Protein color test

Millon’s Test Definition

Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which
is the only amino acid containing the phenol group. Millon’s test is a specific test for
tyrosine, but it is not a specific test for protein as it also detects the phenolic group
present in other compounds as well. Therefore, while performing Millon’s test, it is
essential that other tests like the Biuret test and Ninhydrin test also be performed. As
many proteins consist of tyrosine, the test is useful in the detection of such proteins. The
test was discovered by and named after the French Chemist Auguste Nicolas Eugene
Millon.

Objectives of Millon’s Test

• To detect the presence of tyrosine-containing proteins in a given sample.
• To detect the presence of phenol-containing compounds.
• To differentiate tyrosine from other amino acids.

Principle of Millon’s Test

Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which
then forms complexes with heavy metals like mercury. The reagent used for the test is
called Millon’s reagent, and it consists of mercuric nitrate and mercurous nitrate that is
dissolved in concentrated nitric acid. In the test, the phenol group on the tyrosine molecule
is nitrated by the nitric acid present in the reagent. The nitrated tyrosine then combines
with the mercury ions in the solution to form a red-colored precipitate or solution. In some
proteins containing tyrosine, the initial reaction between mercuric nitrate results in a white
or yellow colored precipitate. After the addition of nitric acid and heating, however, the
residue turns red in color. Both of these results are considered positive results and
indicate the presence of tyrosine in the solution.
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Reagent
§ Millon’s reagent: Millon’s reagent consists of mercuric nitrate and mercurous nitrate
dissolved in nitric acid and distilled water.
§ Preparation of Millon’s reagent: Dissolve 160 grams of mercuric nitrate and 160 grams
of mercurous nitrate in 400 ml concentrate nitric acid solution. The reagent is then
made to 1000 ml by the addition of 600ml distilled water. The formula can be adjusted
to suit the performance parameters.
§ Sample (1% tyrosine)

Materials Required
§ Test tubes
§ Test tube stand
§ Pipettes
§ Water bath

Procedure of Millon’s Test

1. About 2 ml of the sample solution or the 1% tyrosine solution is taken in a test tube.
2. To this, about 2 ml of Millon’s reagent is added. The test tubes are then kept in the
water bath for about 2 minutes if red colored precipitate is not observed immediately.
3. The tubes are then observed for the formation of the colored precipitate.
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Result and Interpretation of Millon’s Test

• Positive result: A positive result in the Millon’s test is demonstrated by the formation of
a red or pink colored precipitate. This indicates the presence of tyrosine or tyrosine
containing protein.
• Negative result: A negative result in the Millon’s test is demonstrated by the absence of
colored precipitate in the test tube. This indicates the absence of tyrosine or tyrosine-
containing protein.
Uses of Millon’s Test
• Millon’s test is used for the detection of tyrosine-containing proteins in a given sample.
• The test also helps in the differentiation of tyrosine from other amino acids.
• The test is useful in the detection of casein protein and the protein found in raw meat.
Limitations
• Compounds like salicylic acid and phenolic compounds give a positive result to this test;
thus, any other phenol compounds that might be present in the test tube should be
avoided. Tests like the Biuret test and Ninhydrin test should be performed for
confirmation.
• The presence of chlorine in the solution might interfere with the reaction; thus, the test
cannot be performed on a sample containing chlorides.
 Protein color test

• The formation of a white or yellow precipitate might be observed immediately after the
addition of Millon’s reagent due to the denaturation of proteins by mercuric ions.
Protein color test

Ninhydrin Test Definition

Ninhydrin test is a chemical test performed to detect the presence of ammonia,
primary/secondary amines, or amino acids. This test involves the addition of ninhydrin
reagent to the test sample that results in the formation of deep blue color, often termed
as Ruhemann’s purple, in the presence of an amino group.

Objectives of Ninhydrin Test

• To detect the presence of amines and amino groups in the test solution.
• To quantify the amino acids present in the sample.
• To distinguish carbohydrates from amino acids.

Principle of Ninhydrin Test

• This assay is based on the fact that two molecules of ninhydrin (2, 2-
dihydroxyindane-1, 3-dione) react with a free alpha-amino acid to produce a deep
purple or blue color known as Ruhemann’s purple.
• In this reaction, ninhydrin acts as an oxidizing agent and causes the deamination and
decarboxylation of the amino acids at an elevated temperature.
• This reaction is then followed by condensation between the reduced ninhydrin
molecules, released ammonia, and the second molecule of ninhydrin.
• By the end of the reaction, a diketohydrin complex is formed which has a deep purple
color.
• In amino acids like proline and hydroxyproline, this test yields an iminium salt, which
is yellow-orange in color.
• Similarly, proteins with a free amind group like asparagine, react with the ninhydrin
reagent to form a brown colored product.
• The intensity of the formed complex is proportional to the concentration of amino
acids in the solution.
• The color intensity, in turn, depends on the type of amino acid present.
Protein color test

Reaction

Reagents
• Ninhydrin reagent: Dissolve 0.35g of ninhydrin in 100 ml ethanol (iso-propanol or
1:1 mixture of acetone/butanol may be used instead of ethanol).
• Diluent solvent (for the quantitative test): Mix equal volumes of water and n-propanol.
• Standard solution (1% protein solution)
• Sample solution
Materials required
• Test tubes
• Test tube stand
• Pipette
Equipment
• Water bath
• Spectrophotometer

Procedure of Ninhydrin Test

1. Take 1 ml of standard protein solution in one test tube and 1 ml of the test sample in
another dry test tube.
2. Add a few drops of ninhydrin reagent to both the test tubes.
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3. Place the test tubes in the water bath for 5 minutes and then allow cooling to room
temperature.
4. Observe the formation of color and note down the result.

Result and Interpretation of Ninhydrin Test

• The presence of a purple-colored complex in the tube represents a positive result and
indicates the presence of amino acid in the sample.
• The absence of the complex in the tube represents a negative result and indicates
the lack of amino acids in the sample.

Uses of Ninhydrin Test

• Ninhydrin test is used to detect the presence of amino acids in unknown samples.
• This test is also used in solid-phase peptide synthesis to monitor the protection for
amino acid analysis of proteins.
• As the ninhydrin test is quite sensitive, it is commonly used to detect fingerprints. It is
possible as the terminal amines of lysine residues in peptides and proteins shed off
in fingerprints react with ninhydrin.
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Limitations of Ninhydrin Test

• Ninhydrin reacts not only reacts with α-amino groups but also with nitrogen in
ammonia and other free amines.
• The Ninhydrin test is not effective to detect high molecular weight proteins as the
steric hindrance limits the ninhydrin from reaching the α-amino groups.
Protein color test

Biuret test:
§ Biuret test is a general test for compounds having a peptide bond. Biuret is a
compound formed by heating urea to 180° C. When biuret is treated with dilute copper
sulfate in alkaline condition, a purple colored compound is formed. This is the basis
of biuret test widely used for identification of proteins and amino acids.

§ This test is given by compounds containing two or more peptide bond (CO-NH group).
Since all proteins and peptides possessing at least two peptide linkage ie. tripeptide
gives positive biuret test.
§ The principle of biuret test is conveniently used to detect the presence of proteins in
biological fluids.
§ Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give
a violet colored product which is due to formation of co-ordination complex of cupric
ions with un-shared electron pairs of peptide nitrogen and O2 of water.

Requirements for Biuret test:

§ 1 % alanine, 5 % egg white (albumin)
§ Biuret reagent
§ Water bath
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§ Dry test tubes

§ Pipettes

Biuret reagents:
§ Copper sulfate (CuSO40
§ Sodium hydroxide (NaOH)
§ Sodium potassium tartarate (commonly known as Rochelle salt)

Preparation Biuret reagent

§ Biuret reagent is prepared by adding NaOH in CuSO4 solution, making it alkaline.

To prepare 1000ml of Biuret reagent

§ Take 1.5 gram of pentavalent copper sulphate (CuSO4) and 6 gram of Sodium
potassium tartarate and dissolve them in 500 ml of distilled water
§ **Sodium potassium tartarate is a chelating agent and it stabilize the copper ion
§ Take 375 ml of 2 molar Sodium hydroxide
§ Mix both the solution in volumetric flask and make it final volume to 1000 ml by adding
distilled water.

Procedure for Biuret test

1. Take 1 ml of test solutions in dry test tubes and in another tube take 1 ml distilled
water as control.
2. Add 1 ml of biuret reagent to all test tubes, mix well.
3. Look for the development of blue colors
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Biuret test positive:

§ color changes to purple
§ all peptides and protein give the test positive
§ Histidine is the only amino acid that give biuret test positive.

Biuret test negative:

§ No color change

Precautions:
§ Presence of magnesium and ammonium ions interfere in biuret test. This can be
overcome by using excess alkali.
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Xanthoproteic test: Objective, Principle, Reagents, Procedure and Result

Objective:
§ This test is used to differentiate aromatic amino acids which give positive result from
other amino acids

Principle:
§ Xanthoproteic test is used to detect amino acids containing an aromatic nucleus
(tyrosine, tryptophan and phenylalanine) in a protein solution which gives yellow
color nitro derivatives on heating with conc. HNO3. The aromatic benzene ring
undergoes nitration to give yellow colored product. Phenylalanine gives negative or
weakly positive reaction though this amino acid contains aromatic nucleus because
it is difficult to nitrate under normal condition. On adding alkali to these nitro derivative
salts, the color change fro yellow to orange.

Reactions:
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Reagents:
§ Test solution: 1 % tyrosine, 1 % tryptophan, 1 % phenylalanine, 5 % egg white
(albumin)
§ Nitric acid
§ 40 % NaOH

Procedures
1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1ml of conc. HNO3 in all test tubes and mix well.
4. Cool the solution under tap water.
5. Now add 2ml of 40 % NaOH to all test tubes.
6. Look for the color development.

Result:
Protein color test

Lead Sulfide Test Definition

Lead sulfide test (or Lead acetate test) is a biochemical test for the detection of amino
acids like cysteine and cystine. The test is a specific test for the detection of amino acids
containing sulfur, S-S group in cysteine, and S-H group in cystine. The test is also called
a lead acetate test as the reagent for the test is lead acetate. Even though the test is
specific for the detection of sulfur-containing amino acids, methionine doesn’t give a
positive result in this test.

Objectives of Lead Sulfide Test

• To detect the presence of sulfur-containing amino acids in a sample.
• To detect protein-containing cysteine and cystine in a given sample.
• To distinguish between sulfur-containing and non-sulfur containing amino acids.

Principle of Lead Sulfide Test

The test is based on the principle of detection of sulfur in a solution by the degradation of
the S-H or S-S group in amino acids under strongly alkaline conditions. Amino acids like
cysteine and cystine release sulfur in the presence of strong alkaline conditions at a high
temperature. The sulfur then combines with the alkali (NaOH) to form Na2 The Na2S thus
formed reacts with lead acetate to form lead sulfide, which results in a black residue. For
the reaction to take place, free sulfur ions should be present in the medium.

Reaction
Cysteine + 2NaOH → Serine + Na2S + H2O
Pb(CH3COO)2 + NaOH → Pb(OH)2 + 2CH3COONa
Pb(OH)2 + 2NaOH → Pb(ONa)2 + 2H2O
Na2S + Pb(ONa)2 + H2O → PbS (black precipitate) + 4NaOH

Reagent
• 2% lead acetate solution in water
• 40% NaOH
• Sample
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Material Required
• Test tubes
• Test tube stand
• Pipettes

Procedure of Lead Sulfide Test

1. In a test tube, 2 ml of the amino acid solution is taken. To this, 2 ml of NaOH is added,
and the solution is boiled for a minute.
2. Once the test tube cools down, a few drops of lead acetate are added to the solution.
3. The test tube is then observed for the formation of a precipitate.

Result and Interpretation of Lead Sulfide Test

• Positive test: A positive test in the Lead sulfide test is represented by the formation
of black precipitate at the bottom of the test tube. This indicates the presence of
cysteine or cystine in the solution.
• Negative test: A negative result in the Lead sulfide test is represented by the
absence of black residue in the test tube. This indicates the absence of cysteine or
cystine.
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Uses of Lead Sulfide Test

• The test is used to detect sulfur-containing amino acids like cysteine and cystine.
• It helps to distinguish between different groups of amino acids.
• The detection of cystine in urine is a pathological symptom of diseases like cystine
stones in the kidneys and bladder.

Limitations
• Methionine doesn’t give a positive result in this test as the sulfur in the thioester bond
in methionine is not released by the treatment with NaOH.
• The addition of excess lead acetate to the solution might result in white-colored
precipitation.