Isolation and Characterization by Color Reaction of the Protein Casein

*B. Ocampo, F. Padilla, K. M. Park, R. Payuyo, D. Ponciano, J. V. Regala

Group 7, 2CMT, Faculty of Pharmacy, UST

Abstract
The objective is to isolate it in order to perform hydrolysis to break its components and color
reaction for its characterization. Acetic acid was added to isolate casein by the formation of a
solid particle called curd. Then, to breakdown its amino acid components acid hydrolysis was
performed by using 6M HCl. Color reaction was performed to characterize the protein and its
functional groups plus the free amino acids. This white solid particle that precipitated after the
addition of acetic acid is confirmed to be casein.
Introduction
Protein--Casein
Protein is a biological molecule which can be classified into two: Fibrous and Globular. Fibrous
protein is a long chain or sheet of amino acids while Globular protein is a compact kind of
spheroidal shape. They are naturally occurring polymer composed of amino acid as its monomer.
Each monomer is connected by peptide bonds. Peptide bonds are amide bonds formed between
the carboxylic acid group of one amino acid and the carboxylic group of a second amino acid.
The solubility of amino acids is largely dependent on the solution pH. The structural changes in
amino acid take place at different pH values that alter the relative solubility of the molecule. In
acidic conditions, both amino and carboxylic groups are protonated. In basic groups, both groups
are unprotonated. The aim of this experiment is to isolate the protein--Casein, an example of
protein found in milk, by adding acetic acid and to identify what amino acids were present in
casein.
Casein (Isolation and Color Reaction)
Casein is phosphoprotein which has phosphate groups attached to the hydroxyl groups of some
of the amino acids side-chains. It exists in milk as a calcium salt, calcium caseinate.  It has an
isoelectric point of pH 4.6. This means it is insoluble in solutions with a pH less than 4.6. The
pH of milk is 6.6; therefore, casein has a negative charge at this pH and is solubilized as a salt.
When hydrochloric acid was added, the casein was neutralized due to the addition of protons to
the phosphate group and the pH is decreased to that of the isoelectric point (point at which the
net charge is zero and the casein is insoluble). Because of this, casein became polar and caused
its precipitation.
Acetic acid, an example of weak acid, was added to isolate casein from milk. Moreover, 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 and their accessibility to
the reagent. The following tests were performed to characterize the protein and determine the
presence of what amino acid:
 Biuret Test was used to detect the presence of peptide bonds. It should produce a violet
color.
 Ninhydrin Test was used for an alpha-amino acid. Amino acids contain free amino group
and free carboxylic acid group that react together with Ninhydrin to produce a blue-violet
solution.
 Xanthoproteic Test detects the side chains of aromatic amino acids which will produce
yellow solution. Xanthoproteic comes from the greek word which means “yellow”.
Hence the name of the test.
 Millon’s test determines tyrosine residues. A red precipitate or red solution indicates a
positive result.
 Hopkins-Cole Test is specific for tryptophan. It should produce a violet solution.
 Sakaguchi test is to detect arginine. The sample to be tested is treated with alpha-
naphthol and sodium hypochlorite. A positive result yields a reddish wine color when
arginine is present. 
 Nitroprusside is used to determine the presence of cystein, the only amino acid containing
a sulfhydryl group (-SH). A red solution should be observed.
 Fohl’s test is used to detect presence of sulfur containing amino acid. A black or brown
solution is a positive result.
 Test for Amides is used to detect asparagines and glutamine. Yellow solution should be
produced.
Materials & Methods
Isolation
To be able to isolate casein from milk, the latter’s pH should be decreased at a point where
casein is insoluble. And that is 4.6. To adjust the pH of milk, 10% acetic acid was added
dropwise to the solution while it is being heated on a 40 degree Celsius temperature. If it happens
that the temperature went beyond the required, casein will disintegrate. When the desired pH was
reached, curds formed and the milk was almost opaque. This only shows that casein, the protein
that gives the milk its milky color, is being separated from it.
Qualitative Color Reaction
Biuret Test :
Twenty drops of 2.5M NaOH then 2-3 drops of 0.1 M CuSO 4 solution.
Everything was mixed into one consistency.

Ninhydrin Test:
Six to ten drops of 0.1% ninhydrin solution to the diluted sample and heated it to
a water bath. The colore produced was noted.
 Xanthoproteic Test:
Ten drops of concentrated HNO3 was slowly added then the color was noted.
After that, 10 drops of concentrated NaOH was added and again another color was
produced.

Millon’s test:
Five drops of Millon’s reagent was placed and a color was produced.

Hopkins-Cole Test:
Twenty drops of Hopkins-Cole reagent was added to the sample. The test tube
was inclined and 20 drops of concentrated H 2SO4 wad added. But this time, the mixture
was not shaken.

Sakaguchi test:
Ten drops of 10% NaOH and 10 drops of 0.02% naphthol solution was added and
was let to stand for 5 minutes. Three drops of NaOBr was added afterwards. The colore
was noted.

Nitroprusside:
Three molar of NaOH, 0.05 ml, was added to 0.5ml sample. Then 0.25 ml of 2%
nitroprusside was added and red solution should be expected to form.

Fohl’s test:
Five drops of 30% NaOH and 2 drops 5% (CH 3COO)2Pb was added then it was
placed on a boiling water bath. Dark brown sediment should be observed.

Test for Amides:

One milliliter of 20% NaOH was added to 10 drops of the sample. It was placed
in a boiling water bath. Moistened litmus paper was placed on top of the test tube. The
result was noted.
Results & Discussion
The expected result from this experiment is a white solid particle which will precipitate from
milk when acetic acid was added. The procedure did produce a white precipitate. The milk is
positive to have casein. It can be concluded that the negativity of casein was neutralized by the
addition of 10% acetic acid. And since the isoelectric point of casein is 4.6, the point at which it
is insoluble, it precipitated when the solution reached this pH.
In this experiment, various color-producing reagents were used to qualitatively detect the
presence of certain functional groups in casein.
 Table 1: Results of the Color reaction

Color Reaction Result

Biuret Violet solution
Blue violet deposit and
Ninhydrin
solution
Xanthoproteic Yellow solution
Millon’s Fleshy solution
Hopkins-Cole Violet solution
Sakaguchi Red solution
Nitroprusside Yellow solution
Fohl’s Black and brown sediments
Basic(red litmus paper to
Test for Amide
blue); yellow solution

The results show that all tests were positive and that all the amino acid that these tests determine
are present:
Biuret confirmed the presence of peptide bonds which produced a violet color. Ninhydrin gave a
positive result for an alpha-amino acid which gave out a blue-violet color. Xanthoproteic
confirmed the presence of aromatic amino acid by producing a yellow solution. Millon’s test
didn’t exactly produce a red solution so tryrosine might not be present. Hopkins-Cole showed
that tryptophan is present when it produced a violet solution. Sakaguchi test yielded a red
solution which is a positive result for the presence of arginine. Nitroprusside is negative for it
yielded a yellow solution. Fohl’s test was positive with the black brown sediment. The test for
amide confirmed the presence of aspargine and glutamine when it produced a yellow solution in
a water bath.
To conclude, a white solid precipitate signified that milk has casein and that it is composed of
several amino acids joined by a peptide bond. By using color reaction, the following kinds of
amino acids were found to be present: aromatic amino acid, tryptophan, arginine, sulfur-
containing amino acid, aspargine, and glutamine.

Reference
Book:

Crisostomo, A. C. (2010). Laboratory Manual in General Biochemistry (p.18). Quezon City:

C&E Publishing, Inc.

Website:

http://homepages.ius.edu/DSPURLOC/c122/casein.htm

http://www.cerlabs.com/experiments/10875404480.pdf
http://www.reference.com/motif/Science/sakaguchi-test-amino-acid-proteins