1. Lab Activity 8 Proteins part II
IUG, 2015
Dr. Tarek Zaida

2. Experiments
A.A can be characterized qualitatively by using several dyes that will react with certain groups of the A.A.
Seven Tests:
Ninhydrin Xanthoproteic Sakaguchi
Biuret 5. Hopkin’s- Cole
Millon’s 6. Sulfur

3. For amino acids containing a free NH2 & free COOH.
1. Ninhydrin Test
For amino acids containing a free NH2 & free COOH.
Reaction with ninhydrin to produce a colored product.
When NH2 is attached to α-C on the amino acid’s carbon chain, the amino group’s N is part of a blue-purple product.
Amino acids that have N-H (a secondary amino group (e.g. proline) also react with ninhydrin, but they yield a yellow product.

4. Reaction of A.A with Ninhydrin

5. Procedure..
Label 6 cleaned, drained test tubes with the names of the following solutions: 2 % glycine, 1 % tyrosine, 2 % proline, 2 % casein, 2 % gelatin, 2 % albumin.
Add 15 drops of each solution in the corresponding test tube.
To each of the test tubes add 5 drops of 0.5 % ninhydrin reagent
solution.
Place the test tubes into the boiling-water bath for 5 minutes.
Remove the test tubes from the water bath and place them in a test tube rack.
Record your observations!

6. 2. Biuret For detecting peptide bonds (hence peptides or proteins)..
How it works?
The copper atoms of Biuret solution (CuSO4 ) in a basic environment will react with peptide bonds (-CO ---NH) to form a chelate of a deep violet color, indicating the presence of proteins.
A light pink color indicates the presence of peptides..

7. Biuret complex with proteins…

8. Procedure..
1. To 1 ml of a solution containing protein add 4 ml of a biuret reagent. 2. Mix well, then let to stand at RT for about 30 min. 3. Record your observations!

9. For the detection of sulfur-containing amino acids such as cysteine.
3. Sulfur Test
For the detection of sulfur-containing amino acids such as cysteine.
Is done by converting S to an inorganic sulfide ( S2-) through cleavage by a base.
When the resulting solution is combined with lead acetate (CH3COOPb), a black precipitate of lead sulfide is formed.
Sulfur-containing protein  ----> NaOH---->  S Pb2+---->  PbS
Cysteine

10. Procedure..
1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0.1 M cysteine into separate, labeled test tubes. 2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution. 3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. 4. Cool and record the results.

11. 4. Sakaguchi
For detection of the amino acid containing the guanidinium group (e.g. arginine).
In basic conditions, α- naphthol and sodium hypobromite/chlorite react with the guanidinium group to form red orange complexes.
Guanidinium
group
Arginine

12. Procedure
Add 1 ml of 3 N NaOH solution to 1 ml of the protein solution, followed by addition of 0.5 ml of 0.1 % α- naphthol solution, and a few drops of 2 % hypobromite solution (NaOBr).
2. The formation of a red color indicates the presence of a guanidinium group in the compound under examination.