Millon’s test: Principle, Reaction, Reagents, Procedure and Result Interpretation

Millon’s test (Cole’s test)

Test For  Detection of Tyrosine

Principle:

Millon’s test is specific test for identification of tyrosine. Tyrosine containing protein when reacts with acidified mercuric sulphate solutions gives yellow precipitate of mercury protein complex. On addition of sodium nitrite solution and heating, the yellow complex of mercury phenolate forms, which is red in colour. Proteins that contain tyrosine will, therefore, yield a positive result.

However, some proteins containing tyrosine initially forms a white precipitate that turns red when heated, while others form a red solution immediately. Both results are considered positive. Note that any compound with a phenol group will yield a positive test, so one should be certain that the sample that is to be tested does not contain any phenols other than those present in tyrosine.

Reaction:

Reagents:

• 10% precent mercuric sulphate in 10% percent sulphuric acid (acid mercuric sulphate solution)
• 1% sodium nitrite (NaNO₂) solution
• Test solution: 1 % arginine, 1 % tyrosine, protein solution, phenol solution

Procedure:

• Take 1 ml of above solutions in different test tubes.
• Add 1 ml of 10% percent of acid mercuric sulphate solution on.
• Boil gently for 30 seconds.
• Cool and add 2 drops of 1% of sodium nitrite solution.
• Mix well (observe color formation)
• Formation of pinkish red color indicates the presence of hydoxy phenyl (phenol) group containing amino acid tyrosine.

Result Interpretation:

• Positive Millon’s test: Brick red color (Tyrosine and phenol solution)
• Negative Millon’s test: no red color ( absence of Tyrosine and phenol solution)

Remember: A yellow precipitate of HgO is NOT a positive reaction but usually indicates that the solution is too alkaline.

Note:

• Salicyclic acid and other phenolic compounds give positive reaction with this test.
• Chloride interferes with this reaction, hence the test cannot be done in chloride containing samples like urine.
• The proteins, on the addition of Millon’s reagent, form a white precipitate first due to denaturation of proteins by mercury salts,

References:

• https://www.onlinebiologynotes.com/millons-test-objective-principle-reagents-procedure-and-result/
• https://www.jbc.org/content/66/2/475.full.pdf
• https://cpb-us-w2.wpmucdn.com/sites.wustl.edu/dist/f/1200/files/2018/10/ Folin_1927-2553row.pdf
• http://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-amino-acids-and-proteins/13065