Introduction

Proteins are organic compound that are arranged in a linear chain. The word
‘protein’ refers to a type of molecule in food that can be broken down into amino
acids.

The body needs twenty amino acids to function as a biological machine and it can
synthesize eleven of these by itself. However there are nine, called ‘essential amino
acids’ that the body cannot create and has to gain through the consumption of
food. When we eat, the body breaks down the protein in food in order to create the
amino acids that it needs. Proteins are large biomolecules, or macromolecules,
consisting of one or more long chains of amino acid residues. Proteins perform a
vast array of functions within living organisms, including catalyzing metabolic
reactions, DNA replication, responding to stimuli, and transporting molecules from
one location to another. Proteins differ from one another primarily in their sequence
of amino acids, which is dictated by the nucleotide sequence of their genes, and
which usually results in protein folding into a specific three-dimensional structure
that determines its activity. (Wikipedia)

Amino acids are biologically important organic compounds containing amine (-NH2)
and carboxylic acid (-COOH) functional groups, usually along with a side-chain
specific to each amino acid. The properties of the side chain are what determines
the classification of the amino acid into weak acid, weak base, hydrophilic and
hydrophobic. (Wikipedia)

The main objective of this experiment is to study the general reactions of proteins
and to study the R groups of the proteins. These objectives are achieved by carrying
out several tests such as Biuret test, Ninhydrin test, Xanthoproteic Test, Millon’s test
and Sakaguchi’s test. (Biochem practical Manual)
Results

Biuret Test

No Sample Observation Interpretation

1 Bovine serum The solution turns from blue Protein is
albumin to dark purple/violet present. It
confirms the
presence of
peptide bonds in
the protein
2 Casein The solution turns from blue Protein is
to light purple/violet present. It
confirms the
presence of
peptide bonds in
the protein
3 Glycine No changes. The solution Protein is absent
remains in blue colour . Glycine is an
amino acid. It
confirms the
absence of
peptide bonds
4 Urea No changes. The solution Protein is
remains in blue colour. absent. Urea is
result of the
breakdown of
amino acid.
Hence the
peptide bonds
are absent
5 H20(water) No changes. The solution Protein is
remains in blue colour. absent. Water
acts as a
negative control
of the
experiment.
Peptide bonds
are absent.

Ninhydrin Test
No Sample Observation Interpretation
1 Glycine A violet blue solution is Free amino acid
formed group is present
in glycine.
2 Proline Dark yellow colour solution Proline is an
with a white precipitate is imino acid. It is a
formed. secondary amino
acid and it has a
ring structure.
Hence the free
amino acid group
is absent.
3 Bovine serum A clear solution is formed
albumin

Xanthoproteic Test

No Sample Observation Interpretation

1 Glycine Colourless solution is formed The benzene ring
is absent. Since
glycine is
aliphatic amino
acid it do not
possess a
benzene ring.
2 Phenylalanine Pale yellow solution is formed Phenylalanine is
an aromatic
amino acid. It
has a benzene
structure .
3 Bovine serum Orange red solution is formed This confirms
albumin thatbovine
serum albumin is
an amino acid
containing a
benzene ring

Millon’s test
No Sample Observation Interpretation
1 Glycine No changes are observed. Protein is absent
Clear solution is formed since glycine is
an amino acid
2 Bovine serum Dark reddish pink precipitate Protein is
albumin is formed present
3 Casein Light reddish pink precipitate Protein is
is formed present

Sakaguchi’s test

No Sample Observation Interpretation

1 Glycine Light yellow Arginine(a type
solution is of amino acid) is
formed absent
2 Arginine Light reddish Arginine is
pink solution is present
formed
3 Urea Light yellow Arginine is
solution is absent
formed

Discussion

Proteins are organic compounds made of amino acids.The amino acids which is the
building block of proteins joined together by the peptide bonds. In a normal functional
protein, the long chain of amino acids is folded into a 3- dimensional shape. Every
protein has a unique 3-dimensional shape that is suited to its biological function in a
living organism. The unfolding of the 3D shape will denature the protein where the
protein will no longer be able to carry out its biological function and will usually be
destroyed by the cell.

There are several quantitative tests for determining whether amino acids or proteins
are present in solution. These tests are specific for the presence of peptide
bonds,certain types of side chains and the type of secondary structure present.
The biuret test is a chemical test used for detecting the presence of peptide bonds.
In the presence of peptides, a copper(II) ion forms violet-colored coordination
complexes in an alkaline solution. The Biuret reagent contains copper ions which
give it a blue color. The copper ions will interact with a compound that contains two
or more peptide bonds, resulting in the formation of a violet/purple-colored product.
The dipeptide bonds in proteins react with the Cu2+ ions to form the complex. The
lone pairs present on the Nitrogen of the peptide linkage bonds with the cupric ions
in the reagent to create a violet purple colour change. When a compound does not
have at least two peptide bonds, it will not react with the Biuret reagent, and no
purple color will appear . The solution will remain a shade of blue due to the copper
ions. Biuret test is specific for testing Proteins, It helps to differentiate between
Proteins (+ve) and Amino Acids (-ve).

In the experiment above, the BSA forms the highest intensity of the purple complex
compared to caseine because of the number of peptide bonds in BSA is higher than
in caseine. The number of peptide bonds present in the protein is proportional to the
intensity of the violet coloured complex formed. Thus, the biuret reaction is the
basis for a simple and rapid colorimetric reagent of the same name for
quantitatively determining total protein concentration The violet coloured complex
formed confirms the presence of proteins, so in BSA and caseine proteins are
present. While glycine shows negative result in the Biuret test because glycine is
the simplest amino acid. A peptide bond (amide bond) is a covalent chemical bond
formed between two amino acid molecules. Since there are no peptide bonds
present in the glycine alone, it shows a negative result in the Biuret test. Urea which
is the breakdown of amino acids shows a negative result in Biuret test since the
peptide bonds are absent .But when the urea is heated it may give a positive result
because the heated urea forms the biuret compound that reacts with the cupric ions
present in the reagent to form the coloured complex.

Although amino acids are the building block of amino acids, but it give negative
results in Biuret test because atleast TWO or more peptide bonds are required for
the formation of the chelate complex. In single amino acids there are no peptide
bonds present While in dipeptides only ONE peptide bond present hence this gives
a negative result.

The biuret test can be extended to quantitatively measure the concentration of total
protein using absorption spectroscopy method. Using a spectrophotometer which
follows the Beer-Lambert law is the instrument which can be used in this method.
Beer Lambert law states that the absorption of the sample is directly proportional to
the concentration of the species. Hence in this case the absorption of the sample is
directly proportional to the number of the peptide bonds.
Ninhydrine test utilized to identify amines in particular alpha amino acids present in
the solution. It is commonly used to detect the lysine in fingerprints. Ninhydrin (2,2-
Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and
secondary amines. When reacting with these free amines, a deep blue or purple
color known as Ruhemann's purple is produced. The ninhydrin reagent will react
specifically with a primary amino functional group on a compound, resulting in the
formation of a violet/purple-colored product. When a compound doesnot have a
primary amino group, it will not react with the ninhydrin reagent, and no purple
color will appear (solution will remain colorless)

In the experiment above , BSA shows positive result by forming violet blue colour
solution. The alpha amino group of the free amino acid react with ninhydrin to form
a blue to purple colour change. However, some amino acids such as the imminoacid
proline react with ninhydrin differently to form a bright- yellow colour change. This
is because proline has a ring structure as shown in the diagram below.

The blueish-purple result is usually associated with primary amino acids. In these
amino acids, the N is free to react with ninhydrin. However, in proline, the N is not
available for reaction as it is locked in the ring structure. Therefore no ammonia is
produced, so no blue color is presented.

The objective of xanthoproteic test is to differentiate between aromatic amino acids

which give positive results and other amino acids which do not possess the benzene
ring.Amino acids containing an aromatic nucleus form yellow nitro derivatives on
heating with concentrated HNO3. The salts of these derivatives are orange in color.

Concentrated nitric acid reacts with the aromatic rings that are derivatives of
benzene giving the characteristic nitration reaction. BSA contain activated benzene
rings which are easily nitrated to orange colored compounds upon heating . The
aromatic ring of phenylalanine dose not react with nitric acid despite it contains a
benzene ring, but it is not activated, therefore it will not react.

Millon’s test is specific for tyrosine, the only amino acid containing a phenol group, a
hydroxyl group attached to benzene ring.The main objective of this test is to detect
the presence of tyrosine in the sample. In Millon's test, the phenol group of tyrosine
is first nitrated by nitric acid in the test solution.Then the nitrated tyrosine
complexes mercury ions in the solution to form a brick-red solution or precipitate of
nitrated tyrosine, in all cases, appearance of red color is positive test which
indicates the presence of proteins. Millon’s test is given by any compound
containing a phenolic hydroxy group. Consequently, any protein containing tyrosine
will give a positive test of a pink to dark-red colour.However ,Millon's test is not
specific for proteins (it detects phenolic compounds), and so must be confirmed by
other tests for proteins such as the biuret test and the ninhydrin reaction.

In the experiment above, glycine shows negative results since it is an amino acid.
And it does not possess phenolic hydroxyl molecule attached to it. While BSA and
caseine give positive result which is confirmed by the formation of dark red
precipitate. This indicates that protein is present.

Sakaguchi test is a specific qualitative test for the detection of amino acid
containing gauanidium group [R-NH-C= (NH2)2+ -NH2]. In other words it’s a test for
guanidines, for an example arginine. In alkaline solution, arginine react with α-
naphthol and sodium hypobromite /chlorite as an oxidizing agent, to form red
complexes as a positive result.In the experiment above only in arginine thelight
reddish pink solution is formed which indicates the positive result.

Conclusion

In conclusion we can state that there are several experimental analysis that can be
done to confirm the presence of protein and also to study the side chain, R group
and structure of the protein. Based on all the reactions above, different protein and
amino acids shows different characteristic difference. Hence different tests must be
done to confirm the specificity of the proteins. By the end of this experiment the
objectives of this experiment were met.