XANTHOPROTEIC TEST

- Test for aromatic groups in proteins

Rgts: Egg white + conc. HNO3 and 50% NaOH

+VR: + conc. HNO3 yellow precipitate + 50% NaOH red-orange solution

Principle: Nitration by Substitution

Compound Detected: Benzene Ring

Compound responsible for +VR: Aromatic Nitro compounds (R-NO2)

Tyrosine

Tryptophan

Phenylalanine

- Benzene ring is stable, therefore, it does not readily undergo nitration

- Its oxidation to tyrosine will allow nitration to occur

Study Guide Questions

What is the chemical composition of Millon’s reagent? What amino acid in a protein is

responsible for a positive Millon’s test?

MILLON’S TEST

- Test especially for tyrosine and proteins containing tyrosine

Rgts: Mercuric and mercurous nitrate [(Hg(NO3)2 + (Hg2(NO3)2]

Nitric acid (HNO3)

Principle involved: Mercuration by substitution

+VR: Brick red color or precipitate

Group responsible: Phenolic compound

Compound detected: Proteins containing tyrosine

Experimental Procedures:

5 mL of milk was placed in a watch glass and heated over a steam bath until a film was

produced.

With the aid of a clean and dry stirring rod, the film was fished-out and a small portion of

the film was places into a clean and dry test tube.

5 drops of freshly prepared Millon’s reagent was added until a change in the color of the

film occurred.

CASEIN

- Main protein is milk

- It is a phosphoprotein, meaning that phosphate groups are attached to the hydroxyl

groups of some of the amino acid side-chains

- Exists in milk as the calcium salt, calcium calseinate

- Has an isoelectronic point of pH 4.6

If acid is added to milk, the negative charges on the outer surface of the casein

micelles are neutralized (by protonation of the phosphate groups) and the neutral

protein precipitates, with the calcium ions remaining in the solution

Ca-caseinate + 2H+ casein + Ca2+

Millon’s test – a specific test for tyrosine, the only amino acid containing a phenol

group, a hydroxyl group attached to a benzene ring

Millon’s reagent - is prepared from mercuric and mercurous nitrate in nitric acid that

when heated with Phenolic compounds give red coloration

Proteins that contain tyrosine will therefore yield a positive result

HOPKIN’S-COLE TEST

- Specific test for the presence of tryptophan in proteins since tryptophan is the only

amino acid having an

EXPERIMENTAL PROCEDURES:

The test is egg white solution (2mL) added with 2mL of Hopkin’s-cole reagent and was

mixed thoroughly.

While the tube was tilted 1mL of conc. Sulphuric acid was carefully poured along the

sides of the tube. Afterwards the tube was brought back to its original upright position.

The phenomenon can be observed when glycoxylic acid is added to certain protein

solutions and conc. Sulphuric acid is poured slowly down the container so that it

forms a layer on the bottom.

Rgts: Glycoxylic acid and conc. H2SO4

Principle involved: Condensation of C-5

+VR: formation of violet ring at the junction of two liquids

Group responsible: Indole group

Compound detected: Presence of tryptophan

Indoles – aromatic heterocyclic organic compound. It has a bicyclic structure,

consisting of a six-membered benzene ring fused to a five-membered nitrogen-

containing pyrole ring.