Reactions of amino acids
1. Ninhydrin test– detection of amino acids
Reaction of amino acids with ninhydrin leads to their decarboxylation, deamination
(formation of CO 2 and ammonia) and formation of aldehyde which has one carbon atom less
in its structure. The ninydrin undergoes reduction:
O
O
OH
C
C
R CH COOH +
R C
H
C
+
C
NH3 +
H
OH
C
NH2
O
CO2 +
C
OH
O
O
Ninhydryna
Zredukowana ninhydryna
Ninydrin
Reduced ninydrin
The reduced ninydrin condenses with ammonia and non- reduced ninhydrin molecule
which leads to the formation of the violet-blue condensation product:
O
H
C
C
C
HO
+ NH3
OH
O
Zredukowana ninhydryna
Reduced ninhydrin
O
O
C
C
C
+
HO
O
C
CH
N
C
+ 3H 2O
C
C
C
O
O
O
Ninhydryna
Ninhydrin
Niebieskofioletowy produkt kondensacji
Violet-blue condensation product
Only proline and hydroxyproline- amino acids molecules which contain a secondary
amino group produce yellow condensation product.
The colour intensity is proportional to the concentration of ammonia from the amino
groups. That is why the reaction with ninhydrin is used for quantitative analysis of amino
acids and free amino groups in proteins.
Ammonium salts, amino sugars and ammonia also give positive ninhydrin test result. In
order to obtain the reliable quantitative results the test solution should not contain the
compounds mentioned above.
Experiment procedure
Add 3-4 drops of ninhydrin solution in acetone to l cm3 of glycine solution. Heat the
mixture in the water bath. The development of violet-blue colour confirms the presence of an
amino acid.
2. Deamination – reaction of an amino acid with HNO 2
When α-amino acids react with nitrous acid they undergo deamination with the
evolution of nitrogen molecule and formation of corresponding α-hydroxyacids:
COOH
H 2N
C
H
COOH
+
HO
N
HO
O
C
H
+
N2
+ H 2O
R
R
α-hydroksykwas
alpha
hydroxyacid
alphaα-aminokwas
amino acid
This reaction is used for gasometric determination of alpha amino acids ( Van Slyke’s
method)
Experiment procedure
Mix 2 cm3 of sodium nitrate (III) with 2 cm3 of 1 mole hydrochloric acid. The evolution of
yellow gas bubbles of strong, irritant smell (nitrogen oxides). Wait until the evolution of gas
bubbles stops and add 2 cm3 of 5% glycine. The evolution of nitrogen gas bubbles is
observed.
3. Detection of sulphur in cystine and cysteine
In a strong basic medium the sulphide ion (S2-) is eliminated from the thiol (–SH) group
of cysteine or disulphide group of cystine. The presence of this ion can be confirmed by
adding lead ions to the test solution (the black precipitate of lead sulphide is formed):
COOH2N
C
H
COO+ 2OH-
C
CH2
O
+
NH3
CH3
SH
Cysteina
Cysteine
Pb2+ +
S2-
PbS
+ H2O +
S2-
Experiment procedure
Add 2 cm3 of 20% of NaOH and some drops of lead (II) acetate to l cm3 of 1% cysteine
or cystine solution. Place the test tube in the water bath for 2-3 minutes. Observe the
formation of black precipitate.
4. Xanthoproteic reaction of aromatic amino acids
Treating aromatic amino acids with the concentrated nitric (V) acid leads to the nitration
of the aromatic ring and formation of yellow nitro-products (nitro derivatives). When the
strong basic solution is added the colour of obtained products turns darker (from yellow to
orange). The positive result of xanthoproteic reaction gives: tyrosine, tryptophan and
phenylalanine (only after extended heating time).
COOH
H2N
Tyrosine
H2N
CH
CH2
COOH
H2N
+
HNO3
CH
CH2
NO2 +
OH
OH
COOH
COOH
CH
CH2
OH
H2N
+
2 HNO3
O2N
CH
CH2
H2O
NO2 + 2 H2O
OH
Experiment procedure
Add 0,5 cm3 of concentrated nitric (V) acid to l cm3 of tyrosine or tryptophan solution.
After heating the test solution turns yellow. After the reaction mixture has cooled down, add l
cm3 of 30% NaOH - the colour will turn orange.
5. Detection of tryptophan - Adamkiewicz–Hopkins’ test
The compounds that have indole ring can condense with aldehydes (more readily with
formic aldehyde) to form colourful condensation products. Among protein amino acids, only
tryptophan undergoes this reaction:
COOH
H2N
CH
CH2
+ H
N
H
Tryptofan
tryptophan
COOH COOH
COOH
HC
H2N
CH
H +
Formaldehyd
formaldehyde
N
H
Tryptofan
tryptophan
HC
N
NH2
CH2
CH2
CH2
O
C
NH2
CH2
N
+ H2O
H
H
Barwny produkt
kondensacji
Colourful
condensation product
Experiment procedure
Add some drops of formalin (40% solution of formic aldehyde in water) to l cm3 of
tryptophan solution. Mix the test tube contents and cautiously introduce down the side of the
tube l cm3 of concentrated sulphuric acid. A purple ring appears between the two layers if the
test is positive for tryptophan.
6. Detection of peptide bond – biuret test
When the diluted solution of copper (II) sulphate and some drops of strong base solution
are added to the solution of a protein or a peptide, the violet colour appears. This reaction
occurs only if the peptide molecule contains at least two peptide bonds (3 amino acids). Free
amino acids and dipeptides do not undergo this test. The false positive test result can also be
observed when diamides of oxalic, malonic and succinic acid are present.
The biuret test generates complex compounds in which copper atom binds to nitrogen
atoms of a peptide chain by coordinate bond. The biuret test is used for quantitative
determination of high protein concentration in serum and other biological fluids as well ( 110 mg/cm3). Determination of lower levels of proteins requires more sensitive methods.
Experiment procedure
Add 1 drop of copper (II) sulphate solution to 1 cm3 of a protein solution, then add 1
cm3 of 2 moles NaOH solution. Observe development of violet colour of test solution.