Biuret Test

The Biuret Test is a general test for proteins. When a protein reacts with Copper (II) sulfate (blue), the positive
test is the formation of a violet colored complex.

Materials:

10% NaOH

0.5% Copper Sulfate

Egg albumin solution

Procedure:

To each of the test tubes, add 5 drops of 10% NaOH solution and two drops of dilute CuSO 4 solution while
swirling. The development of a purplish violet color is evidence of the presence of proteins.

Results:

An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most
often) followed by a few drops of aqueous copper (II) sulfate. If the solution turns purple, protein is present. 5–
160 mg/mL can be determined. A peptide of a chain length of at least 3 amino acids is necessary for a
significant, measurable color shift with these reagents.

Ninhydrin Test
The Ninhydrin Test is a test for amino acids and proteins with a free -NH2 group. When such an -NH2 group
reacts with ninhydrin, a purple-blue complex is formed.

Materials and Reagents:

Ninhydrin Reagent

Test Tubes

Materials for water bath

Procedure:

To each of the test tubes, add 5 drops of Ninhydrin reagent and heat the test tubes in a boiling water bath for
about 5 minutes.
Results:

Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 (carbon dioxide) through a series of
reactions; the net result is ninhydrin in a partially reduced form hydrindantin. Ninhydrin then condenses with
ammonia and hydrindantin to produce a blueish-purple pigment. The typical positive result is a blueish-
purple color. But, there is an amino acid that gives a yellow result. This amino acid is proline.

Xanthproteic Test

Phenyl rings containing an activating group can be nitrated producing a yellow product. The production of a
yellow colored product upon the addition of nitric acid is a test for the presence of tyrosine or tryptophan in a
protein. The addition of strong base will deepen the color to orange. The yellow stains on the skin caused by
nitric acid are the result of the xanthoproteic reaction.

Materials and Reagents:

Test tubes

Concentrated HNO3

Dropper

Watch glass

Hard boiled egg

Procedure:

To each of the test tubes, add 10 drops concentrated HNO3 while swirling. Heat the test tubes carefully in a
warm water bath. Additionally, add a couple of drops of concentrated nitric acid to a dry piece of hard boiled
egg white on a watch glass. Allow the acid to sit on the egg white for at least 5 minutes.

Results:

In the xanthoproteic acid test, albumin gives a distinctly orange color, whereas gelatin gives yellow. Gelatin
lacks tryptophan, and has tyrosine to produce a yellow color.
 Heavy Metal Ions Test

Heavy metal ions precipitate proteins from their solutions by cross-linking free amino groups and carboxylate
groups. Ions commonly used for testing for the presence of proteins include Zn2+, Fe3+, Cu2+, Sb3+, Ag1+, Cd2+,
and Pb2+. Among the metal ions, Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious damage to
proteins (especially enzymes) by denaturing them. Victims who have swallowed Hg 2+ or Pb2+ ions are often
treated with an antidote of a food rich in protein. The protein can combine with the mercury and lead ions and
minimize absorption of these ions. Milk and raw egg white are used most often. The precipitated protein
complexes are then immediately removed from the stomach by an emetic.

Materials and Reagents:

3 test tubes

Milk

Lead

Mercury

Procedure:

To each of 3 clean, labeled test tubes, add 2 mL milk. Add a few drops of each of the following metal ions to
the corresponding test tubes: Pb2+ as Pb(NO3)2 in test tube 1; Hg2+ as Hg(NO3)2 in test tube 2; and Na1+ as
NaNO3 in test tube 3. Dispose of the lead and mercury samples in the appropriate waste containers.

Results:

Heavy metal ions precipitate proteins from their solutions by cross-linking free amino groups and carboxylate
groups.

Millon’s Test

The reaction is due to the presence of the hydroxyphenyl group, C6H5OH in the amino acid molecule; and any
phenolic compound which is unsubstituted in the 3,5 positions such as tyrosine, phenol and thymol will give the
reaction. Solutions of nitric acid containing mercuric nitrate reacts with phenols, producing red colors or yellow
precipitates which react with nitric acid to form red solution. The reaction probably depends on the formation of
a nitro compound; which then reacts with phenol.

Materials and Reagent:

Millon’s reagent

Procedure:

Add 3 to 4 drops of Millon’s reagent to 5 ml of test solution. Mix and bring the mixture gradually to a boiling
point by heating over a small flame. Development of red color is due to the presence of protein. Excess of
reagent should however be avoided since it may produce a yellow color which is not a positive reaction.
 Millon-Nasse reaction

A test developed by Auguste Millon, a French chemist.

Materials and Reagents:

Millon – Nasse Reagent

Procedure:

Add 1 ml of Millon-Nasse reagent to 5 ml of test solution. Place the tube in a boiling water bath for 10 mins.
and cool the contents in water bath for 5 to 10 mins. and add 1 ml of 1% NaNO 2. A deep red color indicates
tyrosine or other 3,5 unsubstitued phenol.

Results:
It detects phenolic compounds. A reddish-brown coloration or precipitate indicates the presence of tyrosine
residue.

Hopkins-Cole reaction

The formation in this test color is due to the presence of indoyl group. Gelatin does not respond to this test due
to lack of amino acid tryptophan. Violet to blue colors develops when a mixture of protein and an aldehyde is
layered over conc. sulphuric acid. A number of tests based on this principle have been suggested; all depends
on the presence of the indoly group of tryptophan which reacts as follows (using glyoxylic acid as an example
of an aldehyde)

This is called Hopkin-Cole test- A similar test was at one time recommended for detection of formaldehyde that
had been as a preservative to milk, the formaldehyde reacting with indolyl groups of milk proteins to give a
color.

Materials and Reagents:

Hopkin-Cole reagent
Conc. H2SO4

Procedure:

Place 2 to 3 ml of test solution and an equal volume of Hopkins-Cole reagent in a test tube and mix thoroughly.
Incline the tube and permit 5 to 6 ml of conc. sulphuric acid to flow slowly down the side of the tube, thus
forming a sharp layer of acid beneath the amino acid solution. When stratified in this manner a reddish-violet
color forms at the zone of contact of the two fluids. If the color does not appear after starting for a few minutes,
the tube may be rocked gently to cause a slight mixing of the liquids are mixed by gentle stirring the precipitate
of protein dissolves and the violet color spread throughout the solution.

Result:
Positive visible result: Purple ring in the junction where two liquids meet.
 Folin test

A phosphomolybdotungstic acid reagent designed by Folin for phenol has been widely used for detection and
analysis of indolyl and phenol groups in amino acids. A characteristic blue color is formed when amino acid
solution is warmed with this reagent. The color so formed is due to the reaction of alkaline copper with the
amino acid and the reduction of phosphomolybdate by tyrosine and tryptophan present.

Materials and Reagents:

- Alkaline Na2CO3 solution (2% in 0.1 N NaOH)

Procedure:

Add 5ml of the alkaline solution to 1 ml of the test solution. Mix thoroughly and allow to stand at room
temperature for 10 mins. Add 0.5 ml diluted Folin-Ciocalteau reagent rapidly with immediate mixing. Observe
for development of color after 30 mins. Development of characteristic blue color indicates presence of indolyl
or phenol group.

Sakaguchi test

Arginine and other guanidyl derivatives (glycocyamine, methylgyanidine etc) react with hypo bromide and
alpha napthol to give a red colored product.

Materials and Reagents:

Sodium hydroxide solution (40%)

Alpha napthol solution (1% in alcohol)

Bromine water (a few drops of bromine in 100 ml distilled water)

Procedure:

Mix 1 ml of sodium hydroxide with 3 ml of test solution and add 2 drops of alpha napthol. Mix thoroughly and
add 4 to 5 drops of bromine water. Note the color formed. Formation of a red color indicates presence of
guanidine group. This is a very sensitive and specific test.

Result:
Presence of red color.

Nitroprusside test

Sodium nitroprusside reacts with compounds containing sulphahydryl groups produce an intensely red but
somewhat unstable color.
Materials and Reagents:

Sulphur amino acids (1.0% cystine, cysteine and methionine)

Sodium nitroprusside (2% prepared fresh)

Ammonium hydroxide

Procedure:
Mix 0.5 ml of a fresh solution of sodium nitropruside with 2 ml of the test solution and add 0.5 ml of ammonium
hydroxide.

Result:

Sodium nitroprusside reacts with compounds containing sulphahydryl groups produce an intensely red but
somewhat unstable color.

References:

Introduction to General, Organic and Biochemistry. 9th Edition.

[Link]
amino-acids
 Bicol University

College of Science

Legazpi City

BIOCHEMISTRY
Chemical Laboratory Tests for Proteins

Mikaela Rome C. Bigay