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Proteins:
Proteins are polyamides and a molecular weight above 5000 Kilo Daltons (K.D)
Polyamides of molecular weight below 5000 K.D are usually called poly peptides.
Amino acids are the building blocks of proteins which consist of an amino group, a
hydrogen atom and a typical R group bonded to carbon atom.
Amino acid
Protein Elements
All proteins contain nitrogen. This fact distinguishes them from most
carbohydrates and fats. Also proteins contain carbon; oxygen; hydrogen and a
smaller quantity of sulfur; iodine and phosphates.
General Color Reactions of Proteins reaction
Color producing tests such as the Biuret, Ninhydrin, Millon`s, Hopkins-Cole and
unoxidized sulfur tests are used to detect proteins in biological mixtures. Some of
these reactions [Biuret and Ninhydrin ]are general tests that give positive results
with all proteins and amino acids.
1. Biuret test:
The biuret test depends on upon the reaction of cupric ions [ Cu+2] in an alkaline
solution with peptide linkages of the protein to produce a purple color which is
apparently caused by the coordination complex of copper atom and four nitrogen
atoms of two peptides bonds as shown below :
protein or poly peptide
Procedure
To 1ml of diluted protein solution add 1ml of 10% NaOH mix then add 4-5 drops
of 0.5% CuSO4 and shake, violet color will be produced indicating the presence of
peptide bonds.
Note:
Excess CuSO4 should be avoided because Cu(OH)2will be formed which has
blue color interfering with the violet or pink color of the biuret reaction.
Why the reaction is called as biuret test?
Biuret compound is formed when the Urea is heated. If alkaline cupric ions are
added to biuret solution a violet color is produced. This is a characteristic color not
only of biuret but also proteins and peptides which contain a structure similar to a
biuret. This test requires the presence of at least two peptides linkages per molecule
to be positive. The biuret reaction can be specific to poly peptide since proteins are
about the only compound found in nature that have poly peptide character;
therefore, the biuret test is remarkably specific to proteins.
H
OH
2H-N-C-N-H
Urea
H OH O
NH3
+ H2N-C-N-C-NH2
Biuret compound
2. Ninhydrin Test:
Ninhydrin reacts with α amino acids to yield a characteristic blue violet
products [decarboxylation]. The overall reaction is shown below :
-3H2O,-CO2
Ninhydrin
amino acid
+ RCHO
blue color
decarboxylated
amino acid
Most amino acids give the same color, except proline makes a pale-yellow
product with ninhydrin. This is due to the fact that proline is an imino acid instead
of having traditional ∝-amino acids structure.
+
Ninhydrin
yellow color
proline
Procedure:
To 1ml of protein solution that should be neutral or slightly acidic pH 5-7 add
2 drops of freshly prepared 0.2% ninhydrin solution. Boil for 1 minute then allow to
cool. A blue color is produced indicating the presence amino acids or any hydrolysis
product .
3. Millon`s test:
This test is done to find tyrosine which present in most proteins. Millon`s reagent
contains Hg ion which forms a complex red color with tyrosine. If the unknown is
protein solution, a red precipitate will be formed due to the heavy metals (Hg) are
precipitating agents, while if the unknown is tyrosine solution then a red solution
will be formed.
Tyrosine
Note:
Excess chloride ions interfere with this test combing with Hg ions present in
Millon`s reagent so this test cannot be used to detect tyrosine or protein in urine
since urine contains a significant amount of chloride ions. Millon`s test will be
conducted with one compound which is not an amino acids nor a protein. This
compound is salicylic acid which a simply a 2-hydroxy benzoic acid.
Procedure:
To 1ml of unknown solution add 1 drop of Millon`s reagent and heat gently. Red
precipitate or solution indicates the presence of protein or tyrosine.
4. Glyoxylic Acid Test(Hopkin`s Cole Reagent)
This test is done to detect the presence of tryptophan amino acid, which contain
indole group. Strong acids such as H2SO4 oxidize glyoxyl (CHO—CHO) to
glyoxylic acid then this compound condenses with tryptophan (indole ring)forming
red, purple, violet or yellow color complex.
Note:
1. glacial acetic acid usually contains some glyoxylic acid as an impurity; therefore,
it is as suitable source glyoxylic acid and glyoxyl.
2. H2SO4 used should be pure, otherwise color will not develop.
Procedure:
To 1 ml of glacial acetic acid which contains glyoxylic acid add 1ml of unknown
solution, mix then incline the test tube and slowly slide 1 ml of concentrated H 2SO4
down its side so that the sulfuric acid forms a distinct bottom of the test tube. A red
or yellow color as ring will appear indicate the presence of tryptophan.
5. Unoxidized Sulfur Test:
This test is positive only in the presence of amino acid containing sulfhydryl
(SH)or disulfide S-S group.
Methionine is the third amino acid which contain sulfur but not in the form of
sulfhydryl (SH)or disulfide S-S group.
When proteins boiled in strong alkali, the SH and S-S groups are converted
into inorganic sulfide. If we add lead acetate solution black precipitate of lead
sulfide PbS is formed.
NH3
NaOH
HS-CH2-CH-COO
Pb (CH3COO)2
inorganic sulfide
PbS
100 C ͦ
Procedure:
1.To about 1 ml of unknown solution add 2 ml of 40% NaOH.
2.Boil for (10min) to convert the organically combined sulfur to inorganic form.
3. Remove from heat and add 10 drop of lead acetate.
4. Brown or black precipitate of lead sulfide will form indicating the presence of
cysteine and cystine.
Cysteine
Cystine
Methionine
Five qualitative color test of proteins are summarized in the following table
Name of test
Reagent used
Biuret
Alkaline CuSO4 in 10%
sodium hydroxide
Specific for material
Poly peptide and
proteins
Ninhydrin
Ninhydrin in water
Amino acid
saturated butanol
Millon
Mercuric and mercurous Phenolic hydroxyl
nitrates in HNO3
Hopkins-cole
group of tyrosine
Glyoxylic acid in H2SO4 Indole group of
tryptophan
Unoxidized
Strong NaOH and
SH and SS groups in
acetate
cystine and cysteine
respectively.
Physical reaction
1. Solubility of protein
2. Precipitation of protein
3. Coagulation of protein
1.Solubility of protein
The solubility of amino acids and proteins is largely dependent on the solution
pH. As amino acids have both an “amino” group and a “carboxylic” group, they are
considered as both “base” and “acid”, i.e. they are amphoteric the protein will
soluble in the basic and acidic medium the protein while in neutral medium the
protein is precipitate.
Albumin
Gelatin
Casein
Pepton
Cold H2O
Not soluble
Not soluble
Not soluble
Not soluble
Hot H2O
Semi soluble
Semi soluble
Semi soluble
Semi soluble
10% NaOH
Soluble
Soluble
Soluble
Soluble
10%HCl
Soluble
Soluble
Soluble
Soluble
2.Precipitation of protein
The charge of protein in the acidic medium will be (+ve ) while in the basic
medium the charge will be (-ve) and the protein is soluble. while in neutral medium
the protein is precipitate .
+
+NH3-CH-COOH
NH3
R
H+
R-C-H
COO -
OH-CH-COO -
R
NH2
Zwitterion
a . Precipitation by Heavy Metal
In most naturally occurring protein solutions the protein molecules are negatively
charged. Neutralization of this charge bring proteins to the isoelectric point. At this
point, maximum precipitation of proteins take place and the protein particles bear
zero net charge. Each protein has its own isoelectric point, since they may be
precipitating by providing the positively charged molecules from another reagent.
salts of heavy metals like iron, copper, zinc, mercury, silver, etc. are very suitable
for this, however, addition of excess amount of salts of heavy metals solutions to
protein hence these particles may become dissolve again.
The precipitation of protein by salts of heavy metals is also due to at least partly to
formation of insoluble compounds of proteins and the metallic cations for example
when we add {Pb+2, Hg+2} to casein or albumin, lead caseinate or s a mercury
albumin may form as a precipitate .
H
R-C-COO - +
H
Ag +
NH2
R-C-COOAg
NH2
silver proteinate
at isolectric point
white PPt
Procedure
To 1ml (alb. or peptone) add 3 drops of CuSO4 or FeCL3 or Pb(CH3COO)2 or
AgNO3
b. Precipitation by alkaloid reagents
The addition of an acid to a protein solution causes the protein particles to acquire
a positive charge. By the cautious addition of an alkaloidal reagent (which provides
complex negatively charged ions) to the acidified protein solution, the protein can
be predicated at the isolelectric point. An excess of the reagent may the precipitate
to disappear by providing the protein particles with an effective negative charge.
Alkaloidal
reagents
include:(sulphosalicylic,
metaphosphoric,
phosophotungtic and trichloro acetic acids.
Procedure
1ml of protein + 1ml of phosophotungtic acid
white ppt
tannic,
c. Precipitation by neutral salts
Protein molecules contain both hydrophilic and hydrophobic amino acids. In
aqueous medium, hydrophobic amino acids form protected areas while hydrophilic
amino acids form hydrogen bonds with surrounding water molecules (solvation
layer). When proteins are present in salt solutions (e.g. ammonium sulfate), some of
the water molecules in the solvation layer are attracted by salt ions. When salt
concentration gradually increases, the number of water molecules in the solvation
layer gradually decreases until protein molecules coagulate forming a precipitate;
this is known as “salting out”. As different proteins have different compositions of
amino acids, different proteins precipitate at different concentrations of salt solution.
1. Half saturation (reversible ppt)
1ml pr.+1ml ammonium sulfate solution
white ppt or turbid
albumin
globin
No ppt
ppt
The reason for the precipitation of globulin and albumin at different ammonium
sulfate concentration could be that the solvation layer around globulin is looser and
thinner than that around albumin. Therefore, globulin needs only half-saturated
ammonium sulfate to lose its solvation layer while albumin loses its solvation layer
in a fully saturated ammonium sulfate solution.
2. Complete saturation
1ml protein + solid ammonium sulfate
white ppt or turbid
3. Denaturation (coagulation):(irreversible precipitation )
Heat disrupts hydrogen bonds of secondary and tertiary protein structure
while the primary structure remains unaffected. The protein increases in size due to
denaturation and coagulation occur.
Denaturation : is a process in which the biological activity of protein is lost
there are many factors such as
1. heating
2. mixing
3.x-ray
4. ultrasonic vibration
Procedure
Add 3 ml of albumin or globulin then 2-3 drops of acetic acid
heating
+ve precipitation
albumin, globulin
casein, pep., gel
.
- ve
Unknown of protein :
Biuret test
+ve
-ve amino acid
protein
or non proteins substance
Denaturation test
Ninhydrin test
+ve turbid or white ppt alb.or glob
+ ve aminoacid
- ve non protein
-Sulfur test (cystein and cystine )
- Hopkin`s cole test(tryptophan)
clear solution (-ve)
pepton, casien, gelatin