Introduction

The food we consume is divided into three main

classes:
1. Carbohydrates: the bodys most readily available
energy source.
2. Lipids: the bodys principal energy reserve.
3. Proteins: the bodys source of energy for growth
and cellular maintenance.
Proteins also make up the second largest portion
of cells, after water.
Proteins consists of amino acid linked together by
peptide bond.

 Amino acids incorporated into proteins are covalently

linked by peptide bonds.
Peptide bonds are amide bonds formed between the
carboxylic acid group of one amino acid and the amino
group of a second amino acid.

 Note: each amino acids consists of:

Carboxylic acid groups (-COOH).
Amino groups (-NH2).
Substituent, or replaceable.
Side chains (-R).

Different side
chain result in
various amino
acid .

 The carbon on all amino acids, except glycine, is a

chiral carbon because it has four different groups
bonded to it.
Glycine: is simple amino acid because R chain is H.

 Twenty different amino acids, which differ only

in the structure of their side chains, are used by
human cells to build proteins.
The side chain structure determines the class of
the amino acid: non-polar, neutral, basic, or
acidic.
Human cells can synthesize most the amino
acids needed to build proteins. However, about
10 amino acids, called essential amino acids
cannot be synthesized by human cells and must
be obtained from food.

Essential Amino Acids in Humans

1.
2.
3.
4.
5.

Arginine
Histidine
Isoleucine
Leucine
Valine

6. Lysine
7. Methionine
8. Threonine
9. Phenylalanine
10. Tryptophan

Non-Essential Amino Acids in Humans

1. Alanine
2. Asparagine
3. Aspartate
4. Glutamate
5. Glutamine
6. Glycine
7. Proline
8. Serine
9. Cysteine
10. Tyrosine

Chemical Reactions of Amino Acids and Protein

Functional Groups
Certain functional groups in amino acids and
proteins can react to produce characteristically
colored products.
The color intensity of the product formed by a
particular group varies among proteins in
proportion to the number of reacting functional, or
free, groups present.
In this part of experiment, various color-producing
reagents (dyes) will be used to qualitatively detect
the presence of certain functional groups in amino
acids and proteins.

Ex (1). Millon Reaction

Principle: Millon's reagent (Hg/HNO3) gives positive results
( pink to dark-red color) with proteins containing the phenolic
amino acid tyrosine
Purpose: To detect the amino acid that have phenol group of
tyrosin.
Note: some proteins containing tyrosine will initially form a
white precipitate that turns red when heated, while others form
a red solution immediately. Note that any compound with a
phenol group will yield a positive test, so one should be certain
that the sample being tested does not contain any phenols other
than those present in tyrosine.

:Procedure
1.
2.
3.

In test tube add 2 ml of protein into separate labeled test

tubes.
Add 3-4 drops of Millons reagent, and immerse the
tubes in a boiling water bath for 5 minutes.
Cool the tubes and record the colors formed.

Result:
A white ppt is formed with
albumin and casein (but not
gelatin) Why???
The ppt gradually turns
into red.

Ex (2). Xanthoprotic Reaction

Some amino acids contain aromatic groups that are
derivatives of benzene.
These aromatic groups can undergo reactions that are
characteristic of benzene and its derivatives.
One such reaction is the nitration of a benzene ring with nitric
acid.
The amino acids tyrosine and tryptophan contain activated
benzene rings and readily undergo nitration.
The amino acid phenylalanine also contains a benzene ring,
but the ring is not activated and therefore does not undergo
readily nitration.

 Principle: Nitric acid gives color when heated

with proteins containing tyrosine (yellow color)
or tryptophan (orange color); the color is due to
nitration.
Purpose: used to identify the presence of an
activated benzene ring.
Note: If one spills a concentrated solution of nitric
acid onto someones skin. The proteins in skin
contain tyrosine and tryptophan, which become
nitrated and turn yellow.

Procedure
1.

Add 2 ml of protein solution in a

test tube and add 2 drops of
concentrated nitric acid.

2.

The formed white precipitate, will

turn yellow upon heating, and
finally will dissolve giving a
yellow color to the solution.

3.

Cool the solution down. Carefully

add 3 ml of 6 N NaOH. Note that
the yellow color turns orange.

Ex (3). Glyoxylic Acid Reaction

: (Hopkins-Col test)
Principle: The indole ring reacts with glyoxylic
acid in the presence of a strong acid to form a
violet cyclic product.
Purpose: The Hopkins-Cole test is specific for
tryptophan, the only amino acid containing indole
group. .

Procedure

Add 1 ml of protein solution in a test tube, add 1 ml

of Hopkins-Col reagent and mix well.
Incline the test tube and slowly add 1 ml of
concentrated H2SO4 on the inner wall of the test tube
thus forming a reddish - violet ring at the interface of
the two layers.

:Result
A reddish violet ring is
formed at the junction
between the 2 layers with
albumin and casein.

Gelatin gives negative

results.

Ex (4). Reduced sulfur test (Test for (-SH) group)

Principle: Proteins containing sulfur (like:
Methionine and cystine) give a black deposit of
lead sulfide (PbS) when heated with lead acetate
in alkaline medium.
Purpose: To detect amino acid which containe
sulfer group.

Sulfur-containing protein ----> NaOH----> S2- ----Pb2+----> PbS

:Procedure
1.

2.
3.

Add 1 ml of protein solution in a test tube, add 2 drops

of 10% sodium hydroxide solution and 2 drops (or few)
of lead acetate.
Stopper the tubes and shake them. Remove the stoppers
and heat in a boiling water bath for 5 minutes.
Cool and record the results.

:Result
A black deposit is formed with albumin
while a slight black turbidity is obtained with
casein due to its lower content of sulfur.
Gelatin gives negative result.

:Ex (5). Sakaguchi Test for Arginine

Principle: - naphthol and sodium
hypobromite/chlorite react with the above mentioned
compound to form red orange complexes.
Purpose: used for the detection of a specific type of
protein with the amino acid containing the
guanidinium group (e.g. arginine).

Procedure
1. To 1 ml of the protein solution add 1 ml of 3 N
NaOH solution and 0.5 ml of 0.1 % - naphthol
solution, and a few drops of 2 % hypobromite
solution.
2. The presence of a guanidinium group in the
compound under examination will be confirmed
by the formation of a red color.

Ninhydrin Test
Ninhydrin is a chemical used to detect free amino acid
and proteins
Amino acids(NH2) also react with ninhydrin at pH=4.
The reduction product obtained from ninhydrin then
reacts with NH3 and excess ninhydrin to yield a blue
colored substance.
This reaction provides an extremely sensitive test for
amino acids.

Procedure
To 1 mL solution add 5 drops of 0.5% ninhydrine
solution
Boil over a water bath for 2 min.
Allow to cool and observe the blue color formed.
Result: all amino acid will give purple or deep blue
with exception Proline gives yellow not violet.