What is Pauly’s Test?

• There is a chemical assay called the Pauly reaction that can determine whether or not a protein contains the amino acids tyrosine or histidine.
• The German scientist Hermann Pauly is credited with describing this process, hence it bears his name. In alkaline circumstances, a coupling reaction between proteins containing tyrosine or histidine and diazotized sulfanilic acid yields a red colour.
• Highly coloured compounds can be formed when diazotized sulphanilic acid combines with amines, phenols, or imidazole.
• Since the diazonium component can only be produced at low temperatures, diazotization requires chilling the solution in ice.^{4, 6}

Purposes of Pauly’s Test

• Tyrosine and histidine, aromatic amino acids, detection

Principle of Pauly’s Test

The presence of tyrosine and histidine can be determined with this test with high sensitivity. Sulphanilic acid is dissolved in hydrochloric acid and used as the reagent in this test. When being exposed to sodium nitrite and hydrochloric acid, sulphanilic acid forms a diazonium salt by the process of diazotization. To produce a red chromogen, the produced diazonium salt combines with either tyrosine or histidine in an alkaline media (azo dye).^6,7

Reaction of Pauly’s Test

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

Diazonium salt + Tyrosine        →        Azo dye (red color)

Requirements for Pauly’s Test

• 1 % tyrosine, 1 % histidine, 1%glycine
• 1 % sulphanilic acid in 10 % HCI
• 5 % sodium nitrite
• 10 % sodium carbonate
• Ice bath

Procedure of Pauly’s Test

1. Take 2 millilitres of test solution in a dry test tube.
2. Take 2ml of distilled water in a separate test tube as a control.
3. Add 1 ml of sulphanilic acid, stir thoroughly, and store in an ice bath.
4. Add 1 millilitre of sodium nitrite solution to each test tube.
5. Place in an ice bath for three minutes.
6. Add 5 ml of sodium carbonate to create an alkaline solution.
7. Watch for the formation of a red complex.

Result and Interpretation of Pauly’s Test

• Positive test: The presence of tyrosine or histidine is indicated by the formation of a red hue.
• Negative test: The absence of red colour production indicates the absence of tyrosine or histidine.

Limitations of Pauly’s Test

• The production of diazonium salt happens at low temperatures; hence, the test should be conducted with ice present.
• The test is incapable of distinguishing between histidine and tyrosine. Histidine produces a negative result in the Millon’s Test, hence the test can be performed.

FAQ

References

1. Tiwari, A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.
2. https://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
3. https://quizlet.com/96596357/pauly-test-flash-cards/
4. https://en.wikipedia.org/wiki/Pauly_reaction
5. https://microbenotes.com/paulys-test/
6. http://biocheminfo.com/2020/04/17/paulys-test-principle-reaction-reagents-procedure-and-result-interpretation/
7. https://www.onlinebiologynotes.com/paulys-test-objective-principle-reagents-procedure-and-result/