ISOLATION AND CHARACTERIZATION OF PROTEINS

(Acid Hydrolysis of Casein)

Carissa Patricia S. Santos, Miguel G. Silvestre, Ellysa Joviel B. Singzon,
Rouville A. Sosa, Chrizanne Irah A. Tagle, Venus L. Tibalao
Group 7 2C - Pharmacy Biochemistry Laboratory

ABSTRACT
Three proteins for isolation were assigned to different groups casein, gluten, and myoglobin. Group 7 isolated casein
from skimmed milk through isoelectric precipitation with acetic acid. This experiment was carried out to isolate casein
from skimmed milk, analyze chemical groups responsible for color reactions and explain the principle involved in each
test, to perform acid hydrolysis on the isolated proteins and enumerate the advantages and disadvantages of each
type of hydrolysis, to determine the amino acid components of the proteins by thin-layer chromatography, and to
quantitatively determine protein concentration in a given sample through Bradford assay. Acid hydrolysis is performed
in order to denature protein and isolate amino acids for color reaction characterization. The intact protein and the acid
hydrolysate were used for colorimetric reactions include Biuret test, Ninhydrin test, Xanthoproteic test, Millons test,
Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohls test, Pauly test and Amide test. The intact protein yielded
positive results in the rest of the tests except for Millons and Pauly test. The acid hydrolysate yielded negative results
in Biuret and Millons tests. After 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. The hydrophobic amino acids were closer to the solvent front. On the other
hand, polar uncharged were in the middle portion, while polar charged amino acids were close to the bottom. After the
performance of paper chromatography, determination of protein concentration was done through the 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 Bovine Serum Albumin concentration to its
absorbance.

INTRODUCTION
Proteins are probably the most important class
of biochemical molecules and are the basis for
the major structural components of animal and
human tissue. Proteins are considered polymer
molecules consisting of amino acids, which may
range from two to several thousands. These
amino acids are linked by covalent peptide bonds
into a linear chain called polypeptide chain. There
are common properties of amino acids due to
relative arrangements of carboxyl and amino
groups while unique physical and chemical
properties depend on the R group.
Isolation of proteins is done to isolate a single
type of protein from a complex mixture
containing various types of proteins. Its
significance is to characterize their solubility,
acid-base property, function, structure, and
interactions. Proteins may be isolated according
to size, shape, charge, hydrophobicity and
physiochemical
properties.
Some
methods
commonly used include isoelectric precipitation,
solubilization, salt-induced precipitation, heat
denaturation, affinity chromatography, and ultracentrifugation. Since amino acids have a great
variety of chemical reactive groups, these
reactions help to identify specific properties
unique to an individual amino acid.
Casein is a protein that is found in milk and is
used independently in many foods as a binding
agent. Casein is part of a group of proteins called

phosphoproteins, a collection of proteins bound

to phosphoric acid. Casein is a salt, meaning it
has no net ionic charge, of the element calcium.

EXPERIMENTAL
A. Sample/s and Compounds used
Skimmed or non-fat milk (casein), 10% acetic
acid, thermometer, pH indicator, funnel and filter
paper, hot plate; acid hydrolysate, 6 M HCl, 1 M
NaOH; amino acid standards, 1-Butanol: acetic
acid: water (4:1:5), 1% Ninhydrin solution in
spray bottle, 12 x 15 cm TLC plate,
chromatography
chamber,
capillary
tubes,
Bradford reagent, Bovine serum albumin (BSA)
standard, test tubes, UV-Vis Spectrophotometer
B. Procedure
1. Isolation of Casein From Skimmed Milk
To isolate casein from skimmed milk at its
isoelectric pH, an acid is used to adjust the pH to
4.6. 20.0 g of powdered non-fat milk and 50.0
mL of water is placed into a 100-mL beaker and
mixed well. The mixture was then heated to
40 and temperature is monitored
about
through the use of a thermometer. Afterwards,
10% acetic acid was added dropwise and the
solution is stirred gently after every 5 drops.
Addition of acetic acid is continued until pH
reached 4.6. The congealed casein was then
filtered through gravity filtration (cheese cloth),
setting aside the decantate for isolation of
albumin. The casein residue was then dried and

was used to calculate the weight% casein

isolated from the powdered milk.
2. Acid Hydrolysis of Intact Protein
5 mL of 6 M HCl was added to 0.5 g isolated
protein in a hard glass test tube. The tube was
stoppered by cotton and submitted to autoclaving
(15 psi for 5 hours). The appearance of the
mixture was noted after autoclaving and 10 mL
of distilled water is then added to the solution.
The mixture is transferred into a 250-mL beaker
and neutralized by adding 1 M NaOH. The
neutralized mixture is used as a sample for
characterization test and chromatography.
3. Qualitative Color Reactions
Biuret Test
20 drops of 2.5M NaOH and 2-3 drops
of 0.1 M CuSO4 was added to the samples
and mixed well. The color of the solution
was then noted.
Ninhydrin Test
6-10 drops of 0.1% ninhydrin solution
was placed into the diluted samples. The
tube was heated in a boiling water bath
and the appearance of a blue-violet
coloration was noted.
Xanthoproteic Test
10 drops of conc. HNO3 was slowly
added to the diluted samples. The solution
was mixed and the color of the solution
was noted. Afterwards, 10 drops of conc.
NaOH was added, the solution mixed, and
the color of the solution was noted again.
Millons Test
5 drops of Millons reagent was added to
the diluted samples and change in color
was noted.
Hopkins-Cole Test
20 drops of Hopkins-Cole reagent was
slowly added to the samples and mixed
well. The test tube was inclined and 20
drops of conc. H2SO4 was added slowly
without shaking. The color of the interface
was noted.
Sakaguchi Test
10 drops of 10% NaOH and 10 drops
0.02% naphthol solution was added to the
samples. The solution was mixed and
allowed to stand for 3 minutes.
Afterwards, 3 drops of 2% NaOBr was
added and the color produced was noted.
Nitroprusside Test
0.5 mL of 3 M NaOH was added to 0.5
mL sample. Then, 0.25 mL 2%
nitroprusside solution was added. The
formation of a red solution was noted.
Fohls Test
5 drops of 30% NaOH and 2 drops 5%
(CH3COO)2Pb was added to the samples.

4.

5.

The tube was placed in a boiling water

bath and the appearance of a dark (black
or brown) sediment was noted.
Test for Amides
1 mL of 20% NaOH was added to 10
drops of the sample. The tube was placed
in a boiling water bath. Test for evolution
of gas during heating is done by placing a
moistened red litmus paper over the
mouth of the tube.
Pauly Test
The diazo reagent was prepared by
mixing 3-5 drops 1% sulfanilic acid with 3
drops 5% NaNO2 solution. 5 drops of the
sample was then added to 3-5 drops 10%
Na2CO3 to the diazo reagent. The
appearance of a red coloration was then
noted.
Separation and Identification of Amino
Acids by Thin-Layer Chromatography
TLC plate was prepared having
measurements of 12 x 15 cm. The origin
was drawn as a pencil line across the plate
with a 1.5-cm margin from the bottom of
the longer edge of the plate. 13 equidistant
points on the line was marked for spotting
of the amino acid standards and 3
hydrolysate samples. The standards were
applied 5 times and sample 10 times using
capillary tubes. The sample was allowed to
dry in between applications. The plate is
placed inside the pre-equilibrated chamber.
The level of the solvent was below the
origin. The chamber was covered and
allowed the solvent to ascend undisturbed.
The plate was removed when the solvent
front is approximately 0.5 cm from the top
edge of the plate. The solvent front was
immediately marked with a pencil line. The
chromatogram was air-dried and 1%
ninhydrin reagent was sprayed lightly. The
chromatogram was placed inside an oven
for 1-3 minutes. The amino acids appeared
as blue, purple, or yellow spots. All spots
were encircled with a pencil and Rf values
were computed.
Protein Assay Using the Bradford
Method
A series of test tubes/cuvettes were
prepared for spectrophotometry. Group 7
prepared 0.35 mL standard mixed with
1.15 mL H2O and an unknown. 1.5 mL of
Bradford reagent was added to each tube
and mixed well. The solution was allowed
to stand for 5 minutes. The absorbance at
595 nm was read within an hour, using
tube 1 as the blank. Cuvettes 2-6 contains
different concentrations of BSA in distilled
water,
each
placed
in
the

spectrophotometer
to
determine
its
absorption.
Afterwards,
the
albumin
standard curve was constructed by plotting
A595
against
concentration
(mcg/mL)
through the resulting values and the
concentration of proteins in the unknown
sample was computed and determined.

RESULTS AND DISCUSSION

Isolation of Casein from Skimmed Milk
The result from the isolation of casein was a
white, curdy solid particle, which precipitated
from milk when acetic acid was added at
40 at pH 4.6 . Thus, producing this curd
signifies that the skimmed milk has a positive
result in containing 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. The percentage casein
present in the skimmed milk was 33.2 % w/w.
Acid Hydrolysis
The solution was subjected to 6 M HCl resulting
to the separation of individual amino acids
present in casein. After autoclaving, the resulting
solution was brown in color and was neutralized
with 1 M NaOH for chromatography purposes.
Qualitative Color Reactions
Visible Results (CASEIN)
Color Test
Intact Protein
Biuret Test
Ninhydrin Test
Xanthoproteic
Test
Millons Test
Hopkins-Cole
Test
Sakaguchi
Test
Nitroprusside
Test
Fohls Test
Test for
Amide
Pauly Test

Purple solution
(+)
Blue-violet
solution (+)
Yellow to
orange ppt. (+)
White solution
(-)
Purple ring at
Interface (+)
Red solution
(+)
Red solution
(+)
Brown ppt. (+)
Red --> Blue
Litmus Paper
(+)
Red solution
(+)

Acid
Hydrolysate
Brown solution
(-)
Blue-violet
solution (+)
Yellow to orange
ppt. (+)
Yellow solution
(-)

NAME OF
TEST
Biuret

Ninhydrin
Xanthoprotei
c
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside

Complexation reaction
Reduction of Cu2+ to Cu+
Hydrolysis of peptides

Oxidative deamination and

decarboxylation
Nitration of benzene rings SEAR
Complexation reaction between the
phenolic group and mercury
Condensation of indole group with
aldehyde
Reaction of guanido group with
alpha naphthol and an oxidizing
agent

Liberation of free SH
groups

Complexation

Cysteine groups reacts with

nitroprusside in alkaline
solution
Fusion followed by ionic interaction
Basic hydrolysis

Fohls
Test for
Amides
Pauly
diazotization
Table 2. Principles Involved for Color Reactions

NAME OF
TEST
Biuret

Ninhydrin
Xanthoprotei
c
Millons

Purple ring at
Interface (+)
Red solution (+)

Hopkins-Cole

Red solution (+)

Sakaguchi

Brown ppt. (+)

Red --> Blue
Litmus Paper (+)

Nitroprusside

Red-orange
solution
(+)
Table 1. Qualitative Color Reactions

PRINCIPLE INVOLVED

Fohls
Test for
Amides

Pauly

PROTEINS
Presence of 2 or more
peptide linkages

All proteins except dipeptides

Peptide bonds by hydrolysis

+ intact protein
Presence of free alpha amino acids
(no P)
+ intact and +2 hydrolyzed
Side chains of aromatic amino acids
(FYW)
+ intact and + 2 hydrolyzed
Phenol group of Tyrosine residues
Tryptophan
Indole nucleus of Tryptophan
residues
+ intact and + basic hydrolyzed
Presence of free or intact arginine
+intact +acid hydrolyzed
Cysteine residues
+intact partially + 2 hydrolyzed
Sulfur-containig amino acids (CM)
+intact +2hydrolyzed
R-groups of asparagine and
glutamine
Primary and secondary amides and
nitriles
+intact +2hydrolyzed
Histidine and tyrosine residues

Table 3. Determination of protein presence

Thin Layer Chromatography

*** MIGS (table, computations for Rf values
and explanations)
Bradford Assay
*** SOSA (graph, computations for linear
regression and explanations)

REFERENCES
From books
[1] C amp be ll, M. an d Far e ll, S. (2 0 08 ) .
Biochemistry (7th ed.). Canada: Brooks/Crole.
[2] Crisostomo, A. C. (2010). Laboratory Manual
in General Biochemistry (p.18). Quezon City: C&E
Publishing, Inc.
[3] The Biochemistry Department (2008).
Laboratory Manual in General Biochemistry.
Manila: University of Santo Tomas.
From the internet
[4] Clark, J. (2004). The hydrolysis of protein.
Retrieved January 10, 2009 from
http://www.chemguide.co.uk/organicpro
ps/aminoacids/proteinhydrolysis.html
[5] Ivanovien, L., Morkniene, R., Banien, R.,
Ivanovas, L. & Borutait, V. Retrieved
January 10, 2009 from
http://www.kmu.lt/nsc/biochemija/Labor
atory_manual_PART%20I.pdf