ISOLATION OF INTACT PROTEIN-ALBUMIN

Maria Andrea Dagala, Elisha de Guzman, Katrina de Lara,

Jes Deiparine, Jorina Diaz, Doxa Espina
Group 3 2A Medical Technology Biochemistry Laboratory

ABSTRACT
Albumin or alpha-lactalbumin, a globular, whey protein found in milk, is isolated by the means of acid treatment of a
50.0mL milk sample to isolate casein and upon filtration, the decantate was subjected to heat denaturation that lead
to the precipitation of albumin. The qualitative analysis of the sample shows that the there is only a scanty amount of
precipitated albumin in the solution, due to whey proteins only take about 15% of milk composition against casein of
80% composition. It is also guaranteed that the precipitated albumin is impure as coaggregation of unfiltered casein
left and whey proteins form a complex at around 75-90 C.

INTRODUCTION
Proteins are large biomolecules made up of
amino acids bonded by peptide bonds (covalent
bonds). Proteins are essential in the life of all
living things on earth as it has the following
functions: it acts as a catalyst (ex. Enzyme), it is
used for regulation (ex. Insulin and Glucagon),
transport (ex. Hemoglobin) and storage (ex.
Casein in milk), it acts as a defender (ex.
Immunoglobin in the plasma), it helps in
movement and motility (ex. Actin and Myosin), it
provides structure (ex. Collagen and elastin) and
lastly it participates in the transmission and
generation of nerve impulses.[1] This experiment
entails the isolation of intact proteins from a
sample and there are 4 major considerations to
be followed. The first one would be the tertiary
structure of a protein, Proteins have four levels of
stucture these are the primary, secondary,
tertiary and quaternary structure. The tertiary
structure is the 3D arrangement of all atoms in
the protein, including the side chains (R-groups).
In this structure, a protein can be differentiated
whether it is globular or fibrous depending on
their characteristics. A fibrous protein has the
following characteristics: an overall elongated,
rod-like shape; insoluble to water and it is stable
to changes in pH and temperature. A globular
protein, on the other hand, exhibits the following
characteristics: it is folded into a spherical shape;
it is soluble in water due to the presence of polar
amino acids on the periphery and lastly, it is
affected by the changes in pH and temperature
due to its allosteric nature. This type of proteins
do not form intermolecular interactions between
protein units as fibrous proteins do, and they are
more easily solubilized as colloidal suspensions.
The second consideration would be interactions
that keep the native conformation of the protein
functional (such as the electrostatic, covalent,
hydrophobic, H-bonding, and van der Waals). The
third consideration would be the acid-base
property and lastly, the fourth consideration
would be the solubility of protein in different

solvents. The solubility of a protein can be altered

by changing the pH of the environment, it was
made possible due to the allosteric nature of
globular proteins. Proteins are insoluble at their
isoelectric pH (pH at which the net charge in
zero). Denaturation happens when the protein is
inactivated from its native conformation( active
conformation) due to changes in the secondary
and tertiary structure causing its alpha helix and
beta pleated sheets to turn into random coil, it
happens when the protein is subjected to
extreme heat or changes in pH.[2]
In this experiment, the group was assigned the
isolation of albumin from skimmed milk. Milk is
composed of 2 major group of proteins. The
casein and whey proteins. There are 3 caseins in
the milk of most species; the different caseins
are distinct molecules but are similar in structure
these are the alpha, beta and kappa casein,
which solubilizes the alpha and beta casein into
the micelle or the solubilized unit. Casein is a
phosphoprotein and it exists in milk as the
calcium salt, calcium caseinate.
All other
proteins found in milk are grouped together
under the name of whey proteins. The major
whey proteins in milk are beta-lactoglobulin, a
fatty acid or lipid binding protein; and alphalactalbumin (albumin) which helps in lactose
synthesis. Lactalbumin or albumin found in milk
is soluble in water and in dilute salt solutions and
can be denatured and coagulated by heat.All
proteins in milk are globular proteins.[3][5][6][7]The
objective of this experiment is to isolate albumin
from skimmed milk and to subject it to qualitative
analysis. The isolated albumin would be then
used for hydrolysis and qualitative color reactions
for amino acid presence, determination of
present amino acid components via Thin-layer
chromatography and protein assay using the
Bradford Method.

EXPERIMENTAL
A. COMPOUNDS USED (or Samples used)
50.0 mL sample of skimmed milk
(Devonsdale skim milk)
10% acetic acid
B. PROCEDURE
This experiment is divided into two procedures, it
would start first with the isolation of casein, to be
able to get the decantate of albumin that was
used in the second procedure.

Figure 1. Chemical Reaction after the acid

treatment of skimmed milk.

50.0mL of skimmed milk was added with 50.0mL

of water and were placed into a beaker. It was
heated upto 40O C. 10% acetic acid was added
dropwise and was stirred gently after 5 drops.
Addition of acetic acid continued until the pH of
4.6 (isoelectric pH) was achieved. Then it was
filtated using a filtration set-up, comprised of a
filter paper, funnel, iron ring and receiver. The
decantate was set aside for the isolation of
albumin.

The decanted albumin then undergone the

second procedure of denaturation via heating
after acid treatment. This is one of the oldest
methods of whey protein recovery and this
resulted to an alpha-lactalbumin to be insoluble
in water.[8]This formed a scanty amount of white,
cloudy precipitate suspended on a yellowish
turbid solution, of which other sugars and
proteins are found. This shows that casein is the
major protein as it had precipitated more
abundantly than the albumin. It is guaranteed
that the albumin precipitate is impure, as heat
can also contribute to coaggregation among
unfiltered casein and whey proteins that forms a
complex at around 75-90 C.[4]

2. Isolation of albumin

REFERENCES

1. Isolation of casein from skimmed milk

The decantate obtained from casein isolation was

heated up to 75O C for 5 minutes in a water bath
and then the liquid was decanted from the
precipitated albumin.

RESULTS AND DISCUSSION

As said in the introduction, Milk is composed of 2
groups of proteins. The first one would be the
casein group comprising the alpha, beta and
kappa caseins and the second group would be the
whey proteins group which is majorly comprised
of beta-lactoglobulin and alpha-lactalbumin. A
fresh milk contains approximately 80% casein,
15% whey protein and 3.5% minor proteins. [9] In
the first procedure, addition of 10% acetic acid to
skimmed milk would cause the water-soluble
calcium caseinate salt to be negative upon
reaching the isoelectric pH or pI which is 4.6, due
to milk having a natural pH value of 6.6 and this
causes the removal of the calcium ion. This
leaves a water-insoluble casein which is seen as a
single large colloid suspended in a yellow, turbid
solution of whey protein. Whey proteins have
hydrophilic properties causing alpha-lactalbumin
to not precipitate from milk at pI. The chemical
reaction is best seen in the figure below.[4][7][10]

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