ISOLATION AND COLOR REACTIONS OF INTACT PROTEINS

Jon Ellis G. Datu, Athina Darla B. Deala, Alan Nathan D. Derige, Mary Kimberly L. Espaldon,
Ma. Theresa Angeli M. Estabillo, and Jemielle Patricia A. Estrada
Group 3 2F Medical Technology General Biochemistry Laboratory
ABSTRACT
In the experiment, the group had isolated casein and albumin from non-fat milk by isoelectric precipitation and heat
denaturation, respectively. Casein was precipitated by heating the mixture up to 40 C and adding 10% acetic acid
until the pH reached 4.6 or until a solid white curd-like substance formed. On the other hand, the albumin was
collected by subjecting the decantate to 75 C water bath for about 5 minutes. After precipitating casein and albumin,
several samples were used for the qualitative color reactions. Intact protein, acid hydrolysate and basic hydrolysate
were used in performing Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi
test, Nitroprusside test, Fohls test, test for amides and Pauly test. The intact protein, casein, yielded negative results
for Millons test and Sakaguchi test and the rest of the tests were positive. On the other hand, the intact protein,
albumin, obtained negative results after subjecting to Ninhydrin test, Xanthoproteic test, Millons test, and HopkinsCole test. Lastly, all test for amides turned the red litmus paper to blue.

INTRODUCTION
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
ultracentrifugation [6].
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 [5].
In heat denaturation, the secondary, tertiary
and quaternary structures of proteins are lost
and proteins are isolated based on their heat
sensitivity. Some proteins denature at certain
temperatures and heating will help isolate the
proteins that easily denature [4].
Specific reactions are used for the purpose of
identifying amino acids and proteins in biological
media, for qualitative and quantitative analysis.
Biuret test is used to determine peptide bonds.
Ninhydrin test is typical for -amino acids.
Xanthroproteic test is a test for the detection of
aromatic proteins in which concentrated nitric
acid reacts with the proteins to form a yellow
color that is intensified to orange-yellow by the
addition of alkali. Millons test is used to
demonstrate the presence of the amino acid
tyrosine. Hopkins-Cole test is specific for
tryptophan group. Sakaguchi test is a test for
guanidines, i.e arginine and peptides that contain
it. Nitroprusside test is a test for cystinuria. Fohls
test is used to know if sulfur-containing amino
acids are present. Test for amides is used to

detect R-groups of asparagines and glutamine

[1].
In the experiment performed, there were two
proteins that were isolated which are casein and
albumin. Casein was isolated through isoelectric
precipitation by acetic acid. Casein exists in milk
as calcium caseinate. On the other hand, albumin
was isolated through heat denaturation. Albumins
are globular proteins that are soluble in water
and in dilute salt solutions. They are, however,
denatured and coagulated by heat [1].
The objectives of this experiment are to isolate
casein and albumin from non-fat milk by
isoelectric precipitation and heat denaturation,
respectively and to analyze chemical groups
responsible for color reactions and explain the
principle involved in each test.

EXPERIMENTAL
A. Compounds tested (or Samples used)
Non-fat milk
Milk is a liquid substance produced by the
mammary glands of mammals. There are several
types of proteins in milk and the major milk
proteins are unique to milk, it is not present in
any other tissue. The primary group of milk
proteins are the caseins. All other proteins found
in milk are grouped together under the name of
whey proteins. The major whey proteins in cow
milk are beta-lactoglobulin and alpha-lactalbumin
[3].

B. Procedure
1. Isolation of Proteins
a. Casein from Non-fat Milk
The group placed 50.0 mL of non-fat milk into
a 100 mL beaker and heated it to about 40 C.
When the mixture had reached 40 C, 10%
acetic acid was added dropwise and the solution
was gently stirred after every 5 drops until the
pH reached 4.6 or until a solid white curd-like

substance formed. Then, the congealed casein

was filtered by gravity filtration and the
decantate was reserved for the isolation of
albumin. Lastly, the casein residue was allowed to
dry and the % weight of the casein isolated from
the powdered milk was calculated.

b. Albumin from Non-fat Milk

In order to isolate the albumin from milk, the
decantate that was reserved from the isolation of
casein was subjected to 75 C heat for about 5
minutes in a water bath. Lastly, the liquid from
the precipitated albumin was decanted.

2. Qualitative Color Reactions

For each test, the group prepared an intact

protein solution that consists of 0.5 g of protein
in 1 mL distilled water in separate test tubes.
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 solution was subjected to
the vortex mixer and the color was noted.
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. After that, the appearance of a blue-violet
coloration was noted.
The third test was the Xanthoproteic test
wherein 10 drops of concentrated HNO 3 was
slowly added into the samples and the solution
was mixed and the color was noted. After adding
HNO3, 10 drops of NaOH was slowly added and
again, the solution was mixed and the color was
noted.
The fourth test was the Millons test wherein 5
drops of Millons reagent was added to the
samples and the change in color was observed.
The fifth test was the Hopkins-Cole test
wherein 20 drops of Hopkins-Cole reagent was
slowly added to the samples. Then each test tube
was inclined and 20 drops of concentrated H 2SO4
was slowly added. The color at the interface was
noted.
The sixth test was the Sakaguchi test wherein
10 drops of 10% NaOH and 10 drops of 0.02%
naphthol solution were added to the samples.
After 3 minutes, 3 drops of 2% NaOBr was mixed
to the samples and the color produced was
noted.
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. Also, the color was noted.
The eighth test was the Fohls test wherein 5
drops of 30% NaOH and 2 drops of 5%
(CH3COO)2 Pb was added to the samples then the
tubes were placed in a boiling water bath and the
appearance of dark (brown or black) sediment
was noted down.

The ninth 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.
The last test performed was the Pauly test
wherein the group prepared the diazo reagent by
mixing 3-5 drops 1% sulfanilic acid with 3 drops
5% NaNO2 solution. Then, 5 drops of the sample
and 3-5 drops of 10% Na2CO3 were added to the
prepared diazo reagent and the appearance of
red coloration was noted.

RESULTS AND DISCUSSION

A. Isolation of Casein from Non-fat Milk
Casein is separated from milk by acidification
to bring it to its isoelectric point. When 10%
acetic acid was added to the non-fat milk mixture
at a controlled pH level, yellowish white
precipitate was produced. That pH value is known
as the isoelectric point of the protein and is
generally the pH at which the protein is least
soluble. For casein, the isoelectric point is
approximately 4.6 and it is the pH value at which
acid casein is precipitated. This process is called
isoelectric precipitation. Precipitation occurred
because the protein has already reached its
isoelectric point wherein its net charge is zero
[5].

Figure 1. Isolation of Casein from Milk

C. Qualitative Color Reaction

Intact proteins albumin and casein were used
to determine which proteins will give a positive or
negative result to a specific reaction test.
Table 1. Qualitative Color Reaction of Intact
Proteins
Color Reaction
Biuret Test
Ninhydrin Test
Xanthoproteic
Test

Figure 2. Casein from Milk

B. Isolation of Albumin from Non-fat

Milk
After the casein was separated, albumin was
extracted next using heat denaturation. To be
able to get the albumin, the extract from the
casein was heated. This would cause destruction
to the tertiary level of the substance enabling the
group to get the albumin. As shown on figure 3
the color of Albumin is slightly yellow.

Figure 3. Isolation of Albumin from Milk

Millons Test
Hopkins-Cole
Test
Sakaguchi Test
Nitroprusside
Test
Fohls Test
Test for Amide
Pauly Test

Intact Protein
(Casein)
Violet
Blue-violet
+ acid: Yellow
+ base:
Orange
Colorless
Violet ring

Intact Protein
(Albumin)
Violet
Colorless
+ acid:
Colorless
+ base:
Colorless
Colorless
Colorless

Colorless
Yellow

White-turbid
Yellow

Brown
sediment
Red-Blue
litmus paper
Red-orange

Brown
sediment
Red-Blue
litmus paper
Yellow-orange

The Biuret Test is a general test done to

proteins to detect peptide linkages. Positive
results show the formation of violet solution. The
principle involved in this test is complexation
reaction which takes place between a metal ion
and a molecular or ionic entity known as a ligand
[7]. Since the test results positive for peptide
linkages, as seen in the results, only the intact
proteins of albumin and casein resulted positive.
Ninhydrin Test is used to detect the free alpha
amino groups. The underlying principle behind
this
test
is
oxidative
deamination
and
decarboxylation. A positive result for the test
would show a blue-violet coloration of the
solution [7]. Based on the results, only the intact
casein resulted positive. The intact albumin
resulted negative indicating that free alpha amino
groups are not present within the protein.
Xanthoproteic test indicates the presence of
active aromatic groups that are derivatives of
benzene in amino acids such as tyrosine and
tryptophan. The principle involved in the test is
the nitration of the activated benzene ring
through nitric acid. The nitration results to a
yellow to orange solution [7]. Based on the
results, the intact protein casein has a positive
result.
Millons test is used to detect the presence of
tyrosine. The principle involved in the test is the
complexation reaction between the phenolic
groups and mercury present in the Millons
reagent. Positive result shows the salmon pink

coloration of the solution [7]. Both intact casein

and albumin showed a negative result.
Hopkins-Cole Test is used to detect the
presence of tryptophan. It is guided by the
principle of condensation of the indole group with
glyoxylic acid and H2SO4. A positive result of the
test shows the formation of a violet ring or the
coloration of two layers. In other cases, a similar
result is produced when sulfuric acid is added to
the solution in presence of formaldehyde [2].
Only the intact casein produced a positive result.
Sakaguchi test is used to detect arginine, the
only amino acid that contains a guanido group.
The principle behind the test is that the guanido
group reacts with naphthol, in the presence of an
oxidizing agent, such as sodium hypobromite.
The reaction produces a red or orange coloration
of the solution [7]. It is negative to both casein
and albumin because they produce a colorless
and white-turbid solution, respectively.
Nitroprusside test is used to determine the
presence of cysteine, the only amino acid that
contains the sulfhydryl group. The principle
behind this test is complexation; the sulfhydryl
group reacts with nitroprusside in presence of
excess ammonia. Cysteine is partially destroyed
and a positive result shows a red or yellow
complex [2]. Both casein and albumin showed
positive results.
Fohls test determines the presence of sulfurcontaining proteins such as methionine and
cysteine. The test is guided by the principle of
fusion followed by ionic interaction. A positive
indicator for the test is the formation of black or
brown precipitate from lead sulfide [2]. A positive
result was observed for both intact proteins.
Test for amides is used to determine the Rgroups of asparagine and glutamine. A positive
result shows the change of red litmus paper to
blue litmus paper [2]. Casein and albumin
resulted positive for the test.
Paulys test detects the presence of tyrosine or
histidine which deals with the formation of azo
dyes. When proteins containing either tyrosine or
histidine are reacted with diazotized sulfanilic
acid, a red to yellow coloration is formed [7]. The
intact proteins of casein produced the strongest
positive result.

1
7

2
3
4
8
9 10

Figure 4. Color Reaction of Casein

10 9
4
3
2

8
1

Figure 5. Color Reaction of Albumin

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