1. Color Tests of Amino Acids
CLS 233
Basic Biochemistry
2nd Semester
Practical # 2:
Color Tests
of Amino Acids

2. Introduction The food we consume is divided into three main classes:
Carbohydrates: the body’s most readily available energy source.
Lipids: the body’s principal energy reserve.
Proteins: the body’s source of energy for growth and cellular maintenance.
Proteins also make up the second largest portion of cells, after water.
Proteins consists of amino acid linked together by peptide bond.

3. Amino acids incorporated into proteins are covalently linked by peptide bonds.

4. Note: each amino acids consists of:
Carboxylic acid groups (-COOH).
Amino groups (-NH2). H.
Side chains (-R).
Different side chain result in various amino acid .

5. Chemical Reactions of Amino Acids and Protein Functional Groups
Certain functional groups in amino acids and proteins can react to produce characteristically colored products.
The color intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional, or free, groups present.
In this part of experiment, various color-producing reagents (dyes) will be used to qualitatively detect the presence of certain functional groups in amino acids and proteins.

6. Ex (1). Millon Reaction
Principle: Millon's reagent (Hg/HNO3) gives positive results ( pink to dark-red color) with proteins containing the phenolic amino acid “tyrosine”
Purpose: To detect the amino acid that have phenol group of tyrosin.
Note: some proteins containing tyrosine will initially form a white precipitate that turns red when heated, while others form a red solution immediately. Note that any compound with a phenol group will yield a positive test, so one should be certain that the sample being tested does not contain any phenols other than those present in tyrosine.

7. Procedure: Result: Tyr: gives red ppt
In test tube add 2 ml of protein into separate labeled test tubes.
Add 3-4 drops of Millon’s reagent, and immerse the tubes in a boiling water bath for 5 minutes.
Cool the tubes and record the colors formed.
Result:
Tyr: gives red ppt
A white ppt is formed with albumin and casein, ppt gradually turns into red.

8. Ex (2). Xanthoprotic Reaction
Some amino acids contain aromatic groups that are derivatives of benzene.
These aromatic groups can undergo reactions that are characteristic of benzene and its derivatives.
One such reaction is the nitration of a benzene ring with nitric acid.
The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo nitration.
Tryptophan has inactivated benzene ring
Phenylalanine still reacts with nitric acid, just not as readily as Tyr or Trp because Tyr and Trp have electron donors (hydroxyl and the nitrogen heteroatom), which make the ring a lot more attractive for electrophilic nitration.

9. Purpose: used to identify the presence of an activated benzene ring.
Principle: Nitric acid gives color when heated with proteins containing tyrosine (yellow color) or tryptophan (orange color); the color is due to nitration.
Purpose: used to identify the presence of an activated benzene ring.
Note: If one spills a concentrated solution of nitric acid onto someone’s skin. The proteins in skin contain tyrosine and tryptophan, which become nitrated and turn yellow.
phenyl ring gets nitrated to give yellow colored nitro-derivatives. At alkaline pH the color changes to orange due to the ionization of the phenolic group.

10. Procedure
Add 2 ml of protein solution in a test tube and add 2 drops of concentrated nitric acid.
The formed white precipitate, will turn yellow upon heating, and finally will dissolve giving a yellow color to the solution.
Cool the solution down. Carefully add 3 ml of 6 N NaOH. Note that the yellow color turns orange.
Tyrosine, Albumin, gelatin (Positive result)

11. Ex (3). (Hopkins-Colé test):
Principle: The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product.
Purpose: The Hopkins-Cole test is specific for tryptophan, the only amino acid containing indole group. .
Tryptophan

12. Procedure:
Add 1 ml of protein solution in a test tube, add 1 ml of Hopkins-Colé reagent and mix well.
Incline the test tube and slowly add 1 ml of concentrated H2SO4 on the inner wall of the test tube thus forming a reddish - violet ring at the interface of the two layers.

13. Result:
A reddish violet ring is formed at the junction between the 2 layers with albumin and casein.
Gelatin gives negative results.

14. Ex (4). Reduced sulfur test (Test for (-SH) group)
Principle: Proteins containing sulfur (like: Methionine and cystine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium.
Purpose: To detect amino acid which containe sulfer group.
Sulfur-containing protein ----> NaOH----> S Pb2+----> PbS

15. Procedure:
Add 1 ml of protein solution in a test tube, add 2 drops of 10% sodium hydroxide solution and 2 drops (or few) of lead acetate.
Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes.
Cool and record the results.

16. Result: A black deposit is formed with albumin
while a slight black turbidity is obtained with casein due to its lower content of sulfur.
Gelatin gives negative result.

17. Ex (5). Sakaguchi Test for Arginine:
Principle: α- naphthol and sodium hypobromite/chlorite react with the above mentioned compound to form red orange complexes.
Purpose: used for the detection of a specific type of protein with the amino acid containing the guanidinium group (e.g. arginine).

18. Procedure
To 1 ml of the protein solution add 1 ml of 3 N NaOH solution and 0.5 ml of 0.1 % α- naphthol solution, and a few drops of 2 % hypobromite solution.
The presence of a guanidinium group in the compound under examination will be confirmed by the formation of a red color.

19. Ninhydrin Test
Ninhydrin is a chemical used to detect free amino acid and proteins
Amino acids(NH2) also react with ninhydrin at pH=4.
The reduction product obtained from ninhydrin then reacts with NH3 and excess ninhydrin to yield a blue colored substance.
This reaction provides an extremely sensitive test for amino acids.

20. Procedure To 1 mL solution add 5 drops of 0.5% ninhydrine solution
Boil over a water bath for 2 min.
Allow to cool and observe the blue color formed.
Result: all amino acid will give purple or deep blue with exception Proline gives yellow not violet.

21. Proline reacts with ninhydrin, but in a different way
Proline reacts with ninhydrin, but in a different way. While most ninhydrin tests result in a purple color, the proline reaction is more yellow due to substitution of the alpha amino group that ninhydrin reacts with carbon rings