albumin also produced yellow solutions when
reacted with lead (II) acetate. Casein, however, produced a dark-red solution when added with lead
(II) acetate. This results of gelatin, albumin, and casein indicate that sulfur was also present in one or
more of the molecules that composed the compounds. While the rest of the samples, alanine gave
colorless or clear solution meaning sulfur was absent in their molecules.
In the last test, different denaturing agents, heat, heavy metal (Ag+ from AgNO3 & Pb2+ from
PbOAc2), ethanol, HNO3, were used to denaturize proteins, which was in the form of casein, placed in
separate test tubes. With heat and PbOAc2, turbid solutions were produced. With AgNO3 and HNO3,
white precipitates were formed right after the solutions were added. And with a cloudy solution was
produced when an ethanol was added to casein. These results indicate that protein was denaturized
by the different denaturing agents. Denaturation occurred because of the presence of hydrogen
bonding and nonpolar-nonpolar bonds which are directly affected upon the addition of the said
denaturing agents. Denaturation results disruption of the secondary, tertiary, and quaternary structural
interactions protein, which led to the disorganization of protein’s characteristic three-dimensional
shape and even loss the biochemical activity of the protein. Denaturation, however, does not affect
the primary structure of protein.
Conclusion
Protein(s) is the result of the combination and is purely made up of amino acids joined
covalently by peptide bonds, which are amide linkages between the -carboxyl group of one amino
acid and the -amino group of another.
Casein can be separated from the milk through heating in a quite high temperature and
addition of 10% acetic acid. These agents aid the protein to reach its isoelectric point, where protein
precipitates out and further becomes insoluble in the solution.
Biuret and Ninhydrin tests are the effective qualitative tests for amino acids and proteins leaving
specific color(s) of solution for each test, pink to violet for Biuret test and blue to violet for Ninhydrin test
respectively. Xanthoproteic and sulfur tests are useful to differentiate whether the amino acid or protein
contains side chain with aromatic ring or sulfur.
Denaturation of proteins occur when white precipitate, cloudy, or turbid solution is formed and
observable upon the addition of strong acids and bases, heavy metals, alcohols, and heat into the
protein solution.

Reference:
Ferrier, Denise R. Lippincott’s Illustrated Reviews Biochemistry. Sixth Edition.
Stoker, Stephen H. General, Organic & Biological Chemistry. Seventh Edition.

Answers to Questions.
1. Write the primary structures for the polypeptide of Ala-Gly-Cys-Phe-Gly.

2. Will the polypeptide in question 1 give a positive or negative result in the following tests for a
protein or amino acid? Explain why or why not.

a. Biuret Test
The above chain is an amino acids chain and since Biuret test is a test for the
presence of amino acids or two or more peptide bonds, thus, a positive result will be
produced.
b. Xanthroproteic Test

Xanthoproteic test is a test for the presence of aromatic ring(s). The amino acids
chain contains an aromatic ring, specifically attached in the phenylalanine amino acid,
therefore, a positive result will be expected.

c. Sulfur Test

Sulfur test is a test for the presence of sulfur in an amino acid or compound. The
above amino acids chain has cysteine, which has a sulfur atom, therefore, a positive
result will be expected.

d. Ninhydrin Test
In this test, the above molecule of amino acids chain will give positive result,
specifically a violet or blue solution since Ninhydrin test is a test for amino acid(s).

3. Why does a protein undergo denaturation?

Protein undergo denaturation because of the presence of H- bonds and
hydrophobic bods in the protein molecule which are greatly affected upon the addition
of denaturing agents.

4. Under what circumstances would the denaturation of protein be useful?

Appendix

Filtration of Casein Biuret Test Ninhydrin Test

Xanthoproteic Test Sulfur Test Denaturation Test