ISOLATION AND CHARACTERIZATION OF CASEIN

Hazel Ann Gianelli Y. Cu, Ma. Athena C. del Rosario, Edward I. Dike,
Kenneth Charles P. Dino, Junelle A. Dumangon and Clarisse Ann H. Duran
Group 3 2H Medical Technology General Biochemistry Laboratory

ABSTRACT
There were three proteins that were isolated in protein isolation which are assigned to different groups. Group 3
isolated casein from low fat milk using isoelectric precipitation with acetic acid. After precipitating casein, several
samples were used for the qualitative color reactions. Intact protein, acid hydrolysates and basic hydrolysates
were used in performing Biuret test, Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, Sakaguchi
test, Nitroprusside test, Fohl’s test and test for amides. The intact protein was negative for Ninhydrin test and
Millon’s test and the rest of the tests were positive. The acid hydrolysate yielded negative results in Biuret test,
Ninhydrin test, Millon’s test and Hopkins-Cole test. Lastly, the basic hydrolysate had negative results in Biuret test,
Ninhydrin test, Millon’s test, Sakaguchi test, and Nitroprusside test. After the qualitative color reactions, paper
chromatography was performed for the separation and identification of amino acid standards based on the
polarities of tryptophan, arginine, proline, cysteine, serine, aspartate, histidine, glycine and alanine. Some of the
group 1 hydrophobic amino acids were closer to the solvent front. Group 2 polar, uncharged amino acids were in
the middle and group 3 polar, charged amino acids stayed at the bottom. After performing the paper
chromatography, protein concentration was determined through Bradford Protein Assay. Albumin standard curve
was constructed and the unknown concentration of protein was determined using linear regression analysis. The
graph studied showed the direct relationship of Bovine Serum Albumin concentration to its absorbance.

INTRODUCTION In chromatography, there are different types

Protein isolation is a process for isolating a
single type of protein from a complex mixture.
The significance of isolating proteins is to
characterize their solubility, acid-base property,
function, structure, and interactions. Proteins
can be separated depending on their size,
shape, charge, hydrophobicity and
physiochemical properties. Some of the methods
that are commonly used are isoelectric
precipitation, heat denaturation, solubilization,
salt-induced precipitation, chromatography, and
ultra-centrifugation.
In isoelectric precipitation, the isoelectric point
must be achieved wherein the net charge of the
protein will be equal to zero. It is done by
precipitating a complex mixture until the protein
is precipitated at a certain pH level.
In heat denaturation, proteins are isolated
based on their heat sensitivity. Some proteins
denature at certain temperatures and heating
will help isolate the proteins that easily
denature.
In solubilization, proteins are separated
through their difference in solubility. Insoluble
proteins are easily isolated, removing other
substances that are soluble by washing.
In salt-induced precipitation, the
physiochemical properties of the protein and the
concentration of the salt are considered in
isolating proteins. Initial salting in at low
concentrations will result in decreasing
electrostatic free energy of the protein and
increasing activity of the solvent, which in turn,
leads to increasing in solubility. In high salt
concentrations, the solvating power of salt ions
and the solubility of proteins are decreased
resulting in precipitation.
that can be used to isolate proteins. They are
gel-filtration, ion-exchange, affinity and high-
pressure liquid chromatography (HPLC). Gel-
filtration chromatography uses beads made from
materials such as dextran, polyacrylamide or
agarose to separate small proteins from larger
ones. Ion-exchange chromatography separates
proteins by net charge using beads that have
carboxylate groups on them. Proteins that stick
to the beads are retained, while those that bind
weakly or not at all flow straight through and
are collected out the other end of the column. It
is also possible to recover the protein that was
bound. This approach is used in affinity
chromatography, which exploits a protein's
affinity for specific chemical ligands. The bound
protein is washed off from the column by
flooding the beads with a solution to decrease
the binding. HPLC is a higher resolution, faster
and much-improved version of chromatography.
Lastly, ultra-centrifugation depends on the
different masses and densities of proteins.
Heavier or denser particles will pellet first, while
lighter or less dense ones will remain dissolved. 
In the experiment performed, there were
three proteins that were isolated which are
casein, gluten, and myoglobin. Casein was
isolated through isoelectric precipitation by
acetic acid. Casein exists in milk as calcium
caseinate. Gluten was isolated through
solubilization wherein the starch was removed
by washing since it is soluble in water while
gluten is insoluble. Myoglobin was isolated from
minced beef muscle by salt-induced
precipitation. It stores protein in muscle cells
and is vital to oxygen transport in vertebrates.
One of the objectives of the experiment is to
perform hydrolysis in preparation for the
qualitative color reactions. This process breaks
covalent bonds of amino acids in the presence of
water and structures that are lost in hydrolysis
are the secondary, tertiary, and quaternary
structures. Hydrolysis can be classified into
three types such as acid, alkaline, and
enzymatic hydrolysis. In acid hydrolysis, there is
complete hydrolysis of peptide bonds and no
racemization. However, one disadvantage is the
destruction of tryptophan to humin (black
precipitate). In alkaline or basic hydrolysis,
there is also complete hydrolysis of peptide
bonds but the disadvantage is arginine would
form ornithine and urea. In enzymatic
hydrolysis, bonds are broken through peptidases
or proteolytic enzymes. These are enzymes that
break long chain-like molecules or proteins into
shorter fragments and eventually into their
components, the amino acids. Common
proteolytic enzymes are pepsin (which breaks all
peptide bonds), trypsin (which breaks arginine
and lysine), chymotrypsin (which breaks
phenylalanine, tyrosine and tryptophan),
aminopeptidase (which breaks aminoterminals),
and carboxypeptidase (which breaks
carboxyterminals). Advantages of performing
the enzymatic hydrolysis are: it is fast and
specific and it does not destroy amino acids. On
the other hand, one disadvantage is that it only
requires certain temperatures.
Another objective is the characterization of
proteins. It involves performing qualitative color
reactions in order to determine the proteins’
composition. Reagents are used to react with
intact and hydrolyzed protein samples for
different kinds of tests.
For the identification and separation of amino
acids, paper chromatography and thin-layer
chromatography can be used. It is a method of
separating the components of a mixture based
on their differential affinity for two chemicals,
which are the stationary phase and the mobile
phase. Polarity of proteins can be determined
through this method.
Lastly, another objective of the experiment is
to determine the protein concentration. It can be
determined by using the Bradford Protein Assay
method. The method is based on the binding of
Coomassie dye to proteins in acidic solution
leading to an increased absorbance of the
sample at 595 nm.
EXPERIMENTAL
A. Compounds tested
Casein, acid hydrolysate, basic hydrolysate,
tryptophan, arginine, proline, cysteine, serine,
aspartate, histidine, glycine, alanine, Bovine
serum albumin (BSA)
B. Procedure
1.Isolation of Casein from Skimmed Milk
Twenty grams of powdered non-fat milk was
mixed with 100 mL of distilled water in a beaker.
The mixture was heated up to 40°C then, 10%
acetic acid was added until casein precipitates.
2.Qualitative Color Reactions
Nine groups of test tubes were prepared for the
test. Each group of test tube contains intact
proteins, acid and basic hydrolysates, added
with 1 mL of distilled water.
The first test performed was the Biuret test
wherein 20 drops of 2.5 M NaOH was added and
mixed to the samples. Then, 2-3 drops of 0.1 M
CuSO4 was added.
The second test was the Ninhydrin test
wherein 6-10 drops of 0.1% ninhydrin solution
was placed into the samples then heated in a
boiling water bath.
The third test was the Xanthoproteic test
wherein 10 drops of concentrated HNO3 was
slowly added into the samples. After adding
HNO3, 10 drops of NaOH was slowly added.
The fourth test was the Millon’s test wherein 5
drops of Millon’s reagent was added to the
samples.
The fifth test was the Hopkins-Cole test
wherein 20 drops of Hopkins-Cole reagent was
mixed to the samples. Then each test tubes
were inclined and 20 drops of concentrated
H2SO4 was slowly added.
The sixth test was the Sakaguchi test wherein
10 drops of 10% NaOH and 10 drops of 0.02%
napthol solution was added to the samples. After
3 minutes, 3 drops of 2% NaOBr was mixed to
the samples.
The seventh test was the Nitroprusside test
wherein 0.5 mL of 3 M NaOH was added to the
samples. Then, 0.25 mL of 2% nitroprusside
solution was added.
The eighth test was the Fohl’s test wherein 5
drops of 30% NaOH and 2 drops of 5%
(CH3COO)2Pb was added to the samples then the
rest were placed in a boiling water bath.
The last test performed was the test for amides
wherein 1 mL of 20% NaOH was added to 10
drops of the sample. The samples were then
placed in a boiling water bath and red litmus
paper was placed and moistened over the mouth
of the tube.
3. Paper Chromatography
For the paper chromatography of the amino
acids, mixture of butanol, acetic acid, and water,
with the ratio of 4:1:5 respectively, was used as
the solvent system. Using a capillary tube,
tryptophan, arginine, proline, cystein serine,
aspartate, histidine, glycine, alanine, acid
hydrolysate and basic hydrolysate was spotted
on the paper chromatogram. When the
chromatography procedure was done, 1%
Ninhydrin solution was sprayed to the
chromatogram to react with the protein and be
identified. The paper was placed over the hot
plate to dry.
4.Bradford Method
Eight cuvettes were prepared for the
spectrophotometry. Each cuvette was cleaned
using ethanol to remove substances that will
contaminate the samples to be placed. In the
first cuvette, 1.5 mL of distilled water was
placed. In the seventh cuvette, 1.5 mL of bovine
serum albumin was placed. In the eighth
cuvette, unknown concentration of BSA was
placed. Cuvettes’ 2-6 contains different
concentrations of BSA in distilled water. Each
cuvette was placed in the spectrophotometer set
at 595nm to determine its absorption. After
getting the values, the concentration of the
unknown sample was computed and the albumin
standard curve was plotted.
RESULTS AND DISCUSSIONS
A. Isolation of Proteins
Casein is the protein that was isolated from
non-fat milk through adding acetic acid. When
acetic acid was added to the non-fat milk
mixture at a controlled pH level, yellowish white
precipitate was produced. That process is called
isoelectric precipitation. Precipitation occurred
because the protein has already reached its
isoelectric point wherein its net charge equalled
to zero.
B. Qualitative Color Reactions
Intact proteins, acid hydrolysates and basic
hydrolysates were all tested to characterize and
determine the functional groups that they
contain. [See table 1]
Each test that was performed has different
concepts behind them. The reactions of the
intact proteins and hydrolysates to the reagents
of each test, depends on their characteristics.
They can be positive or negative in a particular
test. [See table 2]
Biuret test is a general test for proteins and a
test for detecting peptide linkage. Its principle is
complexation reaction. The intact protein should
be positive in this test since its peptide linkage is
not broken unlike the two other samples that
had undergone hydrolysis. As seen in the
results, only the intact protein produced a violet
solution which is a positive indication of the
Biuret test.
Ninhydrin test is a test for detecting free alpha
amino groups. Proline is the only amino acid
that is negative for the said test. Its principle is
oxidative deamination and decarboxylation. A
positive indication of this test would be a blue
violet coloration in the solution. Intact protein
casein and the two other hydrolysates must
yield a positive result in this test. However, due
to possible contaminations in the samples, there
had been errors in the result.
Xanthoproteic test is a test for presence of
aromatic rings which includes tryptophan and
tyrosine. Although phenylalanine is considered
one of them, it will not have a positive result
because it is inactive. Its principle is the
nitration of the phenyl group. To know that the
substance is positive for Xanthoproteic test,
yellow to orange solution must be achieved.
Intact proteins, acid hydrolysates and basic
hydrolysates are positive for this test. In the
result for this test, there had been a slight error
in the color of the result of acid hydrolysate. The
rest were accurate.
Millon’s test is a test for the presence of
tyrosine. Its principle is the complexation
reaction between phenolic group and mercury
that is found in the Millon’s reagent. A positive
indication of this test is old rose or red
precipitate. Intact proteins and the hydrolysates
should be positive in this test. But then again,
there had been errors in conducting the
experiment which affected the result.
Hopkins-Cole test is a test for the presence of
tryptophan. Its principle is the condensation of
indole group with glyoxylic acid and H2SO4. A
positive indication of this test is the formation of
purple ring on the surface of the solution. As
seen in the results, only the acid hydrolysate is
negative for this test because tryptophan cannot
be detected and was destroyed during acid
hydrolysis. It becomes humin (black ppt.).
Sakaguchi test is a test for the presence of
free or intact arginine. Since alkaline or basic
hydrolysis destroys arginine and produces
ornithine and urea, basic hydrolysate must be
negative for this test while all the other samples
are positive. Its principle is the reaction of
Guanido group with α napthol and an oxidizing
reagent. A positive indication for this test is a
red or orange solution. As seen in the results,
only the basic hydrolysate had a light color.
Fohl’s test is a test for sulfur containing
proteins. It also indicates the presence of
methionine and cysteine because those two
amino acids have sulfur in their structures. Its
principle is fusion followed by ionic interaction. A
positive result for this test is the formation of
black precipitate from lead sulfide (PbS). The
dark coloration of the samples caused by the
Fohl’s test indicates that there is sulfur in the
intact protein, acid hydrolyste and basic
hydrolysate.
Nitroprusside test is used for indicating the
presence of cysteine. In this test, cysteine is
partially destroyed and it produced a red
solution. Its principle is complexation. Intact
protein is very positive in this test while the acid
and basic hydrolysate are only somewhat
positive. The cysteine that is partially destroyed
is evident in the results of the experiment.
 The last test that was performed is the test for
amides which indicates primary, secondary and
tertiary amides and nitriles. A positive result for
this test is the change in color of litmus paper
from red to blue. Its principle is basic hydrolysis.
As seen in the result all intact proteins, acidic
and basic hydrolysates have positive results for
this test.
C. Chromatography
Figure 1. Paper Chromatogram
The results of the paper chromatography are
based on the polarity of amino acids.
Tryptophan, a hydrophobic non-polar amino
acid, moved farthest from the base line. On the
other hand, histidine, a polar amino acid, moved
least from the base line. The mobile phase in the
solvent system is the butanol and the acetic
acid. Tryptophan having the farthest traveled
amino acid means that it has a high affinity to
acetic acid and butanol. Histidine has a high
affinity to the stationary phase, which is water.
Looking at the chromatogram, most of the
amino acids that can be seen far from the base
line belong to the 1st group of amino acid, the
hydrophobic non-polar amino acids. On the
other hand, the amino acids seen near the base
line belongs to the 2nd and 3rd group of amino
acid which are the polar uncharged and polar
charged amino acids respectively. Butanol, a
non-polar solvent, carries the non-polar amino
acids up the chromatogram. Acetic acid, a polar
solvent, carries the polar amino acids up the
chromatogram. Due to their quantitative
difference in the solvent system mixture which is
4:1 or four (4) measures of Butanol for every
one (1) measure of acetic acid, non-polar amino
acids are favored than polar amino acids. The
amino acids reacted with the 1% Ninhydrin
solution giving them distinct blue and violet
colors except proline which gives a yellow color.
Cysteine though, had a slight yellow coloration
because it was probably contaminated by proline
during the spotting of amino acid standards. For
the acid and the basic hydrolysate, both of them
can be found near the base line of the
chromatogram which means they have a high
affinity to the stationary phase. When treated
with ninhydrin solution, they gave a bluish
brown color.
D. Bradford Protein Assay
The Bradford assay is commonly used to
determine the total protein concentration of a
sample. The method is based on the binding of
Coomassie dye to proteins. The acidic
environment of the reagent results in a spectral
shift from the reddish brown of the dye, which
has a maximum absorbance of 465 nm, to the
blue form of the dye, which has a maximum
absorbance of 610 nm. The difference between
the two forms of the dye is greatest at 595 nm.
In determining the total protein concentration
of the sample, the dilution formula was used to
compute for the total protein concentration.
C1 V1 = C2 V2
C1= concentration of the sample
V1= volume of the sample
C2= concentration of the sample diluted with
water
V2= volume of the sample diluted with water
Figure 2. Standard BSA Curve
1.4
f(x) = 0.03 x
1.2 R² = 0.94
1
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50 60
Using the linear regression method, the slope
and y-intercept was determined: the slope
having the value of 0.0219, the y-intercept
equal to 0.2072, and the total protein
concentration equal to 31.41. As seen in the
table, there is a direct relationship between the
absorbance and the bovine serum albumin
concentration. [See table 3]
The absorbance of a sample can be related to
the concentration of the absorbing species
through Beer's law:
A = ε cl
where c is concentration, usually measured in
moles per liter; l is the length of the light path,
usually 1 cm; and ε is a proportionality constant
known as the molar extinction coefficient, with
the units of liters per mole per centimeter. The
value of ε is a function of both the particular
compound being measured and the wavelength.
Table 1. Results of Color Reactions

Test Intact Protein Acid Hydrolysate Basic Hydrolysate

(casein)
Biuret Purple solution Pale green solution Grey solution
Ninhydrin Clear solution Black precipitate Black precipitate
Xanthoproteic Orange solution Light brown solution Light yellow solution
Millon’s White precipitate Black precipitate Yellow solution
Hopkins-Cole Violet interference No purple ring Violet interference
Sakaguchi Yellowish brown solution Dark brown solution Light brown solution
Fohl’s Dark brown precipitate Dark brown precipitate Black precipitate
Nitroprusside Yellow solution Yellow solution Black precipitate
Test for Amides Yellow solution Red to blue litmus paper Red to blue litmus paper

Table 2. Expected Result of Samples in Each Tests.

Test Intact Protein Acid Hydrolysate Basic Hydrolysate

(casein)
Biuret +++ - -
Ninhydrin +/- +++ +++
Xanthoproteic ++ + +++
Millon’s +++ +++ +++
Hopkins-Cole +++ - +++
Sakaguchi +++ +++ -
Fohl’s +++ +++ +++
Nitroprusside +++ + +
Test for Amides +++ +++ +++

Table 3. Data of Bradford Assay

TT BSA (mL) Water (mL) Bradford Unknown Absorbance

Reagent
1 0 1.5 1.5 0 0
2 0.1 1.4 1.5 0 0.176
3 0.3 1.2 1.5 0 0.573
4 0.5 1.0 1.5 0 0.774
5 0.8 0.7 1.5 0 0.916
6 1.2 0.3 1.5 0 1.06
7 1.5 0 1.5 0 1.17
8 0 0 1.5 1.5 0.895

Absorbance Total Protein

Concentration
0 0
0.176 3.3
0.573 10
0.774 16.7
0.916 26.7
1.06 40
1.17 50
0.895 31.41
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