Pauly’s Test- Definition, Principle, Procedure, Result, Uses

Proteins are essential biomolecules that perform various functions in living organisms. Proteins are composed of amino acids, which are the building blocks of proteins. Amino acids have different chemical properties and structures that determine their role and function in proteins. Some amino acids have aromatic groups, such as phenol or imidazole, that can react with certain reagents and produce characteristic colors. These reactions can be used to detect and identify these amino acids in proteins.

One such reaction is Pauly’s test, which is a biochemical test for the detection of tyrosine and histidine in proteins. Tyrosine and histidine are two amino acids that have aromatic groups that can undergo coupling with diazonium salts. Diazonium salts are compounds that have a nitrogen-nitrogen double bond attached to an aromatic ring. They are highly reactive and can form colored complexes with other aromatic compounds.

Pauly’s test is named after the German chemist Hermann Pauly, who discovered the test in 1894. He used sulphanilic acid as the source of diazonium salt and observed that it reacted with tyrosine and histidine to produce red-colored compounds. He also noticed that the color intensity varied depending on the pH of the solution. Pauly’s test is a specific test for proteins containing tyrosine and histidine as the reagent undergoes diazotization with aromatic groups. The reaction is performed under cold conditions as the diazonium compound can only form at low temperatures.

Pauly’s test is a simple and rapid method to detect the presence of tyrosine and histidine in proteins. It can also be used to differentiate between these two amino acids from other amino acids that do not react with diazonium salts. The test can be performed in a test tube using a few reagents and materials. The result of the test is indicated by the appearance or absence of a red-colored solution.

In this article, we will discuss the objectives, principle, procedure, result, uses, and limitations of Pauly’s test in detail. We will also explain the reaction involved in Pauly’s test and how it produces the characteristic color change. We hope that this article will help you understand Pauly’s test better and appreciate its importance in biochemistry.

Pauly’s test is a biochemical test that aims to achieve the following objectives:

• To detect the presence of tyrosine and histidine-containing proteins in a given sample. Tyrosine and histidine are two of the 20 amino acids that make up proteins. They have distinctive chemical structures that allow them to react with the pauly’s reagent and form a red-colored complex.
• To differentiate between histidine and tyrosine from other amino acids that do not react with the pauly’s reagent. Other amino acids either do not have aromatic groups or have different functional groups that do not couple with the diazonium salt. Therefore, they do not produce any color change in the test.
• To provide a simple and rapid method for identifying tyrosine and histidine in proteins. The test can be performed in a few minutes with minimal equipment and reagents. The color change is easily observable and does not require any sophisticated instruments or measurements.

The test is useful for studying the structure and function of proteins that contain tyrosine and histidine residues. These amino acids are involved in various biological processes such as enzyme catalysis, protein-protein interactions, signal transduction, and gene regulation. By detecting their presence in proteins, one can infer some information about their roles and properties.

Pauly’s test is based on the principle of coupling between the amino acids and the diazonium ion formed in the reagent. The pauly’s reagent consists of sulphanilic acid dissolved in concentrated hydrochloric acid. The sulfanilic acid undergoes diazotization in the presence of sodium nitrate and hydrochloric acid. The result of the diazotization reaction is the diazonium salt (p-phenyldiazosulphonate).

The diazonium salt thus formed couples with histidine molecule in an alkaline condition to form a dark red or cherry colored compound. The color might decrease in intensity and changes into an orange color when the solution is made acidic. In the case of tyrosine, a similar cherry red-colored complex is formed which then changes into yellow color on dilution and bronze-yellow color on acidification.

Each molecule of histidine and tyrosine reacts with two moles of diazonium compound reacts to form a mole of bis(azobenzenesulphonic acid)histidine or bis(azobenzenesulphonic acid)tyrosine.

The reaction can be represented by the following equations:

Sulphanilic acid + HNO2 + HCl → Diazonium salt + 2H2O

Diazonium salt + Tyrosine → Azo dye (red color)

Diazonium salt + Histidine → Azo dye (red color)

The formation of azo dye indicates the presence of tyrosine and histidine in the sample. The test is specific for these two amino acids as other amino acids do not react with the diazonium salt. The test can also be used to differentiate between proteins containing tyrosine and histidine from other proteins.

Requirements for performing Pauly’s Test

To perform Pauly’s test, the following reagents and materials are required:

Reagents

• 1% Sulphanilic acid (chilled): This is the main reagent that undergoes diazotization in the presence of sodium nitrite and hydrochloric acid to form the diazonium salt that reacts with tyrosine and histidine.
• 10% HCl: This is used to dissolve the sulphanilic acid and to provide acidic conditions for the diazotization reaction.
• 5% Sodium nitrite (chilled): This is used as a source of nitrous acid that reacts with sulphanilic acid to form the diazonium salt.
• 10% Sodium carbonate: This is used to make the solution alkaline, which facilitates the coupling reaction between the diazonium salt and the amino acids.
• Chilled sample (1% tyrosine, 1% histidine): These are the amino acids that are tested for their presence by Pauly’s test. The sample should be chilled to prevent the decomposition of the diazonium salt.

Materials required

• Ice bath: This is used to keep the reagents and the sample at low temperatures, which is essential for the formation and stability of the diazonium salt.
• Vortex: This is used to mix the reagents and the sample thoroughly and quickly.
• Test tubes: These are used to hold the reagents and the sample during the test.
• Test tube stand: This is used to support the test tubes during the test.
• Pipettes: These are used to measure and transfer small volumes of reagents and sample during the test.

• To a test tube, add 1 ml of chilled sulfanilic acid and mix it with a few drops of pre-chilled sodium nitrite in a vortex.
• Immediately add 1 ml of the amino acid sample to the same test tube and mix it in a vortex.
• Add a few drops of sodium carbonate to the test tube drop by drop until a color begins to appear.
• Observe the color change and compare it with the expected results.

The procedure should be performed in an ice bath to maintain the cold temperature required for the formation of diazonium salt. The test tube should be shaken well after each addition to ensure proper mixing of the reagents. The color change should be noted carefully as it might vary depending on the concentration and pH of the solution.

The result of Pauly’s test depends on the formation of a red-colored complex between the diazonium salt and the amino acids tyrosine and histidine. The intensity and shade of the color may vary depending on the concentration and pH of the solution.

• Positive result: A positive result is indicated by the appearance of a red-colored solution, which shows the presence of tyrosine and histidine in the sample. The red color is due to the formation of azo dyes by the coupling reaction between the diazonium salt and the amino acids. The color may change to orange or yellow on acidification or dilution, respectively.
• Negative result: A negative result is indicated by the absence of a red-colored solution, which shows the absence of tyrosine and histidine in the sample. The solution may remain colorless or turn slightly brown due to the decomposition of the diazonium salt.

The following table summarizes the results and interpretation of Pauly’s test for different amino acids:

Pauly’s test is a specific test for proteins containing tyrosine and histidine as these are the only amino acids that can react with the diazonium salt to form a colored complex. The test can be used to detect and differentiate these amino acids from other amino acids in a protein sample.

Pauly’s test is a biochemical test that has several uses in the field of biochemistry and molecular biology. Some of the uses of Pauly’s test are:

• Pauly’s test is used to detect the presence of tyrosine and histidine-containing proteins. These are two important amino acids that are involved in various biological processes, such as enzyme catalysis, protein structure, signal transduction, and gene regulation. By performing Pauly’s test, one can identify whether a protein sample contains these amino acids or not.
• Pauly’s test is also used to differentiate between histidine and tyrosine from other amino acids. This is useful for the analysis and characterization of proteins and peptides. By comparing the color changes of different amino acid samples after adding Pauly’s reagent, one can distinguish histidine and tyrosine from other amino acids that do not react with the reagent.
• Pauly’s test can also be used as a preliminary test for protein purification. Since Pauly’s test is a specific test for proteins containing histidine and tyrosine, it can be used to check the purity of a protein sample before further purification steps. If a protein sample gives a positive result in Pauly’s test, it means that it contains histidine and tyrosine residues, which can be further purified by methods such as affinity chromatography or ion-exchange chromatography. If a protein sample gives a negative result in Pauly’s test, it means that it does not contain these amino acids, and thus may require different purification methods.

• Pauly’s test is a specific test for proteins containing tyrosine and histidine, but it cannot differentiate between these two amino acids. Both tyrosine and histidine produce a red-colored complex with the diazonium salt, which changes color on acidification. To distinguish between them, another test such as Millon’s test can be performed, which gives a negative result for histidine.
• Pauly’s test requires cold conditions for the formation of the diazonium salt from sulphanilic acid and sodium nitrite. The reaction is sensitive to temperature and pH changes, and the color intensity may vary depending on these factors. Therefore, the test should be performed in an ice bath and with chilled reagents to avoid decomposition of the diazonium salt.
• Pauly’s test may give false-positive results with other compounds that have phenolic or imidazole groups, such as phenol, aniline, or tryptophan. These compounds can also react with the diazonium salt and produce colored complexes. Therefore, the test should be performed with pure samples or after separating the amino acids by chromatography or electrophoresis.
• Pauly’s test may give false-negative results with proteins that have low concentrations of tyrosine and histidine or that have these amino acids in inaccessible positions. The reaction depends on the availability of the phenolic and imidazole groups for coupling with the diazonium salt. Therefore, the test may not detect proteins that have tyrosine and histidine buried in their tertiary structure or that have low amounts of these amino acids in their composition.

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