Bicol University

College of Nursing
Legazpi City
S.Y 2015-2016

Research No. 5
(Laboratory)

Test for
Amino Acids &
Proteins
Submitted by:
Mary Grace V. Fuentes
BSN1-B
Submitted to:
Mrs. Noemi Madrid

Qualitative tests of amino acids

Amino acids are molecules containing an amine group, a carboxylic acid

group and a side chain that varies betwen different amino acids. Amino acids

of the general formula RCH(NH2)COOH are amphoteric, behaving as amines

in some reactions and as carboxylic acids in others. At a certain pH known as
the isoelectric point an amino acid has no overall charge, since the number of
protonated ammonium groups (positive charges) and deprotonated
carboxylate groups (negative charges) are equal. Since the amino acids at
their isoelectric points have both negative and positive charges, they are
known as zwitterions.
Amino acids are critical to life. They have particularly important functions like
being the building blocks of proteins and being the intermediates in
metabolism. Amino acids are generally classified by the properties of their
side chain into four groups. The side chain can make an amino acid a weak
acid or a weak base, and a hydrophile if the side chain is polar or a
hydrophobe if it is nonpolar.
Proteins (also known as polypeptides) are organic compounds made of amino
acids arranged in a linear chain. The amino acids in a polymer are joined
together by the peptide bonds between the carboxyl and the amino groups of
adjacent amino acid residues.
Like other biological macromolecules such as polysaccharides and nucleic acids,
proteins are essential parts of organisms and participate in virtually every
process within cells.
Proteins are important in:
- catalyzing biochemical reactions (enzymes)
- structural and mechanical functions (actin and myosin)
- cell signaling
- immune responses
- cell adhesion
- cell cycle
A. Solubility test.
B. The presence of proteins in a solution is often detected by general tests, such as
biuret or specific tests that depend on the presence of a specific amino acid.
1)
2)
3)
4)
5)
6)
7)

1)

Ninhydrin test: for -L amino acids

Xanthoproteic test: for Aromatic amino acids
Lead sulfite test: detection of amino acids containing sulfhydral group (- SH)
Millon's test: for amino acids containing hydroxy phenyl group
Sakaguchi Test.
Hopkins-Cole (Glyoxylic Acid Reaction)
biuret test

SOLUBILITY TEST

The solubility of amino acids and proteins is largely dependent on the solution pH.
The structural changes in an amino acid or protein that take place at different pH
values alter the relative solubility of the molecule. In acidic solutions, both amino

and carboxylic groups are protonated. In basic solutions, both groups are
deprotonated.
Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali
and dilute acid vary from one compound to the other depending on the structure of
their side chains. Apply this test to glycine, tyrosine, glutamic acid and cysteine.
Procedure:
Note the solubility of amino acids in water and alcohol by placing a
small amount in a test tube, adding a few mL of solvent and warming if
necessary.
Determine the amino acid solution is acidic or basic by using a litmus
paper while testing the solubility in water.
Repeat the solubility test using dilute HCl and dilute NaOH.
2)
Ninhydrin Test:
Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammonia
or primary and secondary amines.
Amino acids also react with ninhydrin at pH=4.
The reduction product obtained from ninhydrin then reacts with NH3 and
excess ninhydrin to yield a blue colored substance.
This reaction provides an extremely sensitive test for amino acids.
Apply this test to any of the amino acids you choose.
WARNING: Avoid spilling ninhydrin solutions on your skin, as the resulting stains
are difficult to remove. (Ninhydrin is the most commonly used method to detect
fingerprints, as the terminal amines or lysine residues in peptides and proteins
sloughed off in fingerprints react with ninhydrin).
Procedure:
To 1 mL amino acid solution add 5 drops of 0.2% ninhydrine solution in
acetone.
Boil over a water bath for 2 min.
Allow to cool and observe the blue color formed.
3)
Stability to Alkali:
Amino acids, unlike amides and volatile amines, do not evolve NH3 or alkaline vapor
when boiled with alkali. This method can be used to differentiate amino acids from
amines and amides. Apply this test to the provided amine or amide and also to
glycine.
Procedure:
Pipette 1 mL 1% glycine and the amide or amine solution into separate
test tubes.
Add 1 mL dilute NaOH to each test tube and boil.
Test the vapor from each boiling tube with wet litmus paper.

4) Specific Reactions for Individual Amino Acids:

WARNING: Please DO NOT use vast amounts of solution for these tests,
since most of the amino acids are very expensive!!

a) Xanthoproteic Test:
Some amino acids contain aromatic groups that are derivatives of benzene. These
aromatic groups can undergo reactions that are characteristics of benzene and
benzene derivatives. One such reaction is the nitration of a benzene ring with nitric
acid. The amino acids that have activated benzene ring can readily undergo
nitration. This nitration reaction, in the presence of activated benzene ring, forms
yellow product.
Apply this test to tyrosine, tryptophan, phenylalanine and glutamic acid.
Procedure:
To 2 mL amino acid solution in a boiling test tube, add equal volume of
concentrated HNO3.
Heat over a flame for 2 min and observe the color.
Now COOL THOROUGHLY under the tap and CAUTIOSLY run in sufficient
40%
NaOH to make the solution strongly alkaline.
Observe the color of the nitro derivative of aromatic nucleus.
b) Millons Test:
Millons test is specific to phenol containing structures (tyrosine is the only common
phenolic amino acid). Millons reagent is concentrated HNO3, in which mercury is
dissolved. As a result of the reaction a red precipitate or a red solution is considered
as positive test. A yellow precipitate of HgO is NOT a positive reaction but usually
indicates that the solution is too alkaline. Apply this test to tyrosine, phenylalanine,
glycine and -naphtol.
Procedure:
To 2 mL amino acid solution in a test tube, add 1-2 drops of Millon2s
reagent.
Warm the tube in a boiling water bath for 10 min.
A brick red color is a positive reaction.
Note that this is a test for phenols, and the ninhydrin test should also
be positive if it is to be concluded that the substance is a phenolic
amino acid.
c) Hopkins Cole Test:
The indole group of tryptophan reacts with glyoxylic acid (glacial acetic acid, which
has been exposed to light, always contains glyoxylic acid CHOCOOH as an impurity)
in the presence of concentrated H2SO4 to give a purple color. Apply this test to
glycine, tryptophan and tyrosine.
Procedure:
To a few mL of glacial acetic acid containing glyoxylic acid, add 1-2
drops of the amino acid solution.
Pour 1-2 mL H2SO4 down the side of the sloping test tube to form a
layer underneath the acetic acid.
The development of a purple color at the interface proves a positive
reaction.
d) Lead-Sulfide Test:
When cystine is boiled with 40% NaOH, some of sulfur in its structure is coverted to
sodium sulfide (Na2S). The Na2S can be detected by using sodium plumbate

solution which causes the precipitation of PbS from an alkaline solution. In order to
apply this test, first the sodium plumbate solution should be prepared. Apply this
test to cysteine and cystine.
Procedure:
Sodium Plumbate Solution Preparation:
Add 5 mL dilute NaOH to 2 mL dilute lead acetate.
A white precipitate of lead hydroxide forms.
Boil until the precipitate dissolves with the formation of sodium
plumbate.
Boil 2 mL amino acid solution with a few drops of 40% NaOH for 2 min.
Cool and add a few drops of the sodium plumbate solution.
A brown color or precipitate is a positive test for sulfides.
e) Ehrlich Test:
Aromatic amines and many organic compounds (indole and urea) give a colored
complex with this test. Apply this test to tryptophan, urea and glycine.
Procedure:
Put 0.5 mL of the amino acid solution to a test tube.
Add 2 mL Ehrlich reagent and observe the color changes.
Repeat the test with urea solution.
f) Sakaguchi Test:
The Sakaguchi reagent is used to test for a certain amino acid and proteins. The
amino acid that is detected in this test is arginine. Since arginine has a guanidine
group in its side chain, it gives a red color with -naphthol in the presence of an
oxidizing agent like bromine solution. Apply this test to arginine.
Procedure:
1 mL NaOH and 3 mL arginine solution is mixed and 2 drops of naphthol is added.
Mix thoroughly and add 4-5 drops of bromine solution UNDER THE
HOOD!!
Observe the color change.
g) Nitroprusside Test:
The nitroprusside test is specific for cysteine, the only amino acid containing
sulfhydryl group (-SH). This group reacts with nitroprusside in the presence of
excess ammonia. Apply this test cysteine, cystine and methionin.
Procedure:
Put 2 mL amino acid solution into the test tube.
Add 0.5 mL nitroprusside solution and shake thoroughly.
Add 0.5 mL ammonium hydroxide.
Observe the color change.

4)

Sulfur Test

Sulfur containing amino acids, such as cysteine and cystine upon boiling with
sodium hydroxide (hot alkali) yield sodium sulfide.
This reaction is due to partial conversion of the organic sulfur to inorganic
sulfide, which can detected by precipitating it to lead sulfide, using lead acetate

solution.

5)

S.(protein) + 2NaOH-------- Na2S

Na2S + (CH3COO)2pb ------- PbS + 2CH3COONa
Procedure:
Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine
and 0.1 M cysteine into separate, labeled test tubes.
Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 %
lead acetate solution.
Stopper the tubes and shake them. Remove the stoppers and heat in
a boiling water bath for 5 minutes. Cool and record the results

Nitroprusside Test

The nitroprusside test is specific for cysteine, the only amino acid containing
sulfhydryl group (-SH). This group reacts with nitroprusside in the presence of
excess ammonia.
Procedure:
Added 0.5ml nitroprusside solution into 2ml of amino acid solution
in test tube and shake thoroughly.
Add 0.5ml ammonium hydroxide.
Heat the test tube in water bath for 10-12 min and observe the
change in color.
Result No Red color is appeared I think nitroprusside solution is not
accurate

6)

Paulys Test
Procedure: Cool 2ml of test solution added 1ml sulfonic acid followed by
NaNO2 solution and cold it for 3 mint. Add 2 ml of Na2CO3 and noted the
color changes

7)
Lead acetate test
When sulphur containing amino acid is boiled with NaOH, some of sulfur in its
structure is converted to sodium sulfide .The Na2S can be detected by using
sodium plumbate solution which causes the precipitation of PbS from an alkaline
solution.
Procedure:
Take 2ml amino acid solution added few drops of lead acetate and
boil it with a 1 ml of 40% NaOH for 2 min..
A black color or precipitate is a positive test for sulfides.
Result: Black precipitate appeared which shows positive result.

Tests for Proteins:

1) Biuret Test:
The Biuret Test positively identifies the presence of proteins (not less than two
peptides). The reaction in this test involves the complex formation of the proteins
with Cu2+ ions in a strongly alkaline solution. Apply this test to gelatin, casein and
albumin.
Procedure:
To 2 mL protein solution, add 5-6 drops of dilute CuSO4 (Fehlings
solution A diluted 1/10 with water)

Add 3 mL 40% NaOH solution.

Observe the color change.
If the protein tested is insoluble in water, then apply the procedure
given below:
Measure 3 mL acetone and 1.5 mL water into a test tube.
Add 1 drop of dilute NaOH and a little piece of protein to be tested.
Boil continuously over a small flame for 2 min and cool.
Add 0.5 mL 40% NaOH and 2 drops of a 1/10 diluted Fehlings solution
A.
Observe the color change.

2) Precipitation of Proteins:
The precipitation of a protein occurs in a stepwise process. The addition of a
precipitating agent and steady mixing destabilizes the protein solution. Mixing
causes the precipitant and the target product to collide. Enough mixing time is
required for molecules to diffuse accross the fluid.

I. By Neutral Salts:
The precipitation of a protein by neutral salt is commonly known as salting-out
method. Addition of a neutral salt, such as ammonium sulfate, compresses the
solvation layer and increases the protein-protein interaction. As the salt
concentration of a solution is increased, more of the bulk water becomes associated
with the ions. As a result, less water is available to take part in the solvation layer
around the protein, which exposes hydrophobic parts on the protein surface.
Therefore, proteins can aggregate and form precipitates from the solution. The
amount of neutral salt required to cause protein precipitation varies with the nature
of the protein and the pH of the solution. Apply this test to all the proteins provided.
Procedure:
Add solid ammonium sulfate to about 5 mL of protein solution in a test
tube (the salt should be added in quantities of approximately 1 g at a
time)
Agitate the solution gently after each addition to dissolve the
ammonium sulfate.
II. By salts of Heavy Metals:
Heavy metal salts usually contain Hg2+, Pb2+, Ag1+, Tl1+, Cd2+ and other metals
with high atomic weights. Since salts are ionic, they disrupt salt bridges in proteins.
The reaction of a heavy metal salt with a protein usually leads to an insoluble metal
protein salt. Apply this test to all the proteins provided.
Procedure:
Treat 3 mL of the protein solution provided with a few drops of mercuric
nitrate.
A white precipitate formation should be observed.
III. By Acid Reagents:
The precipitation of a protein in the presence of acid reagents is probably due to the
formation of insoluble salts between the acid anions and the positively charged
protein particles. These precipitants are only effective in acid solutions. Apply this
test to all the proteins provided.

Procedure:
Treat 3 mL of protein solution provided with a few drops of
trichloroacetic acid solution.
Note the protein precipitate formed.

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