Practical Proteins and Amino Acids` Identification

Amino Acid + Amino Acid + Amino Acid
Peptide linkage
Proteins (many peptide linkages)
Proteins
Amino Acids
Casein
Cysteine
Gelatin
Arginine
Albumin
Tryptophan
Peptone
Tyrosine
Cysteine
Tyrosine
Arginine
Tryptophan
Tests to be done:
Ninhydrin’s test (specific to all Proteins & Amino acids)
Biuret’s test (specific to Proteins)
Sakaguchi’s test (specific to Arginine)
Nitropruside’s test (specific to Cysteine)
Millon’s test (specific to Tyrosine)
 Hopkins-Cole’s test (specific to Tryptophan)
Ninhydrin’s
test
Ninhydrin reaction
Principle:
Ninhydrin is Specific for Amino Acids & Proteins – to differentiate between
Carbohydrates (-ve) and Amino Acids & Proteins (+ve).
Ninhydrin Reacts with α-amino acids (–NH2) in proteins giving a purple colored
complex, except Proline and hydroxy proline gives yellow color(no –NH2).
Ninhydrin is most commonly used as a forensic chemical to detect
“fingerprints”, as amines left over from proteins sloughed off in
fingerprints react with ninhydrin giving a characteristic purple color.
Procedure & observation:
− To 1 mL amino acid solution in a test tube, add 2-3 drops of ninhydrin reagent.
- Put in a boiling water bath and observe the formation of a purple color.
Ninhydrin’s
test
2-3 drops of B
Mix
Mix
Heat
2-3 min
1ml of
solution A
C
Result
Unknown
A
Proteins or Amino Acids & Carbohydrates
Reagent
B
Drops of Ninhydrin reagent
Observation
C
Dark purple colour on heat
Proteins or Amino Acids
(free –NH2)
C
No reaction (no change in colour)
Carbohydrates
Biuret’s test
Principle:
Biuret test is Specific for Proteins – To differentiate between Proteins (+ve) and
Amino Acids (-ve). The biuret reagent (copper sulfate in a strong base) reacts with
peptide bonds in proteins to form a violet complex known as the “Biuret
complex”. Two peptide bonds are at least required for the formation of this
complex , this is why amino acids give negative results with Biuret test.
Procedure & observation:
− To 1 mL of protein solution (Albumin – Casein – Gelatin – Peptone) in a test
tube, add 1 mL of 10% sodium
hydroxide solution and 2-3 drops of 1% copper sulfate solution.
− Mix well; a violet color is obtained with albumin, casein & gelatin and a
pinkish violet color with peptone.
Biuret’s test
2-3 drops of CuSO4
1ml of NaOH
Mix
Mix
1ml of
solution A
C
Result
Unknown
A
Proteins or Amino Acids
Reagent
B
2 ml NaOH, then 2 drops of
CuSO4 (1%)
Observation
C
Violet colour
Proteins (reacts with Di-peptide bonds)
C
No change in colour (Blue)
Amino Acids (No di-peptide linkage)
Sakaguchi’s test
Principle:
Sakaguchi test is Specific for free Arginine or proteins containing
Arginine.
Sakaguchi's test is positive for the amino acid containing the guanidine
group in Arginine. Guanidine group present in the amino acid reacts with
α-Naphthol and alkaline hypobromite to give red-coloured complex.
Procedure & observation:
− To 1 mL of protein solution in a test tube, add 40%NaOH (2-3 drops) +
Ethanolic a-Naphthol (2-3 drops) and Bromine water (2-3 drops)
− Mix well; a red-colour complex will be formed with Arginine or protein
containing Arginine.
Sakaguchi’s
test
B
Mix
Mix
1ml of
solution A
C
Result
Unknown
A Proteins or Amino Acids
Reagent
B 2-3 drops 40%NaOH, then 2-3 drops alcoholic
α -Naphthol, mix then
• add 2-3 drops Bromine water or
• (for safety, use Chlorine water)
Observatio
n
C Red colour
Proteins containing Arginine
C Red colour
Arginine (Guanidine gp)
Nitroprusside’s test
Principle:
It is specific for Amino Acids or Proteins containing sulfur , -SH (in
cysteine & cystine) gives a red-purple colour called “Mörner test”.
Procedure & observation:
− To 1 mL of protein solution containing Cysteine/Cystine in a test tube,
add 3 drops of a 5% solution of sodium nitroprusside
- Mix well and add few drops of ammonia solution, a deep red-purple
color appears; called also Mörner test.
l
Nitroprusside’s
test
B
Mix
Mix
1ml of
solution A
C
Result
Unknown
A
Cystine or Cysteine in
Proteins or Amino Acids
Reagent
B
3 drops Sodiun nitropruside,
Then, 3-5 drops Ammonia
soln.
Observation
C
Deep Red – purple colour
Amino Acid: Cystine or Cysteine (-SH gp)
C
Deep Red – purple colour
Proteins containing Cystine or Cysteine (-SH gp)
Millon’s test
Principle:
It is specific for Tyrosine. Millon's reagent (Hg/HNO3) gives positive
results (red – pink colour appears) with proteins containing the phenolic
amino acid “tyrosine”.
Procedure & observation:
− To 1 mL of protein solution in a test tube, add 1ml of Millon’s reagent
[Hg(SO4) + HNO3]
- Heat up for few min
− A red – pink colour appears
Millon’s test
1 mL of B
Mix
Heat
Mix
2-3 min
1ml of
solution A
C
Result
Unknown
A
Tyrosin in Proteins or Amino
Acids
Reagent
B
1 mL Millon reagent [Hg(SO4) +
HNO3] then heat
Observation
C
Red-Pink colour
Amino Acid: Tyrosin (-Ph-OH gp)
C
Red-Pink colour
Proteins containing Tyrosin (-Ph-OH gp)
Aldehyde test
Hopkin-Cole
Principle:
It is Specific for Tryptophan amino acid, whether it is free or in proteins.
Sulphuric acid in presence of mercuric sulphate oxidizes the indole nucleus of
tryptophan. The product formed reacts with aldehydes to form violet-purple
colored complex (ring).
Procedure & observation:
− To 1 ml of protein solution in a test tube, add 2 drops of [0.2 of Formalin + 1
drop of 10% HgSO4] and mix well.
− Incline the test tube and slowly add 2 ml of concentrated H2SO4 on the inner
wall of the test tube to form violet-purple ring at the junction of the 2 layers.
− A reddish violet ring is formed at the junction between the 2 layers with albumin
and casein; gelatin gives negative results.
Aldehyde test
Hopkin-Cole
2 drops of [Formalin + HgSO4]
2 mL of H2SO4
On the side wall
Mix
Tryptophan
Mix
Wait 1 min
1ml of
solution A
C H2SO4
Result
Unknown
A
Tryptophan in Proteins or
Amino Acids
Reagent
B
• 2 drops [Formalin + HgSO4]
• then add 2 mL conc H2SO4
Observation
C
Violet/Purple colour ring
Amino Acid: Tryptophan (Indole ring)
C
Violet/Purple colour ring
Proteins containing Tryptophan (Indole ring)
Some additional tests for qualitative
identification of proteins
A) Heat coagulation test
Place about 5 ml of egg-white solution (albumin solution) in a
test tube and heat the top part of the solution only. Note that it
gradually becomes cloudy and a flocculent precipitate of
coagulated protein is produced.
D) Picric acid test
To 3 ml of gelatin solution in a test tube, add 2ml of saturated
picric acid solution, a yellow gelatinous precipitate is formed.
Proteins & Amino Acids
Procedure
Observation
Unknown
1- Ninhydrin test
Sample solution (l ml) + Ninhydrin reagent (2-3 drops). Observe on
Cold or if not, Boil in a water bath for (5 min) and Cool
Purple colour is
formed
All A.A. having –NH2
except proline &
hydroxyproline
2- Biuret test
Sample solution (1 ml) + Biuret reagent (1 ml)
(Better to warm it)
Violet colour is formed
All Proteins (not
A.A.) as it needs at
least 2 peptide
bonds
3- Millon's test
Sample solution (1 ml) + 1 mL Millon reagent [HgS04 reagent 0.5 ml) +
0.5 ml 2M HNO3]
Boil in water bath for (2 min) and Cool it.
Red colour/precipitate
is formed
Tyrosin
contains a phenolic
group
4- Nitroprusside's test (Mörner test)
Sample solution (1 ml) of protein/Amino acid containing Cysteine
Add (3) drops of sodium nitroprusside
Add (3-5) drops of ammonia solution
Deep dark purple red
colour called also
Cysteine or Cystine
Contain –SH group
5- Sakaguchi's test
Sample solution (1 ml) + 2-3 drops of Sakaguchi reagent [40%NaOH (4
drop) + Ethanolic a-Naphthol]
Add Bromine water (1-2) drops [for safety, use chlorine water]
Red colour is formed
Arginine
Contains guanidine
group
6- Hopkin-Cole test (Aldehyde-test)
Sample solution (1 ml) + 2-3 drops of Hopkin-Cole reagent [Diluted
Formalin (2 drops) + 10% Mercuric sulphate (4 drops)] – Mix well
Add Conc. H2S04 on the side of test tube (1-2) ml
A violet or purplecoloured ring appears
at the junction of two
layers
Tryptophan
Contains Indole
group
Mörner test
Identification of unknown protein solution
Biuret’s Test
Violet Color
Solution is protein
No Violet Color
Solution is not protein
(carbohydrates or amino acids)
Heat coagulation test
Cloudness at top part
of the solution
(Albumin)
No Cloudness at top part
of the solution
(Casein-Gelatin-Peptone)
Reaction to litmus paper
Neutral
Soln. is (peptone or Gelatin)
Alkaline
Soln. is (Casein)
Picric acid Test
No yellow gelatinous ppt.
Soln. is (Peptone)
Yellow gelatinous ppt.
Soln. is (Gelatin)
(Peptone gives pink color with biuret)
Identification of unknown Amino Acids
Biuret Test
Not Violet Color
Solution is not protein
(carbohydrates or amino acids)
Violet Color
Solution is protein
Molisch’s test
Violet ring
Carbohydrate
Nitroprusside’s
test
Cysteine
No violet ring
Amino Acids
(Cysteine – Tryptophan – Tyrosin - Arginine)
Hopkins-Cole’s
test
Tryptophan
Millon’s
test
Tyrosin
Sakaguchi’s
test
Arginine