8 Qualitative Tests for Protein

Proteins are prevalent in every aspect of the living world, present regardless of the size or complexity of the organism, as they form the bedrock of cellular structure and function. There may be situations when identifying these proteins becomes essential. To detect the presence of proteins in an unknown solution, we can employ tests like the Ninhydrin Test, used specifically to identify amino acids and proteins containing free amino groups, or the Biuret Test, which can detect peptide bonds indicative of proteins.

Following the confirmation of proteins in a sample, we can further distinguish the specific amino acids contained within it. For instance, we may perform Millon’s test to specifically pinpoint Tyrosine, an amino acid that plays crucial roles in protein structure and enzyme functions. The Xanthoproteic test can help us uncover the existence of aromatic amino acids, crucial components in protein structure that often interact with each other to form protein’s tertiary structure. To find Tryptophan, a building block of protein that regulates many physiological processes, we could use the Hopkins-Cole test.

For more comprehensive exploration into proteins, one can proceed with various precipitation tests:

• Heavy metal ions, like Na2CO3, AgNO3, CuSO4, HgCl2, etc. are often used in protein precipitation tests. These ions are chosen due to their significant interaction with protein structures, leading to their precipitation.
• Alkaloidal reagents (TCA, Picric acid and meta-phosphoric acid) 
• By salt ( (NH4)2SO4, Na2SO4, NaCl) 
• Organic solvents (Ethanol, Acetone) 

Qualitative Tests for Protein

Given the vast array of tests available to confirm the presence of protein in a sample solution, in this article, we're going to delve into 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

Significant Tests in Protein Detection	Insightful Observations from Protein Testing	Inference
Burnt Test	Burnt hair smell	Proteins may be present
Biuret Test	The appearance of bluish- violet color	Protein Confirmed
Ninhydrin Test	The appearance of deep blue or purple color	Protein Confirmed
Xanthoproteic Test	Formation of a deep yellow or orange color	Aromatic amino acids present
Sakaguchi Test	Development of a red colour	Arginine present
Sulfur Test	Appearance of Black precipitate or brown coloration	Cystine present
Hopkins-Kole Test	Appearance of Intense red color at the junction of the two solutions	Tryptophan present
Millon’s Test	Development of a red precipitate or coloration	Tyrosine 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 of Burnt Test

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 of Biuret Test

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 of Biuret Test

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 of Ninhydrin Test

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 of Ninhydrin Test

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 Test

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

Principle of Xanthoproteic Test

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 of Xanthoproteic Test

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 Test for Arginine

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

Principle of Sakaguchi Test

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 Sakaguchi Test

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 of Sulfur Test

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 of Sulfur Test

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 of Hopkins-Kole Test

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 of Hopkins-Kole Test

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 of Millon’s Test

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 of Millon’s Test

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.

FAQs on Qualitative Test for Proteins

References and Sources

• https://himedialabs.com/td/htbc004.pdf
• https://byjus.com/biology/test-for-protein/
• https://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-amino-acids-and-proteins/13065
• https://www.onlinebiologynotes.com/qualitative-tests-for-amino-acids-and-proteins/
• https://microbenotes.com/xanthoproteic-test/

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