COLOUR REACTIONS OF PROTEINS (AMINOACIDS)

Proteins are complex, specialized molecules composed of carbon, hydrogen, oxygen, and
nitrogen. Many proteins also contain sulfur. The building blocks of proteins are the amino
acids. Proteins Produce Colour in certain reactions.

1. Ninhydrin Test:

Amino acids react with ninhydrin (triketohydrindene hydrate) at pH=4. The reduction
product obtained from ninhydrin then reacts with NH 3 and excess ninhydrin to yield a

blue colored substance. This reaction provides an extremely sensitive test for amino
acids.
Many substances other than amino acids, such as amines will yield a blue color with
ninhydrin, particularly if reaction is carried out on filter paper.

WARNING: Avoid spilling ninhydrin solutions on your skin, as the resulting stains are
difficult to remove.

Procedure:

To 1ml solution add 5 drops of 0.2% ninhydrin solution in acetone. Boil over a water bath
for 2 min. Allow to cool and observe the blue color formed. Test the vapor from each
boiling tube with wet litmus paper.

2. Biuret Test

The biuret test is a chemical test used for detecting the presence of peptide bonds. In a
positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the
nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color
indicates the presence of proteins.
It is possible to use the Biuret reaction to determine the concentration of proteins because
(for most proteins) peptide bonds occur with approximately the same frequency per gram
of material.

Procedure
To 3 ml of protein solution add 1ml of 5% NaOH solution. Mix well and then add few
drops of 1% copper sulphate solution. Note for the formation of Violet colour.

3. Specific Reactions for Individual Amino Acids

a-Xanthoproteic Test

Amino acids containing an aromatic nucleus form yellow nitro derivatives on heating
with concentrated HNO3. The salts of these derivatives are orange in color.

Procedure:

To 2ml of solution in a boiling test tube, add an equal volume of conc. HNO 3. 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.

b-Millon’s Test

Phenols only give this reaction; tyrosine is the only common phenolic amino acid.
Millon’s reagent is concentrated HNO3, in which mercury has been dissolved.

A yellow precipitate of HgO in a test is NOT a positive reaction but usually indicates that
the solution is too alkaline.

Procedure:

To 2ml of amino acid solution in a test tube, add 1-2 drops of Millon’s reagent. Warm the
tube in a boiling water bath for 10min. A brick red color is a positive reaction.
Note that this is a test for phenol, 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 will react with glyoxylic acid in the presence of
concentrated H2SO4 to give a purple color. Glacial acetic acid, which has been exposed

to the light, always contains glyoxylic acid CHOCOOH as an impurity.

Procedure:
To a few ml of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino
acid solution. Pour 1-2ml concentrated 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 Sulfite Test for Cysteine

Some of sulfur in cystine, is converted to sodium sulfide by boiling with 40% NaOH.
The Na2S can be detected by the precipitation of PbS from an alkaline solution. In order

to apply this test, you should first prepare the sodium plumbate solution.

Preparation of Sodium Plumbate:

Add 5ml of dilute NaOH to 2ml dilute lead acetate. A white precipitate of lead hydroxide
will form. Boil until the precipitate dissolves with the formation of sodium plumbate.

Procedure:

Boil 2ml of 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.

Procedure:

To 0.5ml of the amino acid solution add 2ml Ehrlich reagent. Observe color changes

g-Nitroprusside Test:

Thiol groups give a red color with sodium nitroprusside in the presence of excess
ammonia.

Procedure:
-Add 2ml of the amino acid solution into test tubes. Add 0.5ml fresh sodium nitroprusside
solution and shake thoroughly. Add 0.5ml ammonium hydroxide. Observe color changes
 Precipitation of Proteins

1- Heler’s Test

This test is used to detect the presence of proteins in urine. When concentrated acids
reacts with protein, denaturation of proteins takes place. Concentrated acids can even
detect low quantities of proteins. This test is widely used to detect the presence of
proteins in Urine Samples.

Procedure

To 3 ml of concentrated nitric acid, add 1ml of protein solution along the sides of the test
tube. Observe the formation of precipitate.

2-By Neutral salts:

Ammonium sulfate is commonly used for the precipitation of different types of proteins.
The concentration of the salt required to cause precipitation varies with the nature of the
protein and the pH of the solution.

Procedure:
Add solid ammonium sulfate to about 5ml of protein solution in a test tube. The salt
should be added in quantities of approximately 1g at a time. Agitate the solution gently
after each addition to dissolve the ammonium sulfate.

3-By salts of heavy metals:

The heavy metal cations Cu, Ba, Pb and Hg precipitate proteins particularly from weakly
alkaline solutions.
Procedure:

Treat 3ml of the protein solutions provided with a few drops of mercuric nitrate. A white
precipitate should form.

4-By acidic reagents:

The precipitation here 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.

Procedure:

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

5- By Alcohol:

Precipitation of proteins can also be done by the addition of alcohols. The extent of
precipitation will be low when compare to other reagents.

Procedure

To 3 ml of protein solution add 2ml of Ethanol. Observe the formation of Precipitate

6- By Heat

Proteins undergo denaturation when they are heated. It’s a very simple test to detect the
presence of proteins.

Procedure
Take 3ml of protein solution in a test tube. Heat the test tube under flame in a slanting
position. Observe the formation of coagulum.