Precipitation Reactions of

Protein
By Sandip Kanazariya
PRECIPITATION REACTIONS OF
ALBUMIN
• Solubility of protein depends on proportion &
distribution of polar hydrophilic end & non -
polar hydrophobic end in a protein molecule
resulting in a protein dipole moment.
• Ionic polar groups of molecule interact
electrostatically both within the same molecule
and with the surrounding molecule tending to
form aggregates and opposing solubility.
PRECIPITATION REACTIONS OF
ALBUMIN
• In solution, the polar water molecule interacts
with polar group of protein tending to increase
the solubility.
• The solubility of protein molecule is mainly
due to change in particle size, shape and
hydration.
PRECIPITATION REACTIONS OF
ALBUMIN
• Most of the proteins are soluble in dilute acid
& alkali.
• Proteins are very reactive & highly specific in
behavior due to the presence of active groups
in its molecule.
• As they contain both acidic and basic groups in
its molecule, they act as ampholytes or
amphoteric electrolytes.
 PRECIPITATION REACTIONS OF
ALBUMIN

Acidic pH Alkaline pH

Dipole ion or
Cationic Form Anionic Form
Zwitter ion
Protein + Protein -
+ Protein -
PRECIPITATION REACTIONS OF
ALBUMIN
• Isoelectric point is the pH at which protein
contains equal number of positive & negative
charges & protein can exist as dipolar ion or
zwitter ion.
• In dipolar ion total charge is very high & net
charge is zero due to presence of equal number
of both acidic and basic groups in its molecule.
• They dissociate as acidic or basic depending
upon pH of the solution.
PRECIPITATION REACTIONS OF
ALBUMIN
• If acid is added it acts as base and if alkali is
added it act as an acid. These properties are
maximal at isoelectric pH.
• Proteins can be precipitated out from its
solution by a variety of substances.
• Such reactions which precipitate out protein
from their solution are called as precipitation
reaction.
PRECIPITATION REACTIONS OF
ALBUMIN
• Proteins are polymers of alpha amino acids
having large molecular weight and form
colloidal solutions.
• Proteins can be precipitated either by removal
of water layer (Dehydration), denaturation,
adjusting the isoelectric pH or by
neutralization of charge present on protein
molecule.
 PRECIPITATION BY
STRONG MINERAL ACIDS
1. HELLER’S NITRIC ACID TEST

• Reagents required-: Concentrated Nitric acid.

• Reaction- When native protein solution is
treated with Concentrated HNO3, white
precipitate ring is obtained due to denaturation
of protein.
TEST OBSERVATION INFERENCE
1. Heller’s Nitric White ppt ring Albumin is
acid Test: Take is obtained. precipitated out
3ml albumin by strong mineral
solution then add acid
3ml conc. HNO3
test tube take,
from side of the
test tube and do
not mix.

• Note: White precipitate ring is due to denaturation of

albumin by strong mineral acid.
 2. PRECIPITATION BY HEAVY
METAL IONS
• Reagents Required: - Lead acetate and silver
nitrate.
• Reaction: - Proteins are precipitated from their
solution by heavy metal ions. These metal ions
precipitate the protein from their solution. On the
alkaline side of isoelectric pH, Protein dissociates
as protein anion(Pr-) which combines with
positive metal ion (cation) to form insoluble
precipitate of metal proteinate such as lead
albuminate and silver albuminate.
TEST OBSERVATION INFERENCE
2. Lead acetate test: White White precipitate is
Take 3 ml of albumin precipitate is obtained due to
solution obtained formation of
in test tube then add 2 lead albuminate
drops of 2% Na2NO3 therefore albumin is
solution and 1 ml of precipitated
lead acetate solution. by heavy metal ions.

3. Silver nitrate Test White White precipitate is

Take 3 ml of albumin precipitate is obtained due to
solution in test tube obtained formation of silver
then add 1 ml of silver albuminate therefore
nitrate solution. albumin is
precipitated by heavy
metal ions
 3. PRECIPITATION REACTION
OF PROTEIN BY ORGANIC ACID
• Reagents required: Sulphosalicylic acid,
Trichloroacetic acid, Esbach’s reagent and
distilled water.
• Reaction: These organic acids exist as negative
ion i.e. anion. when organic acids are added to
albumin solution proteins are precipitated from
their solution because on acidic side of isoelectric
pH, protein dissociate as cation (protein +ion )
which combine with anions (protein - ion ) of
organic acids to form salt of protein.
TEST OBSERVATION INFERENCE
4. Sulphosalicylic White White precipitate is
acid Test: precipitate is obtained due to
Take 3 ml of albumin obtained formation of
solution in test tube albumin
then add 1 ml of sulphosalicylate
Sulphosalicylic acid therefore albumin is
mix well and observe. precipitated
by organic acid.
5. Trichloroacetic White White precipitate is
acid Test: Take precipitate is due to formation of
3ml of albumin obtained albumin
solution in test tube trichloroacetate.
then add 1 ml of Therefore albumin is
Trichloroacetic acid precipitated by
mix well and observe. alkaloidal reagent.
TEST OBSERVATION INFERENCE
6. Esbach’s Yellow Therefore
Test: Take 3ml of precipitated albumin
albumin solution is obtained precipitated from
in a test tube add its solution by
eual volume of Alkaloidal
Esbach’s reagents.
reagent(1% Picric
acid) to it mix
well
& observe.
 4. PRECIPITATION OF PROTEIN
BY ORGANIC SOLVENTS
• Reagents Required: Absolute Alcohol, Acetone &
chloroform.
• Reaction: Organic solvent such as alcohol, acetone &
chloroform when added to albumin solution, it
decreases the dielectric constant of solvent & displaces
some of the water molecules (dehydration) associated
with protein and decreases the concentration of water.
These effect tends to decreases the solubility of the
protein in solution due to which protein are
precipitated out form their solution.
• The mechanism of precipitation in this case is by
dehydration, denaturation & removal of charges.
TEST OBSERVATION INFERENCE
7. Precipitation White White
by Acetone: Take precipitated precipitated is
3ml of albumin is obtained obtained due to
solution in dehydration,
a test tube add 1 denaturation by
ml of acetone to it acetone.
mix well Therefore
& observe. albumin
precipitated from
its solution by
organic solvents
 5. PRECIPITATION BY SALT
• Reagent Required: Ammonium sulphate.
• Reaction: The high molecular weight compound can be
precipitated from their solution by addition of highly
soluble salt. When protein solution is treated with this
salt, the salt molecule takes up water from the protein
solution for its own solubility.
• Due to which effective concentration of water required
for protein solubility is decreased and proteins are
easily precipitated out from their solution at isoelectric
pH. This process is called salting out.
• The precipitate formed is apparently due to
neutralization & dehydration of molecule & molecule
aggregates in solution.
TEST OBSERVATION INFERENCE
8. Half Saturation No White Albumin is not
Test: Take 3 ml of precipitate is completely
albumin solution in a obtained precipitated from its
test tube add equal solution by half
amount (3ml) saturation with
saturated solution of ammonium sulphate
ammonium sulphate solution.
mix it well and
observe.
Then filter the Filtrate gives violet
solution and perform colour
Biuret test with the
filtrate
TEST OBSERVATION INFERENCE
9. Full Saturation White Albumin is
Test: precipitate is completely
Take 5 ml of albumin obtained precipitated
solution in a test tube from its solution by
saturate it with full saturation with
ammonium sulphate ammonium sulphate
crystals and observe. crystals.

Then filter the Filtrate does not gives

solution and perform violet colour with
Biuret test with the biuret test
filtrate
6. PRECIPITATION OF
PROTEIN BY HEAT
Heat Coagulation Test
 Heat Coagulation Test
• Reagents required: 1% acetic acid
• Reaction: When albumin solution is heated,
white coagulum is obtained because albumin is
denatured by heat (Albumin is a coagulable
protein). After addition of 1drop of 1% acetic
acid, coagulum increases because pH of
albumin solution is shifted towards isoelectric
point. At this pH solubility is minimum and
more protein is precipitated from its solution.
TEST OBSERVATION INFERENCE
10. Heat White coagulum or White coagulum is
Coagulation Test : Turbidity or obtained due to
Fill 3/4 of test tube
th precipitate is obtained denaturation of
with albumin which increases after albumin by heat i.e.
solution. Heat it at the addition of 1 drop of albumin is a
top and observe. Then acetic acid. coagulable protein.
Add a drop of acetic Albumin is
acid to it and again precipitated from
observe. solution by heat.
 Conclusion
1. Albumin is a simple protein.
2. Albumin is denatured by strong mineral acid.
3. Albumin is precipitated by heavy metal ions.
4. Albumin is precipitated by organic acids.
5. Albumin is precipitated by organic solvents i.e.
alcohol / acetone.
6. It is completely precipitated from it solution by
full saturation with ammonium sulphate but not
by half saturation.
7. Albumin is coagulated by heat.