8 Qualitative Tests for Protein | Biology Ideas

Proteins are present in the living world, regardless of the size of the organism, as they form the structural and functional basis of the cell. Under certain circumstances, it may become necessary to identify the proteins. For determining the presence of proteins in the unknown solution Ninhydrin Test and/or Biuret Test can be used.

Once the presence of proteins is confirmed in a given sample, tests for identification of its constituent amino acids can be carried out e.g. Millon’s test for detection of Tyrosine, Xanthoproteic test for presence of aromatic amino acids, Hopkins-Cole test for Tryptophan, etc.

For further studies on proteins using can carry various precipitation tests:

  • Heavy metal ions (e.g. Na2CO3, AgNO3, CuSO4, HgCl2, etc.) 
  • Alkaloidal reagents (TCA, Picric acid and meta-phosphoric acid) 
  • By salt ( (NH4)2SO4, Na2SO4, NaCl) 
  • Organic solvents (Ethanol, Acetone) 

Qualitative Tests for Protein

Their are too many tests to confirm the presence of protein in given sample solution. We are going to perform some of these tests,

  1. Burnt Test
  2. Biuret Test
  3. Ninhydrin Test
  4. Xanthoproteic Test
  5. Sakaguchi Test
  6. Sulfur Test
  7. Hopkins-Kole Test
  8. Millon’s Test
TestObservationInference
Burnt TestBurnt hair smellProteins may be present
Biuret TestThe appearance of bluish- violet colorProtein Confirmed
Ninhydrin TestThe appearance of deep blue or purple colorProtein Confirmed
Xanthoproteic TestFormation of a deep yellow or orange colorAromatic amino acids present
Sakaguchi TestDevelopment of a red colourArginine present
Sulfur TestAppearance of Black precipitate or brown colorationCystine present
Hopkins-Kole TestAppearance of Intense red color at the junction of the two solutionsTryptophan present
Millon’s TestDevelopment of a red precipitate or colorationTyrosine present

Burnt Test

Burnt test is a preliminary test to know whether given sample is carbohydrate or protein. You only observe the smell of that sample after boiling.

Procedure

  1. Take 8 drops of the unknown solution in a test tube.
  2. Boil it directly on Bunsen burner and smell it.
  3. If their is burnt hair smell observed then proteins may present.

Biuret Test

Biuret test is a confirmatory test for protein after performing burnt test. It is optional test for ninhydrin test.

Principle

This is a test for the presence of peptide bonds. A blue to purple color indicates a positive reaction. The reaction is not exactly typical for a peptide bond, as any compound with two carbonyl groups attached to a nitrogen or carbon atom will yield a positive result.

Chemicals and Materials

  • 1 ml of sample solution
  • 2-3 drops of CuSO4 solution
  • 1 ml of 10% NAOH solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 1 ml of the given sample solution into the test tube.
  3. Add 2 ml of sodium hydroxide (CuSO4) in the test tube with the help of dropper.
  4. Add 5 to 6 drops of copper sulfate solution to it.
  5. Shake the test tube gently to blend the ingredients well and allow the mixture to stand for 4 – 5 minutes.
  6. Mix thoroughly and note the color formed.
  7. If there is a bluish-violet color appearance, it indicates the presence of protein.

Ninhydrin Test

Ninhydrin test is a confirmatory test for protein after performing burnt test. It is optional test for biuret test. If this ninhydrin test observed positive then only you have to perform further tests for amino acids.

Principle

This test is positive for amino acids and proteins. The formation of a complex called Rheumann’s purple due to the condensation of two molecules of ninhydrin with one molecule of ammonia from amino acid is responsible for violet color. All amino acids give blue or purple color except proline and hydroxyproline, which give a yellow color under the same conditions.

Chemicals and Material

  • 1 ml of test solution
  • 5 drops of Ninhydrin solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 1 ml of the given sample solution into the test tube with the help of dropper.
  3. Add 5 drops of ninhydrin solution to test tube side by side.
  4. Boil the mixture for 2 minutes and cool.
  5. Note the change in colour.
  6. If there is a deep blue or purple appearance then the presence of protein is confirmed.

Xanthoproteic Reaction

Xanthoproteic test is usually performed to detect the presence of aromatic amino acids present in the given sample.

Principle

Nitration of the phenyl rings in the aromatic amino acids due to the HNO3 gives a yellow color due to the nitro substitution products. Addition of alkali produces orange color due to the formation of Na-salt (nitro derivatives).

Chemicals and Materials

  • 3 ml of test solution
  • 40% of NaOH solution
  • 1ml of conc. HNO3

Procedure

  1. Take a dry and sterile test tube.
  2. Add 3 ml of the given sample solution into the test tube with the help of dropper.
  3. Add a 1 ml of concentrated sulfuric acid (HNO3).
  4. Boil the test solution gently on a Bunsen burner.
  5. The solution turns yellow; then cool the test solution.
  6. Add 40% of NaOH solution till solution turns alkaline.
  7. Observe the change in colur.
  8. Formation of a deep yellow or orange color confirms that test is positive.

Sakaguchi Reaction for Arginine

Sakaguchi test is a specific test performed to detect the presence of Arginine amino acid in the given sample.

Principle

Substances containing guanidine groups (HN=C-NH4) react with the αnaphthol to form a bright red color. Arginine is the only amino acid that contains this group, and hence this test is specific for Arginine.

Chemicals and Materials

  • 3 ml of test solution
  • 1 ml of 5% NaOH
  • A drop of 1% CuSO4 solution
  • 2-3 drops of a 1% alcoholic solution of 1-naphthol
  • Few drops of sodium hypobromite (NaBrO) solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 3 ml of the given sample solution into the test tube with the help of dropper.
  3. Add 1 ml of 5% NaOH solution.
  4. Add a drop of 1% CuSO4 solution
  5. Add 2-3 drops of a 1% alcoholic solution of 1-naphthol, followed by a few drops of sodium hypobromite (NaBrO) solution.
  6. Observe the colouration.
  7. Development of a red colour indicates a positive test.

Sulfur Test for Cysteine and Cystine

Sulfur test specific test for presence of sulfur containing amino acids, i.e. cysteine and cystine.

Principle

Sulfur of the amino acids is the reacting group. Hence when proteins containing cysteine or cysteine are boiled with a strong alkali splits and forms Na2S. Addition of lead acetate causes formation of brown to black lead sulfide precipitate.

Chemicals and Materials

  • 3 ml of the test solution
  • An equal amount of 40% of NaOH solution
  • A drop of 2% lead acetate solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 3 ml of the given sample solution into the test tube with the help of dropper.
  3. Add an equal amount of 40% NaOH solution in it.
  4. Boil the test solution directly on Bunsen burner for 1-2 minutes.
  5. Then add a drop of 2% lead acetate solution.
  6. Observe the colouration.
  7. Black precipitate or brown coloration indicates a positive test.

Hopkins-Kole Test for Tryptophan

It is a specific test performed in laboratory to detect the presence of amino acid Tryptophan.

Principle

Indole group from the tryptophan reacts with glycoxilic acid released by the reaction of conc. H2SO4 on acetic acid to give the red color.

Chemicals and Materials

  • 2 ml of the test solution
  • 2 ml of glacial acetic acid
  • 2 ml of concentrated H2SO4 solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 2 ml of the given sample solution into the test tube with the help of dropper.
  3. Add 2 ml of glacial acetic acid with the help of plastic dropper.
  4. Then add 2ml of conc. H2SO4 carefully with precautions.
  5. Observe the colouration at the junction of the two solutions.
  6. Intense red color at the junction of the two solutions indicates a positive test.

Millon’s Test for Tyrosine

Millon’s test is specific to detect presence of amino acid Tyrosine.

Principle

The reaction is dependent on the formation of colored Hg- compound with phenolic (OH) group. The reaction is not specific for proteins however tyrosine is the only phenolic amino acid in proteins; hence a positive test indicates the presence of  tyrosine.

Chemicals and Materials

  • 1 ml of the test solution
  • 1 ml of Millon’s reagent
  • Few drops of 5% NaNO2 solution

Procedure

  1. Take a dry and sterile test tube.
  2. Add 1 ml of the given sample solution into the test tube with the help of dropper.
  3. Add 1 drop of millon’s reagent in it and shake well.
  4. A yellow precipitate sticking to the sides of the test tube develops due to the precipitation of proteins.
  5. Add a drop of 5% NaNO2 solution.
  6. Observe the change in colouration or precipitation.
  7. Development of a red precipitate or coloration indicates a positive test.

People also ask on Qualitative tests for protein

1. What are the qualitative tests for proteins?

Ans : Qualitative tests for proteins are those test which are being performed in the laboratories to detect the presence of protein and specific amino acids.

2. What are the quantitative test for proteins?

Ans : There are mainly 8 qualitative test for proteins. These 8 tests are: (1) Burnt Test (2) Ninhydrin Test (3) Biuret Test (4) Xanthoproteic Test (5) Sakaguchi Test (6) Sulfur Test (7) Hopkins-Cole Test and (8) Millon’s Test.

3. Is the qualitative test for protein that confirms the presence of arginine?

Ans : Yes. The name of qualitative test for arginine is Sakaguchi Test. This test is established by Japanese Food Scientist Shoyo Sakaguchi.

4. Which test is to confirm the presence of tryptophan?

Ans : Hopkins-Cole Test is one of the test to detect the presence of tryptophan.

5. Which is a confirmatory test to detect presence of protein?

Ans : The confirmatory test for qualitative test for protein is ninhydrin test or biuret test.

References and Sources

Further Readings

  1. Acid Fast Staining
  2. Aseptic Transfer Technique
  3. Bacterial Flagella, Fimbriae and Pili
  4. Growth Curve of Bacteria
  5. Instruments Used in Microbiology Laboratory
  6. MacConkey agar
  7. Monochrome Staining
  8. Negative Staining
  9. Serial Dilution in Microbiology
  10. Spread Plate Technique
  11. Streak Plate Technique

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