DEPARTMENT OF PURE AND APPLIED CHEMISTRY

Visayas State University

CHEM 31a Biochemistry
Laboratory Report

Name: Ellen J. Cerna Date Performed: May 20, 2015

Lab. Schedule: M –F 7:00-10:00 Date Submitted: May 25, 2015

Group No: 4 Score:

Experiment No. 6

Proteins
I. 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 proteins.
 Observe the denaturation of proteins.
II. Data and Results
Separation of a Protein (Casein) from Milk
A1 Mass of flask 113.5 g
Mass of flask and milk 146.4 g
Mass of milk 32.9 g
A2 Mass of Casein product 3.5 g
A3 % Casein in Milk 7%

Tests for Amino Acids and Proteins

Biuret Test
Compound Colour Conclusions
Glycine Clear Amino acid
Alanine Clear Amino acid
Aspartame Yellow Dipeptide
Gelatin Pink Protein
Albumin Violet Protein
Casein Violet Protein

Ninhydrin Test
Compound Colour Conclusions
Glycine Violet Amino acid
 Alanine Violet Amino acid
Aspartame Light yellow Hydroxypyroline
Gelatin Violet Protein
Albumin Violet Protein
Casein Violet Protein
Tyrosine Violet Amino acid

Xanthroproteic Test
Compound Colour Conclusions
Tryphtophan Yellow With aromatic side chain
Tyrosine Yellow-orange With aromatic side chain
Aspartame Colourless With aromatic side chain
Gelatin Yellow With aromatic side chain
Albumin Yellow With aromatic side chain
Casein Yellow With aromatic side chain

Sulfur Test
Compound Colour Conclusions
Alanine Milky white No sulphur in branched-
chain
Gelatine Brown No sulphur in branched-
chain
Cysteine Yellowish No sulphur in branched-
chain
Albumin Brown Sulphur in branched-
chain
Casein Brown Sulphur in branched-
chain

Denaturation of Proteins
Denaturing Agent Observations
Heat Slight precipitation
Heavy Metals
AgNO3 Appreciable amount of white precipitate.
PbAc2 Slight precipitate.
Strong Acid Yellow precipitate
Alcohol Evolution of gas and white precipitate.
III. Discussion
Proteins are large molecules composed of one or more chains of amino acids in a
specific order determined by the base sequence of nucleotides in the DNA coding for the
protein. They are required for the structure, function, and regulation of the body's cells,
tissues, and organs. Each protein has unique functions in which they are essential
components of muscles, skin, bones and the body as a whole.

Separation of a Protein from Milk

Milk is the probably the most nutritionally complete food found in nature. Milk
contains three kinds of proteins: caseins, lactalbumins, and lactoglobulins, all of which are
globular proteins. The main protein in milk is casein. Casein is a phosphoprotein which has
phosphate groups attached to the hydroxyl groups of some of the amino acids side-chains.
Casein exists in milk as a calcium salt, calcium caseinate. It is actually a mixture of three
similar proteins, alpha, beta and kappa caseins which form a micelle. Alpha- and beta-casein
are both insoluble in water and are solubilised by the micelle surrounding them. The kappa-
casein which has a hydrophilic portion is responsible for solubilizing the other two caseins by
promoting the formation of and stabilizing the micelles.

Calcium caseinate has an isoelectric point of pH 4.6. This means it is insoluble in

solutions with a pH less than 4.6. The pH of milk is 6.6, therefore, casein has a negative charge
at this pH and is solubilized as a salt. If an acid such as acetic acid is added to milk, the
negative charges on the outer surface of the casein micelles are neutralized, by protonation of
the phosphate groups. The casein micelles are destabilized or aggregate because the electric
charge is decreased to that of the isoelectric point (pH at which there is no net charge because
there are equal numbers of positive and negative charges present). The casein micelles
disintergrate and the casein (the neutral protein) precipitate because it is no longer polar,
with the calcium ions remaining in solution.

Biuret Test
The biuret test is a chemical assay that detects the presence of proteins and amino
acids in a sample. The test relies on a colour change to confirm the presence of amino acids
and proteins. The biuret test uses an alkaline mixture, or reagent, composed of potassium
hydroxide and copper sulfate. The normal colour of biuret reagent is blue. The reagent turns
violet in the presence of peptide bonds - the chemical bonds that hold amino acids together.
The proteins detected must have at least three amino acids, which mean that the protein must
have at least two peptide bonds. The reagent’s copper ions, with a charge of +2, are reduced to
a charge of +1 in the presence of peptide bonds, causing the colour change. In the experiment,
both alanine and glycine did not give a positive test since they are simple amino acids.
However, compounds such as gelatin, egg albumin and the casein gives a positive result for
this test since they are all proteins which is made up of long polypeptide as been observed by
their colour change from blue to violet.
Ninhydrin Test
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect
ammonia or primary and secondary amines. In the pH range of 4-8, all α- amino acids react
with ninhydrin (triketohydrindene hydrate), a powerful oxidizing agent to give a purple
coloured product (diketohydrin) termed Rhuemann’s purple. All primary amines and
ammonia react similarly but without the liberation of carbon dioxide. The imino acids proline
and hydroxyproline also react with ninhydrin, but they give a yellow coloured complex
instead of a purple one. Beside amino acids, other complex structures such as peptides,
peptones and proteins such as gelatine, egg albumin and casein also react positively when
subjected to the ninhydrin reaction. Shown below is a general reaction for the qualitative test:

Xanthroproteic Test
Xanthoproteic test is used for the detection of proteins in which concentrated
nitric acid reacts with the proteins to form a yellow colour that is intensified to orange-yellow
by the addition of an alkali. Aromatic amino acids, such as Phenyl alanine, tyrosine and
tryptophan, respond to this test as been observed in the experiment. In the presence of
concentrated nitric acid, the aromatic phenyl ring is nitrated to give yellow coloured nitro-
derivatives. At alkaline pH, the colour changes to orange due to the ionization of the phenolic
group.

In the experiment, all the samples except for aspartame give a positive result since
they either both contain tryptophan and tyrosine. Aspartame contains a phenyl group
however the phenyl group has been deactivated towards nitration.
Sulfur Test

Sulfur-containing amino acids include cysteine and methionine. If a sample that

contains one or both of these amino acids is acidified, the gas H2S is produced, which smells
like rotten eggs. When a piece of moistened lead (II) acetate paper is held over the solution as
the H2S is being produced, the H2S reacts with the lead ion, forming a black or gray coating of
PbS (lead (II) sulfide). Appearance of this black colour is taken as a positive test for a sulfur-
containing amino acid. In the experiment, alanine did not give a positive result since it only
has a methyl in its R group while gelatine doesn’t contain the amino groups such as cysteine
and methionine. While egg albumin and casein gives a positive result since both of them
contains the methionine and cysteine amino acids.

Denaturation of Proteins
Proteins are held in their native conformations by a combination of forces:
hydrogen bonds, ionic interactions, disulfide bridges, and hydrophobic interactions.Changing
the conformation of a protein either temporarily or permanently by disrupting these forces is
called denaturation. Denaturation results in a loss of protein activity. It involves the
disruption and possible destruction of both the secondary and tertiary structures. Since
denaturation reactions are not strong enough to break the peptide bonds, the primary
structure (sequence of amino acids) remains the same after a denaturation process.
Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a
random shape. Since the native conformation is usually the most water soluble, disrupting the
secondary and tertiary structures causes changes in solubility and frequently results in the
formation of a solid in the solution. Reagents or conditions that can cause denaturation are
called denaturing agents; these include heat, pH changes, alcohol, and heavy metal salts. In the
experiment, the effect of several denaturing agents on the protein albumin which is a simple
globular protein was observed.

Heat can supply kinetic energy to protein molecules, causing their atoms to vibrate
more rapidly. This will disrupt relatively weak forces such as hydrogen bonds and
hydrophobic interactions. Heat is also used in sterilization to denature and hence destroy the
enzymes in bacteria. A slight precipitation was observed in the experiment since the egg
albumin was broken down by the heat.

Extremes of pH can cause a protein to denature. Addition of strong acids such as

nitric acid can cause extreme pH. The R-groups in the amino acid chain are often charged and
can form ionic bonds with a group of opposite charge. Extremes of pH can change the charges
on these positive and negative groups, disrupting ionic bonds interactions. This can be
observed by an appreciable white precipitate as seen in the experiment.

Some reagents, such as alcohol, are capable of forming hydrogen bonds with protein
molecules which will disrupt the hydrogen bonding present within the molecule. This make
the egg albumin denatured giving a white precipitation as observed in the experiment.
 Salts of metal ions such as mercury (II), lead (II), and silver can form strong bonds
with disulfide groups of the protein. Thus, they disrupt both disulfide bridges and salt
linkages and cause the protein to precipitate out of solution as an insoluble metal-protein salt.
This property makes some of the heavy metal salts suitable for use as topical antiseptics.
However, most heavy metal salts are toxic when taken internally, because they precipitate the
proteins of all the cells with which they come into contact. Substances high in protein, such as
egg whites and milk, are used as antidotes for heavy metal poisoning, because their proteins
readily combine with the metal ions to form insoluble solids. The resulting insoluble matter
must immediately be removed from the stomach by the use of an emetic to prevent the gastric
juices from destroying the protein and once again liberating the poisonous heavy metal ions.

IV. Conclusion
Therefore, protein plays a very vital role in our body by having different function in
depending upon the direction of each. Protein, which are basically a polymers of amino acids
give us an insight on how the different R groups in each amino acids dictate the protein
structure into primary, secondary, tertiary and quaternary structure of proteins. This
experiment also provided a better understanding on the different qualitative test conducted
in which either or both proteins and amino acids can be tested for its presence in a given
sample. Moreover, the experiment gives a closer look to what is the effect of the different
denaturing agents to a protein.

V. Questions and Exercises

1. Write the primary structure 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.
3. Why does a protein undergo denaturation?

When a protein is denatured, it basically loses the structure it has while in its
biologically active form. This is brought about by external factors that disrupt the
interactions that are happening between proteins at the molecular level. The denaturation
is just more or less a reaction to any external stress placed on the molecule, such as heating
or the addition of a strong acid or a base.

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

As has already been mentioned before, proteins are the essential building blocks of all
living organisms, and so bacteria are not exempt from this criterion. The denaturation of
proteins in organisms like bacteria essentially kills them off and could help as a remedy to a
form of bacterial problems and the like. This is in relation to the reason why food is cooked.

VI. References
 http://www.medicinenet.com/script/main/art.asp?articlekey=15380
 http://education.seattlepi.com/biuret-test-mean-biology-4659.html
 http://www.ukessays.com/essays/biology/quantitative-tests-for-aminoacids-
and-proteins-biology-essay.php
 http://www.ukessays.com/essays/biology/quantitative-tests-for-aminoacids-
and-proteins-biology-essay.php
 http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
 http://www.mrgscience.com/uploads/2/0/7/9/20796234/protein_denaturati
on_lab.pdf
 http://users.uma.maine.edu/SBaker/bio%20dentaure.htm