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Protein Color Reactions

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Group No. 1




Date Performed
: Feb/06/2012
Chemistry Laboratory 204 A


Facilitator's Initials
:___________
Members:






1. Alcuizar, Timothy James F. - technician


2. Catingub, Cherrielyn C. - Recorder
3. Famor, Razmine S. - manager


Data Sheet
Activity
Proteins and Amino Acids I:
10
Color Reactions



Objectives
:4 1. To determine reactive functional groups of different proteins and
amino acids using different chemical tests
2. To Explain the observed colors of the proteins and amino acids
3. To Identify chemical and physical properties of proteins from natural
sources and some chemical reactions of amino acid residues in these
proteins
Data:


Name of Test Test
Reagents
Result
Explanation
Solutions
Added
A. Biuret
1% Albumin 10% NaOH
violet
albumin is a typical
Reaction

1% CuSO4
protein, then it is


proteins that wil
normally give the biuret
reaction. In fact it is
given by all substances
having at least two
peptide linkages (-CO-
NH-) which includes all
proteins and most
peptides.


1% Casein
violet
Casein is a water

soluble protein since

the reagent binds with
the pepdide bonds, so
it should give a positive
test result with Biuret.








1% Glycine
No reaction
. Glycine, being a free,

simple amino acid, does

not possess any
peptide bonds.
1% Peptone
pink
Pepsin is also positive

but is less than that of

albumin, this can be
explained by the fact
that peptone is a
polypeptide chain and
has fewer peptide
bonds than albumin,
therefore the color
change is less dramatic
Urea (s)

Biuret is a condensation

compound of urea


Data Interpretation:
3Many proteins contain sulfur. They are complex with molecules composed of
carbon, hydrogen, oxygen, and nitrogen. Amino acids are the result of these blocks of
proteins and they are connected by peptide bonds. There is a lot of similarity between
these amino acids and the biuret molecule and both of them react in the same way.
Biuret reagent is a light blue solution, which turns purple when mixed with a solution
containing protein. A purple color complex is formed when the copper ions of the Biuret
Reagent react with peptide bonds in the polypeptide chains.
The biuret reaction is based on the reduction of Cu2+ to Cu+ which then
complexes with the nitrogen atoms on the peptide bonds. This reactionis carried out at
high pH and gives a violet colour and pink when combined with short chained-
polypeptides. It is the amide bond (common to all proteins) which is resposible for the
colour.
Since proteins are made up of amino acids, presence of peptide bonds during
Biuret test for proteins wil always give a positive result for all kinds of protein based
foods.

O
O
O
heat
2 H2N C NH2
H2N C NH C NH2 + NH3
urea
biuret


When urea is heated to above its melting point, it is converted into biuret, as
ammonia is liberated:












Two peptide bonds at least are required
For the formation of this complex












Structure of casein for biuret test



Peptide bond:





B. Millon's

1% Albumin Mil on's
Flesh to Red Protein containing
Test

Reagent
Tyrosine wil yield a

[HgNO3 and
positive result from
Hg(NO3)2
pink to dark red.
1% Casein
added with Red
Caseine is one of the



conc. HNO3]
protein that makes up
milk. The protein is
tyrosine, which is the
only protein that yields
a positive result.
1% Gelatin
No reaction
Gelatin wil not react

with mil on's test since

it is a protein.
1% Phenol
Red
Millon's Test is used to

precipitate
test for the presence of

phenols (an alcohol
consisting of an OH
group attached to a
benzene ring.

Data Interpretation:


Rgts: Mercuric and mercurous nitrate [(Hg(NO3)2 + (Hg2(NO3)2]Nitric acid (HNO3)
Principle involved: Mercuration by substitution
Positive reaction: Brick red color or precipitate
Group responsible:Phenolic compound
Compound detected: Proteins containing tyrosine

Chemical structure of albumin:


Mil on's test is used to detect the presence of hydoxyphenyl group (benzene ring with -
OH). It is a specific test for tyrosine (since tyrosine is the only amino acid that has a
hydroxyphenyl group) and gives a red precipitate or a red solution as a positive result.
This happens when the phenol group of tyrosine is nitrated and then the nitrated phenol
complexes with mercury (I) and mercury (II) ions. Nevertheless, it is important to make
sure that no other phenols aside from the ones from tyrosine are present because the
Mil on's test gives a positive result for any compound with phenol groups. Phenols
containing more than one hydroxyl group do not give the typical red color. The fact that
aniline and other primary aromatic amines give this test is due to the presence in the
reagent of free nitric and nitrous acid. The red color observed in this test is probably due
to a mercury salt of nitrated tyrosine.







O
H
O
O

N
HgNO3 + HNO3

O
O
H

NH2

OH


O

NH
O
H
2





It is composed of salts of mercury dissolved in nitric acid. Tyrosine (both free and
constitutiveof proteins) reacts with reagent and produces a purple-red salt of mercury.


Name of Test Test
Reagents
Result
Explanation
Solutions
Added
C.
1% Albumin conc. HNO3
YELLOW
a reaction due to the
Xanthoproteic
conc. NH4OH
prescence of phenyl
Reaction


group (-C6H5) in the


protein molecules. it

involves the hnitration

of the phenyl rings
presnt in the aromatic
amino acids such as
tyrosine, phenylalanine,
and tryptophan,
forming yel ow nitro-
substitution products
that turn orange when
alkali is added
1% Casein
Pale yel ow The Xanthoproteic test

solution
uses nitric acid to

produce an intense
yel ow color in samples
containing proteins. It
works by nitrating
(adding a NO2 group
to) the aromatic ring in
certain amino acid
groups (tyrosine,
tryptophan, and
phenylalanine). Casein
contains amino acids
with these rings and
would give a positive


result.






1% Gelatin
clear
Gelatin lacks

tryptophan, and has

tyrosine to produce a
yel ow color.
1% Phenol
yel ow
phenol has a hydroxyl

group (-OH) which is

bonded to a phenyl
ring. It yields the same
positive result like
tyrosine which has a
hydroxyl group bonded
to its phenyl ring.



DATA INTERPRETATION:

Reagents: Egg white + conc. HNO3 and 50% NaOH
Positive reaction : + conc. HNO3 yel ow precipitate + 50% NaOH red-orange solution
Principle: Nitration by Substitution
Compound detected: Benzene ring
Compound responsible for +VR : Aromatic Nitro compounds (R-NO2)

Concentrated nitric acid (HNO3) wil react with phenyl groups (aromatic rings) to
produce a yel ow color. The amino acids tyrosine, tryptophan and phenylalanine and any
proteins that have significant numbers of tyrosine and phenylalanine in their primary
sequence wil generate a yel ow color.. To 2 ml of the test sample add an equal volume
of concentrated nitric acid. The appearance of a deep yel ow colour signifies the
presence of protein.


NO2
NO2
HONO2
OH
protein
OH
protein
OH
protein
O
tyrosine
yellow
intense yellow


Some peptides and protein chains contain amino acids which have aromatic rings.
These rings undergo nitration to give derivatives which are yel ow. In basic solution, the
yel ow color intensifies.







STRUCTURAL FORMULA OF PHENOL



D. Test for

1% Albumin 10% NaOH
Black
The product of this
Sulfur

5%
precipitate
reaction is black lead

Pb(CH3COO)2
(II) sulfide. It forms
upon the reaction
between lead (II)
acetate and the sulfur
in the egg.
Gelatin (s)
No black
it lacks essential sulfur-

precipitate
containing amino acids

and the essential amino
acid tryptophane.
Peptone (s)
black
If there is any

blackening of the

medium, it indicates the
reduction of sulfur and
is a positive result

Some amino acids contain sulfur (e.g. cystine, cysteine, and methionine). When these
are present in a peptide or protein the disulfide bonds may be cleaved in basic solution
to give inorganic sulfide ions. In the presence of lead ions, black lead sulfide may
precipitate:

STRUCTURAL FORMULA FOR PEPTONE





NaOH
Pb2+
S
S
S2-
PbS
disulfide
black ppt





E. Hopkins-
1% albumin conc. H2SO4
Violet ring
Tryptophan has an
Cole Reaction
indole nucleus which is

responsible for the
violet ring found at the
junction between the
two layers.
1% casein
White ring
Hopkins-Cole test only

reacts with proteins

containing Tryptophan.
Its indicator is the
violetcyclic product.
Casein is negative for
hopkns-cole reaction
test.
1% gelatin
Violet ring
Gelatin is not supposed

to react with the

Hopkins cole test and
should produce a clear
solution, however in
our activity, it produced
a violet ring, because
we used the wrong test
solution.

2This test is carried out by mixing the test solution with glyoxylic acid and then layering
the mixture with concentrated sulphuric acid. In the presence of tryptophan a purple
ring appears at the interphase of two liquids. This test only works for proteins containing
tryptophan, which is the only amino acid which contains and indole ring (a 5 membered
ring, including one nitrogen, in the ring, and one double bond) (much the same as
pyridine, but with a double bond). The glyoxylic/oxalic acid reacts with this indole ring to
form a voilet product. Tryptophan has an indole nucleus which is responsible for the
violet ring found at the junction between the two layers.

Structural formula of albumin





Tryptophan:


Indoles aromatic heterocyclic organic compound. It has a bicyclic structure,consisting
of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrole
ring.


F. Ninhydrin

0.2%
0.1%
Bluish
Albumin is a protein
Reaction
Albumin
ninhydrin
solution with with water solubility

solution
purple
that contains amino
cloudy layers acids so we wil get a
positive result
Ammonia
Purple
Ninhydrin test is used
water
solution
to detect ammonia, so

a solution containing
ammonium ion wil
yield a positive result
for this test.
0.2% Urea
Purple
Ninhydrin and urea

combine to form a

stable crystal ine ureide
of the
composition
CLoHloOsNz, 1 mole of
urea combining with 1
mole of ninhydrin
without loss of water.
The compound readily
loses 1 mole of water
at 56"
to form a substance of
the composition
C&H804N2.
02%
Purple
Since it is a primary
Glycine
solution
amine, it wil be

positive for the
ninhydrine test.

1Ninhydrin (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 evolved. Ninhydrin is also used in amino
acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with
ninhydrin except proline; Also, certain amino acid chains are degraded. treatment with


ninhydrin should result in a dramatic purple color if the solution contains this species. In
the analysis of a chemical reaction by thin layer chromatography (TLC), the reagent can
also be used. It wil detect, on the TLC plate, virtually all amines, carbamates and also,
after vigorous heating, amides. When ninhydrin reacts with amino acids, the reaction
also releases CO2. The carbon in this CO2 originates from the carboxyl carbon of the
amino acid The carbon atom of a carbonyl bears a partial positive charge, so the central
carbon of a 1,2,3-tricarbonyl is less stable and more electrophilic than a simple ketone.
In most compounds, a carbonyl is more stable than the dihydroxy (hydrate) form.
However, ninhydrin is a stable hydrate of the central carbon because this form does not
have the destabilizing effect of adjacent carbonyl partial-positive centers. Indane-1,2,3-
trione reacts readily with nucleophiles. Note that in order to generate the ninhydrin
chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff
base. Thus only ammonia and primary amines can proceed past this step. At this step,
there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an
amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The
reaction of ninhydrin with secondary amines gives an iminium salt, which is also
coloured, and this is generally yel ow-orange in color.


STRUCTURAL FORMULA FOR GLYCINE



Document Outline

  • Group No. 1 Date Performed : Feb/06/2012
  • Chemistry Laboratory 204 A Facilitators Initials :___________ Members:
    • Proteins and Amino Acids I:
    • Color Reactions
  • Activity
  • 2 BO0043-Experiment 2 Tests for Carbohydrates, Proteins and Lipids

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