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Millon’s Test for Tyrosine

In this Millon’s test for tyrosine post we briefly summarise about: principle, reagents requirements, Millon’s test for tyrosine procedure, result, application and limitations of Millon’s test for tyrosine.

Definition

Millon’s test is only applicable to structures that contain phenol (tyrosine is the only common phenolic amino acid). Millon’s reagent is a concentrated HNO3 solution containing mercury. A red precipitate or a red solution is considered a positive test as a result of the reaction. 

A yellow HgO precipitate is not a positive reaction, but rather a sign that the solution is excessively alkaline. Tyrosine, phenylalanine, glycine, and β-naphtol can all be tested with this method.

Objective

To detect amino acid containing phenol group (hydroxyl group attached to benzene ring) i.e. Tyrosine.

Principle

Millon’s test is a tyrosine-specific identifying test. When tyrosine-containing proteins react with acidified mercuric sulphate solutions, a yellow mercury protein complex is formed. 

The yellow complex of mercury phenolate, which is red in colour, formed when sodium nitrite solution is added and heated. Tyrosine-containing proteins will therefore produce a positive result. 

Some tyrosine-containing proteins, on the other hand, generate a white precipitate that turns red when heated, while others form a crimson solution right away. Both outcomes are regarded as positive. 

It’s worth noting that any substance containing a phenol group will result in a positive test, so be sure the sample you’re testing doesn’t contain any phenols other than those found in tyrosine.

Millon’s Test

Tyrosine and Tryptophan

Requirements

Reagents

1. Millon’s Test Reagent: Mercuric and mercurous nitrates are dissolved in nitric acid and distilled water to make Millon’s test reagent. In a 400 mL concentrate nitric acid solution, dissolve 160 grams of mercuric nitrate and 160 grams of mercurous nitrate. After that, 600 mL of distilled water is added to the reagent to make it 1000 mL.

Materials

1. Test tubes

2. Test tube stand

3. Pipettes

4. Water bath

Procedure

1. To 2 ml of protein solution in a test tube add 2 ml of 10% mercuric sulfate (HgSO4) in 10% sulphuric acid. Boil for 30 seconds.

2. A precipitate may form at this stage. Add a few drops of 1% Sodium nitrite (NaNO2) and gently warm.

Observation

Millon’s Test

Millon’s Test Positive Result

Red precipitate forms and solution turns red. An amino acid solution gives red color without a precipitate. Protein contains the amino acid tyrosine which contains a phenolic radical.

Applications

1. Millon’s test is used for the detection of tyrosine-containing proteins in a given sample, kind of contrary to popular belief.

2. Millon’s Test also generally helps in the differentiation of tyrosine from basically other amino acids.

3. Millon’s Test really is useful in the detection of casein protein and the protein actually found in raw meat in a fairly major way.

Limitations

1. Salicylic acid and other phenolic compounds produce a positive response in this test; hence any other phenolic substances found in the test tube should be avoided. For confirmation, tests such as the Biuret test and the Ninhydrin test should be conducted.

2. Because the presence of chlorine in the solution may interfere with the reaction, the millon’s test cannot be done on a chloride-containing material.

3. Due to the denaturation of proteins by mercuric ions, a white or yellow precipitate may appear shortly after the addition of millon’s Test reagent.

Further Readings

Reference

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