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Anneliese Y. Abalos, 

, Lia Carmela S. Aranton,

Grace Marie Nicole Biso, and Zillah Glarilyn Calaunan
Group 1 2G Medical Technology Biochemistry Laboratory




The experiment revolved mainly on the reactions of intact casein and acid hydrolyzed casein to the different tests.
Positive results were manifested by both of the samples upon subjecting them to Biuret Test, Xanthoproteic Test,
Millon¶s Test, Fohl¶s Test, and Test for Amides. The intact protein obtained a positive result in Ninhydrin Test, and a
negative result in Hopkins-Cole Test, while the hydrolyzed casein obtained a positive result in Hopkins-Cole Test, and
a negative result in Ninhydrin Test.


  
Casein is a protein that is found in milk and
used independently in many foods as a binding
agent. The experiment shows the qualitative
tests done in intact casein and acidic hydrolyzed
casein using different reagents.
The objective of the experiment is to
characterize the properties of the protein casein
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by subjecting it to different color reactions as well
as to compare the reactions of the intact casein
and the acidic hydrolyzed casein with the same
qualitative tests.
 In 2 dry test tubes, 10 drops of each of the




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The compounds tested were the intact casein
and hydrolyzed casein.
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#!
In this test, 20 drops of 2.5 M NaOH was
*+ ,-(
In two separate test tubes containing the
samples, 20 drops of Hopkins-Cole reagent was
slowly added. Then, the test tubes were inclined
and 20 drops of concentrated H2SO4 was added.
The test tubes were not shaken. Only the colors
of the interface were noted. 
.
In this test, 5 drops of 30% NaOH and 2
drops of 5% (CH3COO)2Pb to each of the samples
in separate test tubes
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"
samples were placed separately. To each test
tube, 1 mL of 20% NaOH was placed and the
solutions were subjected to a boiling water bath.
A moistened red litmus paper was placed over
the mouth of each test tube and the changes in
color of the litmus papers were noted down.

 
  
introduced to the samples and mixed well. Then,
3 drops of 0.1 M CuSO4 solution was added to the
solutions. The test tubes were then shaken to
mix the solution well and the colors were noted
down.
$%"
 In two separate test tubes containing the
samples, 10 drops of 0.1% Ninhydrin solution
was added. The test tubes were then heated in a
boiling water bath and the appearance of a blue-
violet coloration was noted.
  
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In two separate test tubes containing the
samples, 10 drops of concentrated HNO3 was
slowly added and well mixed. The colors of the
each solution were noted. Then, 10 drops of
concentrated NaOH was slowly added into the
solutions. Lastly, the test tubes were mixed well
and the changes in color were noted.
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In this test, 5 drops of the Millon¶s reagent
was added to each of the samples in separate
test tubes. Changes in color were noted.


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Biuret test is used for detecting the presence
of peptide bonds. The biuret test relies on the
reaction between copper (II) ions and peptide
bonds in an alkaline solution. A violet coloration
indicates the presence of proteins.
Both the intact and the hydrolyzed casein
indicated a positive result in this test.
1(#Results obtained from Biuret Test
(
+%"%(
Biuret Test Pale purple Blue suspension
solution in purple
solution
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Amino acids contain a free amino group and
free carboxylic acid group that react together
with ninhydrin to produce a colored product.
When an amino group is attached to the first, or
alpha, carbon on the amino acids¶ carbon chain,
the amino acids¶ nitrogen atom is part of the
blue-purple product. Proteins also contain free
amino groups on the alpha-carbon and can react Millon¶s Test White cloudy White cloudy
with ninhydrin to produce a blue-purple product. solution solution
A blue-violet coloration was observed in the
intact protein upon adding ninhydrin reagent. No *+ ,-(
changes were obtained from the protein The Hopkins-Cole reagent only reacts with
hydrosylate when in fact there should be a blue- proteins containing tryptophan. The protein
violet coloration because it still contains free solution is hydrolyzed by the concentrated
amino groups. In this part, there was an error sulfuric acid at the solution interface. Once the
committed by the group which can be an error tryptophan is free, it reacts with glyoxylic acid to
from the preparation of samples or reagents, or it form the violet product.
can be an error in the execution of this part of A negative result was given by the intact
the experiment. protein because the tryptophan was not free or
 was not hydrolyzed enough for it to react with
1($Results obtained from Ninhydrin Test the glyoxylic acid, while the hydrolyzed protein
(
   gave a positive result which is a violet coloration
+%"%( at the interface.
Ninhydrin Test Blue-violet Cloudy solution
coloration 1(*Results obtained from Hopkins-Cole Test
(
  
&  +%"%(
This test is used to determine the presence Hopkins-Cole Yellow coloration Violet coloration
of aromatic rings in a given sample or the Test at the interface at the interface
presence of residues of tyrosine or tryptophan.
The solution to be tested is treated with .
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concentrated nitric acid, which will nitrate the Fohl's test is used to know if sulfur-containing
benzene rings of those residues. The nitrated amino acids are present. The positive result for
aromatic rings are yellow in color and are called this test is the appearance of dark (black or
Xanthoproteic acids (Xantho, = yellow, Greek). brown) sediment.
Both the intact protein and the protein Upon subjecting the samples to this test,
hydrolysate turned to a yellow solution which both manifested a positive result.
indicates a positive result for the presence of
aromatic rings. 1(.Results obtained from Fohl¶s Test
(
  
1(& Results obtained from Xanthoproteic Test +%"%(
(
   Fohl¶s Test Brown cloudy Brown Solution
+%"%( solution
Xanthoproteic Clear yellow Yellow solution
Test solution /0
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As the title suggests, this test is used to
'(() determine the presence of amides. Both samples
Millon¶s test is specific for tyrosine, the amino showed positive results. However, the litmus
acid containing a phenol group, a hydroxyl group paper used for the hydrolyzed protein took longer
attached to a benzene ring. In this test, the to change in color than that of the litmus paper
phenol group of tyrosine is first nitrated by nitric used for the intact protein.
acid in the test solution. Then the nitrated
tyrosine complexes mercury (I) and mercury (II) 1(/Results obtained from Fohl¶s Test
ions in the solution to form either a red (
  
precipitate or a red solution. Proteins that contain +%"%(
tyrosine will therefore yield a positive result. Test for Amides Red litmus Red litmus
(Litmus paper paper to blue paper to blue
However, some proteins containing tyrosine
test) litmus paper litmus paper
initially form a white precipitate that turns red
when heated.
In the case of casein, both the intact and the




hydrolyzed casein resulted to a white cloudy Milio, Frank R., Towson State Universityand
solution which means that both samples contain Loffredo, William M., East Stroudsburg University
tyrosine. http://www.cerlabs.com/experiments/108754044
80.pdf 1/08/2011
1('Results obtain from Millon¶s Test McGuigan, Brendan
(
   http://www.wisegeek.com/what-is-casein.htm
+%"%( 1/08/2011
Palleros, Daniel R., Experimental Organic
Chemistry
http://www.uwlax.edu/faculty/koster/newlactose.
htm 1/08/2011
http://www.mondofacto.com/facts/dictionary?biu
ret+test 1/08/2011