Download Lab activity 8 Proteins 2 Alaa S Baraka Islamic university of Gaza

Lab activity 8
Proteins 2
Alaa S Baraka
Islamic university of
Gaza
March2013
Color test of amino acids and
proteins
• A.A can be characterized
qualitatively by using several dyes
that will react with certain groups of
the A.A.
Lab activity
• Sulfer test
• Sakaguchi test
• Ninhydrin test
• Biuret test
Sulfur test (Test for (-SH) group)
(To detect amino acid which containe sulfur group)
Principle
• Proteins containing sulfur (cysteine
and cystine and Methionine) give a
black deposit of lead sulfide (PbS)
when heated with lead acetate in
alkaline medium.
• Sulfur-containing protein ----> NaOH---->
S2- ----Pb2+----> PbS
Procedure
1. Add 1 ml of protein solution in a
test tube, add 2 ml of 10% sodium
hydroxide solution and 5 drops of
lead acetate.
2. labble the tubes and shake them.
then heat in a boiling water bath for
5 minutes.
3. Cool and record the results.
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.
Sakaguchi Test for Arginine
(detection of a specific type of protein with the amino acid containing the guanidinium group )
guanidinium group
Principle:
• α- naphthol and sodium
hypobromite/chlorite react with
guanidine group to form red orange
complexes.
• NaOBr+
>>>>>>>>>>>>
Quinonَ
Quinon
+ guanidine
>>>>>>>>>>>
Red
complex
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 % αnaphthol solution, and a few drops of
2 % hypobromite solution (NaOBr).
(For
Biuret
detecting peptide bonds )
Principle
• 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..
Biuret complex with proteins
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.
Ninhydrin Test
(general test for compounds with free a – amino groups)
• 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.
Reaction of A.A with Ninhydrin
O
O
O
OH
OH
+
R
H
+
OH
RCHO
+
CO 2
+
NH3
OH
NH2
O
O
O
O
OH
OH
O
+
2 NH3
+
H
+
NH4 O
O
HO
O
+
-
N
O
O
H2O
Procedure..
1.
2.
3.
4.
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
then in a test tube rack.
Record your observations!
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