39

Ex eriment no:
9
IONS OF PROTEINS
1. BIURET TEST: It is general Test ror
Proteins
Experiment
Test: To 2ml Of protein Observation Inference
solution, a. Violet colour
add 2ml of is seen a. Proteins present
Na0H and 2
drops of 1% copper sulphate (presence of peptide
linkages)
(CUS04)_Mix well
Control: To 2ml of water, add 2mI b.Blue colour is seen b. Proteins absent
of 10%NaOH and 2 drops of 1%
CuS04. Mix well

principle: In alkaline medium, cupric ions

chelates(attachmentor metallic ions to nonmetallic
ions, by covalent bonds) with peptide linkages
in proteins to give the violet color. A
minimum of 2 peptide bonds are required for a positive
test.
The name of the reaction is derived from the organic compound biuret,
which is
formed by the condensationof two urea moleculesat high temperature.When biuret
is treated with dilute CuS04 in an alkalinemedium,a violet color is obtained.
Significance: General test for Proteins. It can also be used for quantitative estimation and
identification of proteins.

2. PRECIPITATION REACTIONS OF PROTEINS

Proteins are precipitated from solution by salts of heavy metals like mercuric chloride, silver
nitrite, copper sulphate and lead acetate. They are also precipitated by certain acids called as
alkaloidal agents like Trichloroacetic acid, phosphotungsticacid, tannic acid etc.
Proteins can also be precipitated by concentratedsolutions of salt like ammonium sulphate,
sodium chloride and organic solvents like ethanol, methanol and acetone.

Clinical significance of precipitation of proteins:

a) Purification of enzymes and proteins present in blood.
b) During estimation of biochemical parameters in blood like glucose, urea etc, proteins
by precipitationreactions.
may interfere. Thus proteins free filtrate is obtained
c) Tanning in leather industry
d) Used in the treatment of heavy metal poisoning.
Precipitationby AmmoniumSulphate
a) HalfSaturationtest:

Ex eriment Observation Inference

Take 2ml of proteinsolutionand a)Violetcolouris seen a)Protein are not
add 2m] of saturatedammonium precipitated by half
sulphate solution.Mix well and saturationtest
leave for 5 min. Filter and perform Ex: Albumin
Biuret test with 2m! Of the filtrate,
add Iml of 40%Naom & 2drops b) Violet colour not b)Proteins are precipitated
of 1%CuS04Solution.Mixwell seen by half saturationtest. Ex:
Casein
b) Full Saturation Test :

Ex eriment Observation Inference

Violet colour not Proteins are precipitated
Add ammoniumsulfatecrystals seen by full saturationwith
with mixing till solution is Ammonium sulfate. Ex:
saturated.(Thereshouldbe some Albumin
un-dissolved salt crystals at the
bottomof the test tube). Leave
for 5min. Filter & perform Biuret
test with the filtrateusing40%
NaOHand 2 dropsof 1%Cus04.
Mix well

Principle:Ammoniumsulphateleadsto dehydrationandprecipitation
Ofproteins(SALTING
OUT). The concentrationof the salt required to precipitate the
protein depends upon
the molecularweightof the proteins.Higherthe molecularweightof
the protein
lower the salt concentrationrequired to precipitatethe proteinand
vice versa.
Significance:This propertycanbe usedfor separationofalbumin& casein
 41

COAGULATION TEST :
11. HEAT

Ex criment Observation Inference

Take of given protein a) Coagulum is a) Presence of heat
solutionin a test tube. Hold the seen in the upperpart of coagulable proteins
test tube in a slanting position and the Solution Ex: Albumin
boil upper pan of the solution. b) Coagulum is not b) Presence of non
seen coagulable protein. Ex:
casein
Add 10 acetic acid drop Turbidity' intensifies Presence of heat
wise.(only if coagulum is seen) coagulable protems
confirmed.
Lower part serves as a control.

principle: On heating, proteins are denatured. The denatured proteins are less soluble and hence
forma thick, semisolid viscous material called coagulum. On addition of dilute acetic acid, there
is a decrease in pH which promotes further coagulation.
Significance: This test is commonly done to detect the presence of albumin in Urine.

111. PRECIPITATION BY ORGANIC SOLVENTS:

Ex eriment Observation Inference

To 2mI of protein solution White precipitateis formed Proteins are precipitated
adds 2ml ethanol. Mix well by organicsolvents
& let it stand

Principle: Addition of organic solvents, such as acetone & alcohol decreases the dielectric
constant & displaces water molecules associated with proteins. This precipitates proteins.

IV. PRECIPITATION BY ALKALOIDAL REAGENTS:

a) Precipitation by sulphosalicylicacid:
Ex eriment Observation Inference
To 2mI of protein solution Whiteprecipitateis seen Proteins are precipitated
add 3-4 drops of by alkaloidalreagents
sulphosalicylic acid.
42

b) Precipitationby Picricacid
Eyp€rirnent Observation Inference
To 2ml of protein solution Yellow precipitateis seen Proteins are precipitated
add 3-4 dropsof picricacid. by alkaloidalreagents

c) Precipitation by Trichloroacetic acid:

Ex eriment Observation Inference
To 2m! of protein solution Whiteprecipitateis seen Proteins are precipitated
add 3-4 drops of TCA by alkaloidalreagents

Principle: Proteins are positively charged at acidic pH which is neutralized by negatively

charged anions of alkaloidal reagents (like phosphotungsticacid, phosphornolybdicacid,
Trichloroaceticacid &sulphosalicylicacid) & hence are precipitated.

V. PRECIPITATION BY SALTS OF IIEAVY METALS:

Ex eriment Observation Inference

To 2ml of protein solution White precipitate is seen Proteins are precipitated
add 5-10 drops of 10% by heavy metals like
mercuric sulphate (Hg SO*' mercury.
& mix well
To Iml of protein solution Whiteprecipitateis seen Proteins are precipitated
add 5-10 drops of lead by heavy metals like lead.
acetate solution and mix well

Principle: Proteins are negatively charged in alkalinepH, whichare neutralizedby positively

charged cations of heavy metal salts & hence are precipitated.
43

3. REACTIONS FOR PROTEINS

General Jest for Proteins

TEST.
Eyperiment
Test: To 2ml of protein solution Observation
Violet colour is seen
Inference
add 2ml of 10% NaOH and 2 drops Proteins present
of 1% CuS04 Mix well (presence of peptide
linkages)
Control: To 2m! of water add 2mI
of 10%NaOHand 2 drops of 1%
CuS04 Mix well Bluecolouris seen Proteinsabsent

principle: In alkaline medium, cupric ions

chelates chelates (attachmentof metallic ions to
nonmetallic ions, by covalent bonds)
with peptide linkage in proteins to give the
violet color
The name of the reaction is derived
from the organiccompoundbiuret, which is
formed by the condensation of two urea molecules
at high temperature.When biuret
is treated with dilute CuS04 in an alkalinemedium,a violet color is obtained.
Significance: General test for Proteins and also can be used for quantitativeestimation and
identification of proteins.

COLOUR REACTIONS FOR PROTEINS

1. XANTHOPROTEIC TEST:

• To 3ml of protein solution • A whiteprecipitateis formed Indicatesthe presence

add Iml of conc. HN03. of Aromatic amino
Boil the above solution for • Yellow coloursolutionis seen acids (tyrosine &
I min tryptophan)
Add excess of 40% NaOH. • Yellow colourchangesto orange
Mix well.

Principle: This test is specific for aromatic amino acids tyrosine & tryptophan.The benzene ring
undergoes nitration at high temperature & gives yellow colored nitro benzene. The colour
changes to orange when the solution is made alkaline.
Note: phenylalanine does not answer this test.
44

11. MILLON'S TEST:

coagulum in Indicates the
To 2ml of protein solution, add Deep red presence
red solution is seen. phenol group
2mI of 10% HgS04 (Millons faint
amino acid (tyrosine)
contain'
Reagent) Heat it to boil, cool it.
Add 3-4 drops of SodiumNitrite
solution.

Principle: Phenol group of Tyrosine present in proteins will give a red color, due
formationof nitrated mercuricphenolateions.

111. SAKAGUCHI TEST.

To 2ml of protein solution, add Brightred colouris seen. Indicates the presence
2drops of —napthol (molisch's Guanido group con
taming
reagent), add 8-10 drops of sodium amino acid (Arginine).
hypobromite

Principle: In alkaline medium, guanido group Of arginine. reacts with (1 —napthol& sodium
hypobromite to give red colour complex.

IV. ALPEUYUE-I.ESJ-(HOPKINCOLESTEST)

To Iml of protein solution,add I Violet ring is seen at the Indicates the presenceof.
drop of I :500 formaldehyde, add I junction of two liquids Indole group containingamino
drop of 10% HgS04. Mix well acid (Tryptophan).
then add Iml conc H2S04 along
the sides of test tube, holdingthe
test tube in slanting position

Principle: This reaction is due to the presence of indole ring of tryptophan. H2S04with
HgS0:
oxidizes the indole ring of tryptophan.AIdehydesreact with oxidized products
of theind0iE
nucleus to give a violet colour complex.

V. SULPHUR TEST: (LEAD ACETATE TEST)

To 2ml of protein solution, add Blackprecipitateis seen

Indicates the presence
2ml of 40% NaOH & boil for
sulphur containing aminoacid
2min, cool it, then add 2-3 drops
cysteine or Cystine.
of lead acetate
45

principle: when cystein or cystine amino acids are boiled with a strong alkali, organic sulphur is
converted to sulphidc. Addition of lead acetate gives black colored precipitate of lead sulphide.
Note: a. This test is answered by albumin. Casein does not answer this test as it does not contain
cysteine or Cystine.
b. Methionine does not answer th sulphur test as methionine does not contain free sulphur
group.
VI. PAULY'S DIAZO TEST
To 0.5ml Of 0.5% Sulphanilic Cherry red colour seen Indicates the presence of
acid, add 0.5mI of 1% sodium imidazole group containing
nitrite. Mix it and wait for amino acid (histidine).
Imin. Add Iml of protein soln,
mix it and add 1m! 10%
sodium carbonate.

principle: Diazotized Sulphanilic acid reacts with imidazole ring of histidine to form a cherry
red coloured diazotized product under alkaline conditions.
Questions:

1. Define proteins. What are they made up of?

2. (a) Name the various agents used to precipitate proteins. Give two examples for each.
(b)What is the principle of precipitation of proteins by each agent?
3. What is denaturation? Name any two denaturation agents.
4. Name two heat coagulable proteins.
5. What is the clinical significance of heat coagulation test?
6. Name four color reactions of proteins.
7. Name the amino acids which answer (a)Xanthoproteic Test (b)Sakaguchi's test
(c)PauIy's test
8. Name (a) Aromatic Amino Acids (b) Sulphur containing amino acids.
9. Why sulphur test does not answer for methionine?
10. Name the amino acid with imidazole ring. Which test you will do to detect it.
11. What is the isoelectric pH of casein?
12. Name the test to detect phosphorus in casein.
13. Name the color reaction not answered by (a) Casein (b) Gelatin.
14. Why does egg albumin answer Molish's test?
15. Why is the test for protein called as biuret test?