Test for Proteins Introduction

Proteins are the most abundant organic compounds present in nature. They are present in every living organism. Life without proteins is not possible. 

Proteins may be present in solutions along with other biological molecules like proteins present in milk etc. Some biological fluids do not contain any proteins, for example, urine. The presence of proteins in such fluids is of biological importance. Proteins present in any solution can be easily identified by performing different biological tests.

Tests to identify proteins can be divided into two categories;

1. General tests
2. Confirmatory tests
3. Differentiating tests

Different tests that fall under these categories are discussed below.

General Tests

If you are given a solution in the biological lab to identify the proteins present in it, your first step would be to confirm the presence of proteins. Only after this confirmation, you would be able to identify the type of protein present in the solution. This is done by performing the general tests. 

General tests are positive for all types of proteins. They are usually based on the feature that is common in all proteins like peptide bond, amino group, etc. Most of the general tests are colour tests. Proteins are identified based on the colour changes of the solution. 

A good practical approach is to rule out the presence of any other biological compound before the confirmation of proteins. However, this approach may not be valid in the case of solutions that contain multiple biological compounds. For example, milk contains lipids, carbohydrates, and proteins. If you try to rule out the presence of carbohydrates and lipids before confirming proteins, your results will be misleading. 

In the case of both types of solutions, general tests will tell you whether proteins are present in them or not. The following are the general tests for proteins. 

Biuret test

This test indicates the presence or absence of peptide bonds and is positive for all types of proteins. The minimum requirement for this test to be positive is the presence of two peptide bonds.  

Principle

Biuret test is based on the reaction of the cupric ions Cu²⁺ with peptide bonds in an alkaline solution. These ions react with the nitrogen of the peptide bond to form a purple or violet coloured complex. In an alkaline medium, cupric hydroxide is generated from copper sulfate of biuret reagent that helps in chelating the peptide bond with cupric ions to give violet or purple colour.

Apparatus 

• Test tube
• Dropper or pipette
• Solution to be tested

Reagents  

• Biuret reagent: It contains sodium potassium tartrate and copper sulfate

Procedure

1. Take 2 ml. of the solution to be tested in a test tube
2. Add 2 ml. of 5% sodium hydroxide solution
3. Mix the solutions
4. Add two drops of 1% copper sulfate solution

Observation

The solution will turn violet or purple. 

Results

The violet colour indicates the presence of peptide linkage in the solution. 

Points to remember

The following points should be kept in mind about the Biuret test.

• It is positive for all the compounds that contain more than one peptide bond like proteins, proteoses, peptones, polypeptides, etc. 
• The amino acids and dipeptides don’t give this test. 
• Non-protein compounds like oxamide and biuret also give positive Biuret test. 
• Biuret is a compound formed by condensation of two urea molecules at 180-degree Celsius. 

Precautions

• Do not add more than 2 drops of copper sulfate. 
• Do not perform this test with salts of magnesium or ammonium. 

Ninhydrin test

It is another colour test for proteins that indicates the presence of amino acids in a solution. Only alpha-amino acids give a positive Ninhydrin test. 

Principle

Ninhydrin is a powerful oxidizing agent that causes oxidative decarboxylation of the alpha-amino acids forming an aldehyde, ammonia, and Hydrindantin (reduced form of Ninhydrin). This Hydrindantin reacts with ammonia and another Ninhydrin molecule to form a bluish-purple coloured complex.  

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

• Solution of 0.2% Ninhydrin in acetone

Procedure

• Take 1 ml. of test solution in a test tube
• Add 10 drops of Ninhydrin solution in the above test tube 
• Hold the test tube on flame
• Boil the solution

Observation

Bluish-purple colour will be formed in the solution. 

Results

The formation of bluish-purple colour indicates the presence of free alpha-amino acids in the solution. 

Points to remember

• This test is positive only for alpha amino acids having a free amino group or carboxylic group. Therefore, all the proteins, peptides, polypeptides, free amino acids give positive Ninhydrin test.
• Proline and hydroxyproline do not have an alpha-amino group, and thus don’t give positive Ninhydrin test. These amino acids from brown color with Ninhydrin. 
• It is an additional test to detect proteins. 
• If the Biuret test is negative while the Ninhydrin test is positive, it indicates the presence of free amino acids in the solution. 

Precautions

• Do not add more Ninhydrin solution. 
• Do not overheat. 

Differentiating tests

The general tests listed above confirm the presence or absence of proteins in the given solution. Once the presence of proteins has been confirmed, the next step in the identification is to differentiate them from other proteins. For this purpose, differentiating tests are performed. 

The following are important differentiating tests for proteins.

Solubility test

This test is used to differentiate keratin from the rest of the proteins. 

Principle

The solubility test is based on the ability of proteins to dissolve in water other than keratin. Keratin is a large fibrous protein having numerous disulfide bonds. These disulfide bonds make it insoluble in water while the rest of the proteins are highly soluble due to hydrophilic amino acids present in them. 

Apparatus

• Test tube
• Distilled water

Reagents

• Distilled water

Procedure

1. Take 10 ml. distilled water in a test tube. 
2. Add the given powder into the water
3. Shake the test tube

Observation

The given powder is insoluble in water. 

Results

The insoluble protein is keratin. 

Points to remember

• Most of the proteins are soluble in water due to hydrophilic amino acids present on their outer surface. 
• Fibrous proteins are usually insoluble in water. Keratin is the most important fibrous protein present in hair, nails, and other similar structure.  

Heat coagulation test

Some proteins coagulate upon heating like albumin and globulin. These are the two most common proteins present in plasma. Heat coagulation test is used to differentiate albumin and globulin from other proteins. 

Principle

When proteins are exposed to heat or high temperature, they lose their structure and become denatured. These denatured proteins form a coagulum. The addition of acetic acid precipitates the coagulum by providing suitable conditions. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

• 1% acetic acid solution

Procedure

1. Take the solution to be tested up to 3/4^th of the test tube
2. Hold the test tube on the flame in a tilted position so that the solution in the upper portion of the test tube can be heated
3. Boil the solution in the upper portion of the test tube
4. Add 1% acetic acid solution to the test tube drop by drop 

Observation

A white coagulum is formed in the upper portion of the test tube. The coagulum gets intensified upon adding the acetic acid solution. 

Results

The white coagulum indicates the presence of a heat coagulable protein i.e. albumin or globulin. 

Points to remember

• Proteins have specific secondary or tertiary structure that gets denatured upon heating.
• The primary structure of proteins is preserved even after heating. 
• Albumin and Globulin are the proteins that can be easily identified by the heat coagulation test.  

Precautions

• Don’t heat the bottom of the test tube. The solution at the bottom serves as control.
• Don’t add more than 10 drops of acetic acid. 

Salt saturation test

A salt saturation test is used to differentiate albumin from globulin. Globulin undergoes precipitation upon half-saturation while albumin only precipitates upon full-saturation. 

Principle

Proteins are kept dissolved in solution by water molecules surrounding them. These water molecules make a hydration shell around the protein molecules. When neutral salts are added to the solution, the ions present in the slat have more affinity for water molecules. They attract the water molecules, removing the hydration shell around the proteins, and rendering them insoluble. As a result, the proteins precipitate out of the solution. This process is known as ‘salting out’ of proteins. 

Saturation test can be performed in two ways; 

1. Half saturation, in which protein precipitates when the solution is only half saturated with salt. 
2. Full saturation, in which precipitation only occurs upon full saturation if solution with a neutral salt. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Filter paper
• Solution to be tested

Reagents

• Saturated solution of Ammonium Sulfate
• Solid Ammonium Sulfate
• 40% Sodium Hydroxide solution
• 1% Copper Sulfate solution

Procedure

• For Half Saturation;

1. Take 3 ml. of test solution in a test tube
2. Add 3 ml. saturated solution of ammonium sulfate to the test tube
3. Mix the solution and allow to stand for 5 minutes
4. Filter the above solution
5. Perform Biuret test on the filtrate

• For Full Saturation;

1. Take 3 ml. of test solution in a test tube
2. Add small amount of solid Ammonium Sulfate in the test tube
3. Mix the solution by shaking the test tube
4. Keep adding solid Ammonium Sulfate to the solution until it doesn’t dissolve any more 
5. Allow to stand for five minutes
6. Filter the solution
7. Perform Biuret test on the filtrate

Observation

• For Half Saturation;

1. White precipitate is formed
2. White precipitate is not formed 
3. Filtrate gives purple color 
4. Filtrate does not give purple color

• For Full Saturation;

1. White precipitate is formed
2. White precipitate is not formed 
3. Filtrate gives purple color 
4. Filtrate does not give purple color

Results

• For Half Saturation;

If a white precipitate is formed, it indicates that globulin protein is present in the solution. 

If filtrate form purple colour on the Biuret test, it shows other proteins are also present that are not precipitated yet. If the Biuret test is negative, it indicates that all the proteins present in the solution have been precipitated.  

• For Full Saturation;

The formation of a white precipitate indicates the presence of albumin in the solution.

If the filtrate gives a positive Biuret test, it contains additional proteins. The reverse is true if the Biuret test is negative for filtrate. 

Confirmatory tests

These are the final tests that confirm the presence of a particular protein in the solution.  The following are confirmatory tests for some important proteins.

Salt Saturation test

This test has already been discussed. It is a differentiating test as well as a confirmatory test for albumin and globulin proteins. 

Isoelectric pH test

Isoelectric pH is defined as the pH at which amino acids carry no net charge and is electrically neutral. It has an equal number of positive and negative charges at this point. It is a confirmatory test for mil protein, Casein. 

Principle

At isoelectric pH, protein has minimum solubility and tend to precipitate out. the reason for this precipitation is the absence of electrostatic repulsive forces among the protein molecules due to their neutral charge. The protein molecules come together and form a precipitate at this pH. The addition of acetic acid to the solution of protein drops the pH to 4.6, the isoelectric pH of Casein, and it precipitates out. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Bromocresol green Indicator
• Solution to be tested

Reagents

• 1% acetic acid solution

Procedure

1. Take 3 ml. test solution in a test tube
2. Add 3 drops of indicator (bromocresol green)
3. Add 1% acetic acid solution to the above test tube drop by drop
4. Keep adding acetic acid until a light green color appears indicating isoelectric pH
5. Allow it to stand

Observation

A curdy green precipitate is formed at the top of the test tube.

Results

The protein present in the solution is Casein. 

Points to remember

• Bromocresol turns green when the pH in between 4 to 4.6.
• Isoelectric pH of Casein is 4.6.
• The precipitate of Casein formed at pH 4.6 will re-dissolve in a highly acidic or alkaline environment. 

Precautions

• Don’t add more than 3 drops of indicator to avoid false results.
• Stop adding acetic acid when the solution turns light green. Otherwise, casein will dissolve again. 

Sulfur test

It is a confirmatory test for keratin. The sulfur atoms present in cysteine and cysteine residues are detected in this test. Keratin is rich in cysteine residues from string disulfide bonds between them rendering it insoluble in water.

Principle

When a solution containing keratin is boiled with sodium hydroxide, the sulfur present in its amino acids (cysteine) is converted to inorganic sodium sulfide. It reacts with lead to form lead sulfide that forms black coloured precipitates inside the solution.  

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

• 40% Sodium Hydroxide solution
• Lead acetate solution

Procedure

1. Take 2 ml. of test solution in a test tube
2. Add 2 ml of 40% sodium hydroxide solution to the test tube
3. Hold the test tube on flame and boil for one minute
4. Cool the test tube under tap water
5. Add 5 drops of lead acetate

Observation

Black or brown coloured precipitate is formed. 

Results

The protein in the solution is abundant in cysteine residues. It might be Keratin.

Points to remember

• This test is specific for amino acids containing thiol group. 
• The sulfur of methionine will not give this test as it cannot be cleaved by sodium hydroxide. 
• Albumin can also give this test positive. 

Precautions

• Do not add too much lead acetate. Otherwise, it may lead to the formation of white lead acetate crystals giving false-negative results. 

Summary

Proteins perform several functions that are essential for all forms of life. They also act as important structural molecules. 

The proteins in a solution can be identified in the laboratory by performing three types of tests.

1. General tests
2. Differentiating tests
3. Confirmatory tests

General tests confirm the presence of a protein in a solution. They are positive for all types of proteins. These include:

• Biuret test: It is positive if two or more peptide bonds are present in a compound.
• Ninhydrin test: It is positive in the presence of an alpha amino acid.

Differentiating tests are performed to separate certain proteins from the rest. Important differentiating tests include:

• Solubility test: It differentiates Keratin. 
• Heat Coagulation test: It differentiates albumin and globulin from the rest of the proteins. 
• Salt Saturation tests: These tests are used to differentiate albumin and globulins from one another. 

Confirmatory tests confirm the presence of a particular protein in the solution. These include:

• Salt Saturation tests: These are also confirmatory tests for albumin and globulin. 
• Isoelectric pH test: It is a confirmatory test for Casein.
• Sulfur test: It is a confirmatory test for Keratin.

All these tests must be performed considering the precautions listed along with them. 

Frequently Asked Questions

What are the two general tests for proteins?

The two general tests for identifying proteins are the Biuret test and the Ninhydrin test. All proteins give a positive result in these tests. If a molecule does not test positive in them, it is not a protein. 

What is the principle of the Biuret test?

The principal of this test is the reaction between cupric ions Cu2+ and nitrogen of peptide bonds in an alkaline solution. These ions react with the nitrogen of the peptide bond, and a  purple or violet coloured complex is formed.

What is heat coagulation test?

This test is based on the principle that when exposed to high temperatures, larger proteins lose their structure and undergo coagulation. This test helps in differentiating albumin and globulin from other proteins

What is Ninhydrin test?

This test is a colour test used to detect the presence of proteins in a solution. Only proteins having alpha amino acids give a positive Ninhydrin test and a bluish-purple coloured complex is formed. 

References

1. “Chemistry of Protein Assay” Thermo Scientific Protein Methods Library. http://www.piercenet.com
2. Ninfa, Alexander; Ballou, David; Benore, Marilee (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Wiley. p. 111. ISBN 978-0470087664.
3. Fenk, C. J.; Kaufman, N.; and Gerbig, D. G. J. Chem. Educ. 2007, 84, 1676-1678.
4. Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85
5. https://commons.wikimedia.org/wiki/File:Biuret_test.PNG
6. https://commons.wikimedia.org/wiki/File:Ninhydrin_Test.svg