PBC 2101 Bio Chemistry

Lecturer : Miss Wong Yuen Nee

Experiment 1: Identification Test for Amino Acids

NAME : MOHAMMAD MARJAN B. MOHAMED AYYOOB

ID : I10006649

COURSE : DIPLOMA IN PHARMACY

DATE : 11 FEB 2011

Name : Mohammad Marjan

ID : I10006649

Partner : Lim Sok Bin

Introduction :

Proteins are biochemical compounds consisting of one or more polypeptides typically folded

into a globular or fibrous form in a biologically functional way. A polypeptide is a single
linear polymer chain of amino acids bonded together by peptide bonds between
the carboxyl and amino groups of adjacent amino acid residues. Amino acids are the building
blocks of protein. 20 amino acids are needed to build the various proteins used in growth,
repair and maintenance of body tissues. 11 of these amino acids can be made by the body
itself, while other 9 amino acids that are called essential amino acids come from the diet. The
essential amino acids are isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan and valine. Another amino acid, histidine, is considered semi-essential because
the body does not always require dietary sources of it. The nonessential amino acids are
arginine, alanine, asparagines, aspartic acid, cysteine, glutamine, glutamic acid, glycine,
proline, serine, and tyrosine. Other amino acids, such as carnitine, are used by the body in
ways other than protein-building and are often used therapeutically.

Protein from animal sources, such as meat and milk, is called complete, because it contains
all nine of the essential amino acids, most vegetable protein is considered incomplete because
it lacks 1 or more of the essential amino acids. This can be a concern for someone who
doesn’t eat meat or milk products. But people who eat a vegetarian diet can still get all their
essential amino acids by eating a wide variety of protein-rich vegetable foods.

Structure of albumin: Structure of tyrosine

Structure of phenylalanine structure of arginine

Structure of cystine structure of methionine

Structure of proline
Aim : To identify the presence of amino acids and to determine types of amino acids
presence.

Procedure :

A. Biuret Test
1. 3mL of water, albumin and 1% tyrosine solution were transferred into their
appropriate labelled test tubes.
2. Into each test tube, 3mL of NaOH and 3-4 drops of 1% Copper sulphate solution
were added.
3. The observation was recorded.

B. Millon’s Test
1. 1mL of water, albumin and tyrosine was filled in three test tubes that are labelled.
2. Then, 1mL of 10% mercuric sulphate was added and the test tube was boiled
gently for half a minute.
3. After cool down the test tubes, 3 drops of 1% NaNO2 solution was added and the
test tube were mixed well.
4. The observation was recorded.

C. Xanthoproteic Test
1. 3mL of water, albumin, phenylalanine and tyrosine were transferred into their
appropriate labelled test tubes.
2. Then, 1mL of concentrated HNO3 was added and the test tube was boiled gently
for half a minute.
3. After the tubes was cooled , the test tube was observed for the fo0rmtio of yellow
colour.
4. Then 2mL of concentrated ammonia was added and the test tubes was observed
for changes after the addition of alkali.
5. The result was recorded.

D. Sakaguchi’s Test
1. 5mL of water, albumin and arginine were transferred into their appropriate
labelled test tubes.
2. Then 5 drops of 5% NaOH and 4 drops of Molisch’s reagent was added into each
tube.
3. After that, 10 drops of bromine water solution is added after mix the contents
thoroughly.
4. The result was recorded.
 E. Sulphur Test
1. 3mL of water, albumin, methionine, cysteine/cystine were transferred into their
appropriate labelled test tubes.
2. Then 3mL of 40% NaOH was added and the solution was mixed thoroughly and
was boiled for 3 minutes.
3. 1mL of lead acetate solution was then added after the solution was cool down.
4. The result was record down.

F. Ninhydrin Test
1. 3mL of proline, 1% tyrosine and water were transferred into their appropriate
labelled test tubes.
2. Then the solution was heated to boil after adding 3 drops of ninhydrin reagent
(0.1% in acetone).
3. The result was recorded.

Results:

Test Sample Solution Observation

Albumin Purple
Biuret Test Amino acid:Tyrosine Light blue
Water Light blue
Albumin Brick red ppt
Millon’s Test Tyrosine Brick red suspension
Water No change
Albumin Deep yellow
Xanthoproteic Test Phenylalanine No change of colour
Tyrosine Light yellow ->Deep
yellow
Water No change
Albumin Light purple (polluted)
Sakaguchi’s Test Arginine Dark purple (polluted)
Water Dark purple (polluted)
Albumin Brown
Sulphur Test Cystine / Cysteine Light grey
Methionine light grey
Water Light grey
\ Amino acid:Tyrosine Violet colour
Ninhydrin Test Proline Light yellow
Water Colourless
Discussion

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 a violet-colored complex in an alkaline solution.
For the Biuret Test, the albumin solution and tyrosine solution turn to purple colour while
there is no change in water. Purple colour in albumin and tyrosine indicates that the test is
positive. At least two peptide bonds (a tripeptide) must be present for a positive test.

Millon's reagent is an analytical reagent used to detect the presence of soluble proteins. For

Millon’s test, the albumin and tyrosine each form brick red precipitate and brick red
suspension while there is no change in water. The phenolic group of tyrosine reacts with the
Millon’s reagent to give a brick-red solution. The appearance of red colour is a positive test
for tyrosine.

 Xanthoproteic test is a method that can be used to determine the amount of protein soluble in
a solution, using concentrated nitric acid. The test gives a positive result in those proteins
with amino acids carrying aromatic groups, especially in the presence of tyrosine. If the test
is positive the proof is neutralized with an alkali, turning to dark yellow. In this case, albumin
and tyrosine show positive result while phenylalanine and water show negative result.

The Sakaguchi test is a specific qualitative test for the detection of a specific type of protein
with the amino acid containing the guanidinium group. In basic conditions, alpha naphthol
and sodium hypobromite/chlorite react with the aforementioned compound to form red-
orange complexes. For the test that my group conduct, the albumin, arginine and water give light
purple and dark purple colour respectively as the solution is contaminated.

For the sulphur test, albumin and cystine/cysteine give brown and light grey colour
respectively which is positive test while methionine and water is colourless which means its
negative test. The test is positive because when cystine and cysteine are boiled with strong
alkali, organic sulphur is converted to sulphide. This sodium sulphide reacts with lead acetate
to form a black grey precipitate and amino acids in cystine and cysteine contain sulphur.

Ninhydrin is a chemical used to detect ammonia or primary and secondary amines. When

reacting with these free amines, a deep blue or purple colour known as Ruhemann's purple is
produced. Proline and hydroxyproline will give yellow colour due to the NH2 group. In this
case, tyrosine and proline both give violet colour and light yellow respectively which is a
positive test while water is colourless which means its a negative test
Conclusion.

Albumin, tyrosine, cysteine/cystine and proline give positive result for the test that have been
conducted.

Reference

http://en.wikipedia.org/wiki/Biuret_test

http://chemistry.olivet.edu/classes/chem100/pdf/Labs/Simple%20Color%20Tests%20for
%20Amino%20Acids%20and%20Proteins%20Lab.pdf

http://wiki.answers.com/Q/What_is_indication_of_violet_color_in_biuret_test_of_enzymes

http://answers.yahoo.com/question/index?qid=20090507142525AADR3XD