• General information about amino acids.
• Qualitative tests of amino acids.
 Food are divided into three classes :
1- Carbohydrate
Source of energy.
2- Lipid
Principal of energy reserve.
3- Proteins
Energy for growth and cellular maintance.
(Building blocks of proteins which linked to peptide bond )
 Each amino acid consists of :
1. Central carbon atoms
2. An amino acid
3. Carboxyl group
4. Side chain (All amino acids found in proteins have this
basic structure, differing only in the structure of the Rgroup or the side chain.)
• Essential amino acids: Humans incapable of
forming requisite and must be Required in diet.
• Non essential amino acids: Not required in diet.
• The simplest, and smallest, amino acid found in
proteins is glycine for which the R-group is a
hydrogen (H).
• Proline It is unique among the 20 protein-forming
amino acids in that the amine nitrogen is bound to
not one but two alkyl groups, thus making it a
secondary amine.
1. Non-polar (Hydrophobic amino acid) : are amino
acid that contain C,H in their side chain (hate
water, normally buried inside the protein core)
2. Uncharged polar.
3. polar amino acids with a charge: amino acid that
contain in their side chain O,N and they can
dissolve in water ( like dissolve like ) hydrophilic
(love water),tend to found on surface
A. Basic polar (positively charged).
B. Acidic polar (negatively charged).
At acidic pH, the carboxyl
group is protonated and
the amino acid is in the
cationic form
At
neutral pH, the
carboxyl
group
is
deprotonated but the
amino
group
is
protonated. The net
charge is zero; such ions
are called Zwitterions
At alkaline pH, the amino
group is neutral –NH2
and the amino acid is in
the anionic form.
 Iso electric point (PI) : It is the pH
value at which concentration of anionic
and cationic groups are equal (i.e. the net
charge of this molecule equals zero).
 Each amino acid have a different PI
 There are number of tests to detect the
presence of amino acid
 This is largely depend on the natural of side
chain
1.
2.
3.
4.
Ninhydrin test: for α-L amino acids
biuret test
Xanthoproteic test: for Aromatic amino acids
Lead sulfite test: detection of amino acids
containing sulfhydral group (- SH)
5. Millon's test: for amino acids containing hydroxy
phenyl group
6. Sakaguchi Test.
7. Hopkins-Cole (Glyoxylic Acid Reaction)
Principle:
• Ninhydrin degrades amino acids into aldehydes (on pH range
4), ammonia and CO2 through a series of reactions.
• The reducont product obtained from ninhydrin
(hydrindantin) then reacts with NH3 and excess
ninhydrin to produce an intensely blue or purple pigment,
sometimes called ruhemann's purple.
• This reaction provides an extremely sensitive test for amino
acids.
• alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final
complex(BlUE) + 3H2O
Note
• The amino acid proline and hydroxyproline also react with ninhydrin, but
they give a yellow colored complex instead of a purple one.
• Besides amino acids, other complex structures such as peptides, peptones
and proteins also react positively when subjected to the ninhydrin
reaction.
Ninhydrin_ethanol reagent is flammable. Toxic, and irritant. Keep away
from Bunsen burner flames prevent eye, skin, clothing contact. Avoid
inhaling the vapors or ingesting the reagent.
With all amino acid will give purple or deep blue with
exception Proline gives yellow not violet (why)?
The secondary amino group (imino group) of proline
residues is held in rigid conformation that reduces the
structural flixibility of polypeptide regions containing
proline. Proline does not give ninhydrin reaction as this
reagent requires free alpha amino group but proline have
imino group
 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.
 Results:
 Biuret structure:
it is result of condensation of two molecule of urea
 Principle:
• The biuret reagent (copper sulfate in a strong base)
reacts with peptide bonds in proteins to form a blue
to violet complex known as the “biuret complex”.
• This
color
change
is
dependent
on
the number of peptide bonds in the solution, so the
more protein, the more intense the change.
• The NaOH is there to raise the pH of the solution
to alkaline levels; the crucial component is
the copper II ion (Cu2+) from the CuSO4.
• When
peptide
bonds
are
present
in
this alkaline solution, the Cu2+ions will form a
coordination complex with 4 nitrogen atoms from
peptide bonds.
• N.B. Two peptide bonds at least are required for the
formation of this complex.
A chelate is a chemical compound composed of a metal ion and a
chelating agent. A chelating agent is a substance whose
molecules can form several bonds to a single metal ion. In other
words, a chelating agent is a multidentate ligand.
Procedure
• To 2 ml of protein solution in a test tube, add
4ml of reagent incubation 30 min
• Result :
Observations
No change ( solution
remains blue )
The solution turns from
blue to violet( purple)
Interpretation
Proteins are not present
Proteins are present
The solution turns from Peptides are present (
blue to pink Peptides or peptones are
short chains of amino acid
residues)
Objective:
• to differentiate between aromatic amino acids which give positive
results Yellow color and other amino acids.
Principle:
• Concentrated nitric acid react with aromatic nucleus present in the amino
acid side chain [nitration reaction] giving the solution yellow color.
Note:
• Amino acids tyrosine and tryptophan contain activated benzene rings
[aromatic nucleus] which are easily nitrated to yellow colored
compounds.
• The aromatic ring of phenyl alanine dose not react readily with nitric
acid despite it contains a benzene ring, but it is not activated, therefore it
will not react
 Procedure
• To 2 mL amino acid solution in
a boiling test tube, add equal
volume of concentrated HNO3.
• Heat over a flame for 2 min and
observe the color.
• Now COOL THOROUGHLY
and CAUTIOSLY run in
sufficient 3ml NaOH (why)
• Observe the color of the nitro
derivativative
of
aromatic
nucleus.
objective:
Specific for tryptophan (the only amino acid containing indole
group)
Principle:
• Reacting with a glyoxylic acid in the presence of a strong
acid, the indole ring forms a violet cyclic product.
• The protein solution is hydrolyzed by conc. H2SO4 at the
solution interface.
• Once the tryptophan is free, it reacts with glyoxylic acid to
form violet product.
Procedure
1. In a test tube, add to 2 ml of the
solution an equal volume of
Hopkins- Cole reagent and mix
thoroughly.
• Incline the tube and let 5 to 6 ml
of conc. H2S04 acid flow slowly
down the side of the test tube, thus
forming a reddish - violet ring at
the interface of the two layers.
That indicates the presence of
tryptophan
Objective:
• This test is specific for tyrosine. Because it is the only amino
acid containing a phenol group.
• Note: phenol group, a hydroxyl group attached to benzene
ring.
Millon’s reagent contains mercury and
HNO3 and is very toxic, corrosive a
strong oxidant, an irritant, and can
cause burns
Principle:
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 , appearance of red color is
positive test.
Note:
all phenols (compound having benzene ring and
OH attached to it) give positive results in Millon’s
test.
Procedure
• To 2 ml of protein solution in a
test tube, add 3 drops of Millon’s
reagent.
• Mix well and heat directly on a
small flame. BWB 5 min
• A white ppt is formed with
albumin and casein (but not
gelatin);
• the ppt gradually turns into
brick red.
Objective:
detection of amino acid containing gauanidium group. In other
words it’s a test for, arginine.
Principle :
In alkaline solution, arginine react with α-naphthol and sodium
hypobromite /chlorite as an oxidize agent, to form red
complexes as a positive result.
Procedure
1. Add 1 ml of 3 N NaOH
solution to 1 ml of the
protein solution, followed by
addition of 0.5 ml of 0.1 %
α- naphthol solution, and a
few drops of 2 % sodium
hypobromite
solution
(NaOBr).
2. The formation of a red color
indicates the presence of a
guanidinium group in the
compound
under
examination.
Objective:
This test specific for–SH [sulfhydral group ] containing amino
acid (Cysteine).
 Principle:
- Sulfur in cystine, is converted to sodium sulfide by boiling with
10% NaOH.
- The Na2S can be detected by the precipitation of PbS (lead
sulfide) from an alkaline solution when adding lead acetate Pb
(CH3COO)2.
Methionine and cysteine contains sulfur group
1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2%
gelatine and 0.1 M cysteine into separate, labeled test tubes.
2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of
10 % lead acetate solution.
3. Stopper the tubes and shake them. Remove the stoppers and
heat in a boiling water bath for 5 minutes. Cool and record the
results.
Now to
practical part
Ninhydrin Test
1 mL solution•
5 drops of 0.5% •
ninhydrine soln
Boil over a water
bath for 2 min.
Millon’s Test
Biuret test
1 ml of •
protein soln
2 ml of •
biuret
reagent
incubation 30 •
min .
2 mL solution in a •
boiling test tube
2 ml of conc. HNO3. •
Heat over a flame for 2 •
min.
COOL•
3ml NaOH •
Add 2ml of •
soln + 2 ml
of Hopkins
reagent.
5-6 ml conc. •
H2S04
Lead sulfite test
protein •
solution
3 drops of Millon’s •
reagent.
Mix well and heat
directly on a small
flame, BWB 5 min
2 ml of
1 ml of the protein •
solution
1 ml of 3 N NaOH •
0.5 ml of α- •
naphthol
a few drops of 2 % •
(NaOBr).
1ml cysteine•
2 ml of 10 % •
NaoH
Add 5 drops of •
10 % lead acetate
soln.
Put in BWB for 5 •
min.