Collegedunia Team Content Curator
Content Curator
Proteins are organic molecules containing long chains of amino acid residues joined by peptide bonds. They are large macromolecules and can be divided into three parts fibrous, globular, and membrane plaing an important role in the development and nourishment. Plants produce a protein with the help of carbon dioxide, water, and minerals whereas animals produce proteins by eating the plants.
The tests for Proteins such as are the Biuret test, Xanthoproteic test, Millon’s test and Ninhydrin test help to decide the presence of protein in substances. In the Biuret test, the compound containing the peptide linkage undergoes this test. In the Xanthoproteic test protein is treated with nitric acid for nitration and gives a yellow precipitate which when treated with alkali changes into orange. The phenolic group of tyrosine of protein is treated with mercuric sulfate in the presence of sodium nitrite and sulfuric acid to give red color in Millon’s test. In the Ninhydrin test, the Pyridine solution of Ninhydrin changes its color into a deep blue, violet-pink, or red in certain cases.
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Aim of the Protein Tests
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Protein is the most important constituent of living cells and also is the building block of the body. Proteins also play an important role in the development of muscles and tissues in the body.
Proteins have high molecular mass, long-chain polymers, are composed of amino acids, and contain carbon, hydrogen, nitrogen, and oxygen. The aim of this experiment is to run several simple tests to determine the presence of proteins in the provided sample.
Read more: differences between catabolism and anabolism
Theory of the Experiment
Proteins have a larger molecular mass than long-chain polymers made up of amino acids. Proteins are found in all living bodies since they are found in cells. Proteins are made up of carbon, oxygen, nitrogen, hydrogen, and occasionally sulfur and phosphorus. A few tests are performed to determine the presence of proteins in the samples provided.
These protein laboratory tests are as follows:
- Biuret Test
- Millon’s test
- Xanthoproteic Test
- Ninhydrin Test
Each of the tests, as well as the chemical reaction involved in the test, is detailed below.
Some tests help find the presence of protein in the given food sample and are as follows –
Read More:
| Important Topics | ||
|---|---|---|
| lipoproteins | Endomembrane System | Plastids |
| Eukaryotic Cells | Mitochondria | Cytoskeleton |
Biuret Test
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The compounds which have the peptide linkage are supposed to undergo this test. The proteins are the polypeptides of the amino acids that are connected by peptide bonds.
The alkaline solution of the protein is treated with the drop of the aqueous copper sulfate and a bluish violet color residue is the resultant.
The Biuret test is useful for the estimation of protein in the food sample. Consequently, the biuret is a kind of chemical that is produced when the urea gets heated to 180 degrees Celsius.
During the reaction, two molecules of urea are combined to condense and form a biuret molecule. The reagent of biuret reagent forms a residue of violet color with copper ions that confirms that the protein is present in the food sample.
Biuret Test: Procedure
- Take the sample in a clean and dried test tube.
- Add 2ml of sodium hydroxide and some drops of copper sulfate in the test tube and shake it well.
- Leave the test tube for about 4-5 minutes.
- The appearance of bluish violet color confirms the presence of proteins.
Read more: difference between cilia and flagella
Xanthoproteic Test
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When the treatment of protein takes place with nitric acid, it gives yellow or orange color. For nitration, we have to use nitric acid. In the treatment of proteins with nitric acid, we get yellow precipitate, which changes into orange color when treated with alkali.
Xanthoproteic Test: Procedure
- Take 2ml of the sample in the clean and dry test tube.
- Take any food sample in the test tube and add some drops of sulfuric acid, afterward heat the test tube.
- After this, if you get to see the yellow precipitate then the appearance of protein is confirmed in the food.
Read more: cell biology
Millon’s Test
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In the presence of sodium nitrite and sulfuric acid, the phenolic group of tyrosine of proteins acts with mercuric sulfate to give red color. Gelatin is not acquired to give this test.
The proteins holding phenolic amino acids have to give the millions test. Firstly, when the proteins are treated with millions of reagents it gives white precipitate, after boiling it turns into brick-red color which confirms the presence of proteins.
Millon’s Test: Procedure
- In a clean and dried test tube, take 2ml of the sample.
- Add some drops of the million’s chemical in the test tube and then shake it well.
- If the white precipitate occurs and after boiling it changes into brick-red color, then the existence of proteins is confirmed.
Read More:
| Chapter Related Topics | ||
|---|---|---|
| Microbodies | Amino Acids | Vitamin B |
| Mycology | diagram of mitochondria | Discovery of Cells |
Ninhydrin Test
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The reaction of pyridine solution of ninhydrin changes its color into deep blue and violet-pink or also gives red color in some cases. By dissolving 0.1gm of ninhydrin into approximately 100ml of distilled water, a ninhydrin solution is prepared. But this solution of ninhydrin is highly unsteady and can be kept for 2 days only.
Ninhydrin Test: Procedure
- Take the sample solution in a clean and dried test tube.
- Add about 1-2ml of ninhydrin test into the test tube and shake it well.
- Then boil the mixture to see if any changes occur or not.
- If the solution turns into deep blue or violet-pink or sometimes into red color, then the presence of proteins is confirmed.
Read more: Centriole
Apparatus Setup
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- Sodium hydroxide
- Pyridine solution
- Ninhydrin reagent
- Copper sulfate solution
- Nitric acid
- Sodium nitrite
- Sulfuric acid
- Mercuric sulfate
- Distilled water
- Dropper
- Test tubes
- Test tube holder
- Stirrer
- Water bath
Observations and Inference
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| Test’s Name | Observations |
|---|---|
| Biuret test | The appearance of violet color confirms the presence of proteins. |
| Xanthoproteic test | The formation of yellow or orange color confirms the presence of proteins. |
| Millon's test | The solution of the brick red color confirms the appearance of proteins. |
| Ninhydrin test | The formation of deep blue and violet-pink color or even red sometimes confirms the appearance of proteins. |
Result
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The test helps in finding out whether the food sample contains proteins.
Read more: Lipids
Precautions
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We have to take some precautions while doing the tests for the identification of protein and some are mentioned below -
- Don’t heat the bottom of the test tube. The solution at the bottom serves as the control.
- Don’t add more than 10 drops of acetic acid.
- Do not add more than 2 drops of copper sulfate in the Biuret Test
- Do not perform a Biuret test with salts of magnesium or ammonium.
- Handle chemicals with care while performing the experiment.
- Use hand gloves and aprons during the experiment.
- Use droppers to take the reagents every time from the bottles.
Read more: Plasma Membrane
Previous Years Questions
- A bivalent consists of...[NEET 1989]
- A cell organelle containing hydrolytic enzymes is...[NEET 2016]
- Water soluble pigments found in plant cell vacuoles….[NEET 2016]
- Which of the following are not membrane - bound?….[NEET 2015]
- Electron microscope has a high resolution power…. [NEET 1990]
- Flagella of prokaryotic and eukaryotic cells….[NEET 2004]
- A major site for synthesis of lipids is….[NEET 2013]
- which one of the following colours of light so as to….[NEET 2005]
- According to fluid mosaic model, plasma membrane is….[NEET 1998]
- Acetabularia used in Hammerling's nucleocytoplasmic...[NEET 1988]
- All types of plastids possess essentially….[NEET 1992]
- An outer covering membrane is absent over….[NEET 1992]
- Balbiani rings are the sites of...[NEET 1993]
- the Golgi apparatus is also concerned with the...[NEET 1994]
- Biological organisation starts with….[NEET 2007]
Things to Remember
- The important biological compounds that contain long chains of amino acids are known as proteins.
- Albumin and globulin present in our blood play an important role in our lives.
- Proteins play a vital role in the development of muscles and tissues in the body.
- Proteins contain carbon, hydrogen, nitrogen, and oxygen.
- All the tests are implemented to find the presence of proteins in a food sample.
- There are 4 types of tests namely the Biuret test, Xanthoproteic test, Million’s test, and Ninhydrin test.
Also Read: Proteolytic Enzymes
Sample Questions
Ques: How is polypeptide formed? (2 marks)
Ans. When a water molecule is lost, the two amino acids are connected. The chain of the polypeptide is formed when the amino acids are joined by the peptide bonds. The unit of amino acids present in the polypeptide is mentioned as the residue.
Ques: Describe the monomer and polymers of the proteins. (2 marks)
Ans. A molecule that forms bigger polymer molecules is called a monomer. It is also known as the building block of proteins. For example- the building blocks of the proteins are amino acids. Similarly, the polymer is defined as the protein of the monomer series.
Ques: What will happen when the protein undergoes the hydrolysis process? (2 marks)
Ans. The protein evolves into alpha-amino acids when undergoing the hydrolysis process.
Ques: What is the composition of the Biuret reagent? (2 marks)
Ans. The biuret reagent is collectively formed by the Hydrated copper sulfate, Potassium Hydroxide solution, Potassium sodium tartrate.
Ques: Name the technique that is used to separate proteins according to their sizes? (2 marks)
Ans. The process or technique that is used to separate the proteins according to their size is Gel Electrophoresis.
Ques: What exactly are proteins? (1 mark)
Ans. Proteins are made up of smaller components known as amino acids that are linked together in the form of longer chains. A protein molecule can be formed by combining twenty different types of amino acids.
Ques: What are the distinctions between the two types of proteins? (1 mark)
Ans. Fibrous proteins and globular proteins are the two types of proteins.
Ques. What happens when a protein is hydrolyzed? (1 mark)
Ans. When proteins are hydrolyzed, -amino acids are formed.
Ques: How do amino acids combine to produce a polypeptide? (2 marks)
Ans. When a water molecule is lost, two amino acids become bonded together. The amino acids that are linked together by peptide bonds create a chain of polypeptides, and each unit of amino acids in the polypeptide is referred to as a residue.
Ques. What do you mean by protein monomer and polymer? (2 marks)
Ans. A monomer is a molecule that joins together to generate larger polymer molecules. It is recognized to be a protein-building block. Amino acids, for example, are the building blocks of proteins. A protein of the monomer series is referred to as a polymer.
Ques: What is done in the biuret test for proteins? (2 marks)
Ans. In a test tube, add an equal volume of 10% NaOH solution to 2-3 ml of protein solution. Add a few drops of 0.5 percent copper sulfate solution and thoroughly mix. If protein is present, the colour changes to purple-violet.
Ques: What are the precautions that we need to take during this experiment? (3 marks)
Ans. The precautions that must be taken are as follows:
- Handle chemicals with care while performing the experiment.
- Use hand gloves and aprons during the experiment.
- Use droppers to take the reagents every time from the bottles.
Ques: Why does the skin turn yellow when it inadvertently comes in contact with HNO3? (2 marks)
Ans. A yellow stain is often observed on the skin when it comes in contact with nitric acid. The reason for the yellow stain is the xanthoproteic reaction.
Ques: What happens if our body lacks protein? (2 marks)
Ans. Protein is essential for the growth of the human body. Protein shortage has been linked to malnutrition and insufficient protein intake. Protein deficiency can result in major health problems such as porous bones that are weak and thin, swelling (stomach bloating), stunted growth in youngsters, fatty liver, increased infection severity, and skin deterioration, among other things. A lack of protein may also result in muscle loss, as well as an increased risk of bone fractures. The amount of protein required is determined by the individual's age.
Ques: Describe the amino acid. (2 marks)
Ans. Amino acids are crystalline, colorless solids. Instead of ordinary amines or carboxylic acids, these acids are water-soluble, have a high melting point, and behave like salts. Dipeptide, tripeptide, and polypeptide connections are produced depending on the relative location of the amino group with regard to the carboxyl group.
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