Transcript Document 7731007

Prepared by
D.r Huda Hania
Introduction

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
Amino acid and protein

Amino acid are the building block of proteins

There are about 300 amino acids occur in nature.
Only 20 of them occur in proteins.

Structure of amino acids:

Proteins consists of amino acid linked to
peptide bond

4.
Each amino acid consists of :
Central carbon atoms
An amino acid
Carboxyl group
Side chain

Different side chain result in various amino acid
1.
2.
3.
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

Proline
 It is unique among the 20 proteinforming amino acids in that the amine
nitrogen is bound to not one but two
alkyl groups, thus making it a secondary
amine

an imino acid is any molecule that
contains both imino (>C=NH) and carboxyl
(-C(=O)-OH) functional groups
Essential Amino Acids in Humans
Required in diet
 Humans incapable of forming requisite


Arginine
Histidine
Isoleucine
Leucine
Valine
Lysine
Methionine
Threonine
Phenylalanine
Tryptophan
Non-Essential Amino Acids in Humans

Not required in diet
Alanine
Asparagine
Aspartate
Glutamate
Glutamine
Glycine
Proline
Serine
Nonpolar amino acid
Hydrophobic amino acid: are amino acid
that contain C,H in their side chain
 They tend way from water (hate water)
 Hydrophobic (normally buried inside the
protein core):

Polar amino acid
also called hydrophilic (love water)
 Tend to found on surface
 That amino acid that contain in their side
chain O,N and they can dissolve in
water ( like dissolve like ) (covalent
bond = hydrogen bond causing folding
protein )

Acidic amino acid

Basic amino acid
Uncommon Amino Acids
Hydroxylysine, hydroxyproline which is the
building block of collagen
 Carboxyglutamate which is the building
block of blood-clotting proteins

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.
Qualitative test for amino acids
There number of test to detect the presence
of amino acid
 This is largely depend on the natural of side
chain

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.


With all amino acid will give purple or deep
blue with exception Proline gives yellow not
violet (why)

Proline reacts with ninhydrin, but in a
different way. While most ninhydrin tests
result in a purple color, the proline reaction
is more yellow due to substitution of the
alpha amino group that ninhydrin reacts with
carbon rings
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.

biuret test :
Biuret structure:
it is result of condensation of two molecule
of urea

peptides containing three or more amino acid
residues form a colored chelate complex with
cupric ions (Cu2+) in an alkaline environment
containing sodium potassium tartrate.
 Single amino acids and dipeptides do not give
the biuret reaction, but tripeptides and larger
polypeptides or proteins will react to produce the
light blue to violet complex that absorbs light at
540nm
 One cupric ion forms a colored coordination
complex with four to six nearby peptides bonds.
The intensity of the color produced is
proportional to the number of peptide bonds
participating in the reaction..

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 working range for the biuret assay
is 5-160mg/mL.
 What is chelation?
chelation involves the formation or
presence of two or more separate
coordinate bonds between a polydentate
(multiple bonded) ligand and a single
central atom.

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”.
 N.B. Two peptide bonds at least are
required for the formation of this complex.


Procedure:
To 2 ml of protein solution in a test tube,
add 4ml of reagent incubation 30 min
 Result :

Interpretation
Observations
No change ( solution
remains blue )
The solution turns from blue
to violet( purple)
The solution turns from blue
to pink
Proteins are not
present
Proteins are present
Peptides are present
( Peptides or
peptones are short
chains of amino acid
residues)
Millons test :
It is specific for tyrosine, the only amino
acid that contain a phenol group on
which a hydroxyl group is attached. It
gives red precipitate.
 Consequently, any protein containing
tyrosine will give a positive test of a pink
to dark-red color.

Principle:
A test for protein, the tyrosine (phenolic
group) of which reacts with nitrite after
treatment with mercuric ion in acid to give a
red color.
 The reagent used in this test is called
Millon's reagent and it contains mixture of Hg
++, Hg2 ++, HNO3 and HNO2


Procedure & observation:
 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.

Xanthoproteic Test:
Some amino acids contain aromatic groups
that are derivatives of benzene. These
aromatic groups can undergo reactions
 One such reaction is the nitration of a
benzene ring with nitric acid. The amino
acids that have activated benzene ring can
readily undergo nitration.
 In the presence of activated benzene ring,
forms yellow product. Apply this test to
tyrosine, tryptophan, phenylalanine and
glutamic acid.

Why benzene ring in phenylalanine is inactive
 Phenylalanine still reacts with nitric acid, just
not as readily as Tyr or Trp because Tyr and
Trp have electron donors (hydroxyl and the
nitrogen heteroatom), which make the ring a
lot more attractive for electrophilic nitration.
 To get Phe to react, you may have to boil it
with nitric acid

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.
Hopkins-Cole (Glyoxylic Acid Reaction)
• Specific for tryptophan
(the only amino
acid containing indole group)
• 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.
Indole
Glyoxylic acid
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.
Sulfur test:
Sulfur containing amino acids, such as
cysteine and cystine upon boiling with
sodium hydroxide (hot alkali) yield
sodium sulfide.
This reaction is due to partial conversion of
the organic sulfur to inorganic sulfide,
which can detected by precipitating it to
lead sulfide, using lead acetate solution.
S.(protein) + 2NaOH-------- Na2S
Na2S + (CH3COO)2pb ------- PbS + 2CH3COONa

Methionine and cysteine contains sulfur group
Procedure:
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.
Sakaguchi test

For detection of the amino acid
containing the guanidinium group (e.g.
arginine).

In basic conditions, α- naphthol and
sodium hypobromite/chlorite react with
the guanidinium group to form red
orange complexes.
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 %
hypobromite solution (NaOBr).

2. The formation of a red color indicates
the presence of a guanidinium group in
the compound under examination.
Arginine