Lab Activity 8
Proteins part II
IUG, Spring 2014
Dr. Tarek Zaida
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Experiments
• A.A can be characterized qualitatively by using
several dyes that will react with certain groups
of the A.A.
 Seven Tests:
1. Ninhydrin
2. Biuret
3. Millon’s
4. Xanthoproteic
5. Hopkin’s- Cole
6. Sulfur
7. Sakaguchi
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1. Ninhydrin Test
• For amino acids containing a free NH2 & free
COOH.
• Reaction with ninhydrin to produce a colored
product.
1. 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.
2. Amino acids that have N-H (a secondary amino group (e.g.
proline) also react with ninhydrin, but they yield a yellow
product.
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Reaction of A.A with Ninhydrin
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Procedure..
1.
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.
2. Add 15 drops of each solution in the corresponding test tube.
3. To each of the test tubes add 5 drops of 0.5 % ninhydrin reagent
solution.
4. Place the test tubes into the boiling-water bath for 5 minutes.
Remove the test tubes from the water bath and place then in a
test tube rack.
Record your observations!
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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..
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Biuret complex with proteins…
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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!
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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.
Cysteine
Sulfur-containing protein ----> NaOH----> S2- ---Pb2+----> PbS
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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. Cool and record the results.
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7. 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
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Procedure
1. 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.
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