Laboratory Test of Proteins

Proteins are a class of biologically important compounds having high molecular weight. Proteins are perhaps the most complex organic materials produced in nature. Plants build up their proteins from carbon dioxide, water and minerals in the presence of sunlight. Animals derive proteins from plants.

Aim:

To perform some simple tests to identify the presence of proteins in the given sample.

Theory:

Proteins has high molecular mass long chain polymer composed of α-amino acids. Proteins are constituents of cells and hence present in living bodies. Proteins contain carbon, hydrogen, nitrogen, oxygen and sometimes phosphorus and sulfur.

The following are the tests carried to find the presence of proteins in a given sample.

  1. Biuret test
  2. Xanthoproteic test
  3. Millions test
  4. Ninhydrin test

(a) Biuret Test:

The compounds with peptide linkage undergoes this test. Proteins are polypeptides of amino acids linked together by peptide bonds. An alkaline solution of protein is treated with a drop of aqueous copper sulfate when a bluish violet colour is obtained.

Biuret Test

This test can be used for the identification of proteins and also for protein estimation. Biuret is the chemical product formed when urea is heated to 180oC. In this reaction two molecules of urea condense to form a bi-urea or biuret molecule. Biuret reagent in the presence of copper ions forms a violet colour complex.

Note: Formation of violet colouration confirms the presence of Proteins.

(b) Xanthoproteic Test:

Proteins on treatment with nitric acid gives a yellow or orange colour. Concentrated nitric acid is used for nitration. On the treatment of nitric acid, proteins give yellow precipitate which turns to orange colour on treatment with alkali.

Xanthoproteic Test

Note: The appearance of yellow colour solution confirms the presence of proteins.

(c) Millon’s Test:

Phenolic group of tyrosine of proteins react with mercuric sulfate in the presence of sodium nitrite and sulfuric acid to give red colour. Millon’s test is given by proteins containing phenolic amino acids. Gelatin does not give this test. First white precipitate is formed when proteins treated with millon reagent and turns to brick red on boiling confirms the presence of proteins.

Millon's Test

Note: The appearance of brick red colour solution confirms the presence of proteins.

(d) Ninhydrin Test:

Proteins reacts with pyridine solution of ninhydrin gives coloured solution from deep blue to violet pink sometimes even red colour in some cases. Ninhydrin solution is prepared by dissolving 0.1gm of ninhydrin in about 100ml of distilled water. But this solution of ninhydrin is unstable and can be kept for two days.

The chemical reaction is given below.

Ninhydrin Test

Note: The appearance of violet colour solution confirms the presence of proteins.

Materials Required:

  1. Copper sulfate solution
  2. Sodium hydroxide
  3. Nitric acid
  4. Mercuric sulfate
  5. Sodium nitrite
  6. Sulfuric acid
  7. pyridine solution
  8. Ninhydrin reagent
  9. Distilled water
  10. Test tubes
  11. Test tube holder
  12. Dropper
  13. Water bath
  14. Stirrer

Apparatus Setup:

Test of Proteins

Procedure:

(a) Biuret Test:

  1. Take the given sample to be tested in a clean test tube.
  2. Add 2ml of sodium hydroxide solution to it.
  3. To that add 5 to 6 drops of copper sulfate solution to it.
  4. If there is the appearance of bluish violet color indicates the presence of protein.

(b) Xanthoproteic Test:

  1. Take 2ml of given sample compound in a test tube.
  2. Add a few drops of concentrated sulfuric acid and heat.
  3. If there is formation of yellow precipitate then the presence of protein is confirmed.

(c) Millions Test:

  1. Take 2ml of given sample solution in a clean test tube.
  2. Add 2-3 drops of Millon’s reagent and shake well.
  3. Observe the change.
  4. If there is formation of white precipitate and changes to brick red on heating then the presence of protein is confirmed.

(d) Ninhydrin Test:

  1. Take the sample solution to be tested in a clean test tube.
  2. Add 1-2ml of ninhydrin solution to it.
  3. Boil the mixture and observe the change.
  4. If there is the appearance of blue colouration then the presence of protein is confirmed.

Observation and Inference:

Biuret test

Formation of violet colouration confirms the presence of Proteins.

Xanthoproteic test

The appearance of yellow colour solution confirms the presence of proteins.

Millions test

The appearance of brick red colour solution confirms the presence of proteins.

Ninhydrin test

The appearance of violet colour solution confirms the presence of proteins.

Results and Discussions:

The given sample contains _________ (proteins).

Precautions:

  1. While performing the experiment handle chemicals with care.
  2. Use lab aprons and hand gloves during the experiment.
  3. Use droppers to take the reagents from the bottle.

Viva Questions for Laboratory Test of Proteins

What are Proteins?

Proteins consist of hundreds or thousands of smaller units called amino acids that are attached in long chains to each other. It is possible to combine 20 different types of amino acids to make a protein.

What are the two types of proteins?

The two types of proteins are globular proteins and fibrous proteins.

What happens when proteins undergo hydrolysis?

Proteins undergo hydrolysis resulting in the formation of α-amino acids.

How do amino acids form a polypeptide together?

Two amino acids are linked together with the loss of a water molecule. A number of amino acids joined by peptide bonds form a chain of polypeptides, and each unit of amino acids in a polypeptide is called a residue.

What is the monomer and polymer of proteins?

Monomer, a molecule that form a larger polymers molecules and is a building block of protein. For instance, an amino acid acts as the protein building blocks. Polymer of protein is a monomer series.

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