Concepcion, Jenny P.

Date due: December 19, 2016

Lorenzo, Marianne Angelina R. Date submitted: December 19,
2016
Nalupta, Jamie Patrice A.

EXPERIMENT 6
ISOLATION AND PURIFICATION OF PROTEINS

ABSTRACT

Protein isolation and purification techniques serve its relevance in the study of
proteins with respect to its structure, function and interactions with other
molecules. The experiment was focused on casein, albumin and vegetable seeds.
These were extracted and their concentration were determined. Casein had a
protein concentration of 1.9477mg/ml. Vegetable seeds had a percentage recovery
of 40.33%. Concentration in egg albumin was not computed due to limitations in
the experiment. Isolation and purification would have been performed using a
more effective method to yield accurate results.

INTRODUCTION

Proteins are the most abundant organic molecules of the living system. Proteins on
complete hydrolysis yield L-α amino acids, the polymer of proteins (Satyanarayana, 2013).
Amino acids being the building blocks of proteins play critical role. It is composed of an amine
group, a carboxylic group and a varying side chains that differs between different amino acids
(Milio and Lofredo, n.d.).

Proteins are essential constituents of all organisms. It can be isolated from their native
source and then purified by different methods. These techniques depend on the differences in
the molecular weight, sizes, charges, solubility and their acid-base behavior.

Protein isolation is a method of separating a single type of protein from its natural source
or from a mixture of several types of proteins. This process is important in studying the function
of a specific protein, its structure and its interaction with other materials in the human body.
Extraction of protein from its source requires breaking the tissue or cell containing it and
immersing it into the solution. In this process, the tissue cell undergoes different procedures like
freezing, sonication, homogenization, filtration, and permeabilization by an organic solvent and
after the soluble protein has been separated from the insoluble type, the protein of interest can
be isolated from the cell membrane or DNA by centrifugation. Protein can be further purified by
using different techniques such as chromatography, centrifugation, filtration and electrophoresis.
They must be stabilized to avoid denaturation, hence they must be kept at a fairly cool
temperature and not on high temperature. The pH should also be maintained to inhibit
proteases that may destroy the small peptide bonds. Moreso, proteins must be kept at a high
concentration because many proteins are unstable at air-water interfaces at low-temperature.
(Aguida, et. al.,n.d.).

This experiment endeavors to isolate casein from milk, albumin from egg and crystalline
globulin from vegetable seeds using some methods employed in protein isolation.
 Egg white is the common name for the clear liquid contained within an egg. It is formed
from the layer of secretions of the anterior section of the hen’s oviduct during the passage of
egg. It forms around either fertilized or unfertilized egg. It consists mainly of about 15%
proteins dissolved in water. Its primary natural purpose is to protect the egg yolk and provide
additional nutrition for the growth of the embryo as it is rich in proteins and also of high
nutritional value. It just well could be that the egg white is like an organic solvent due to
the amount of proteins and fat in it. With this, the ionic properties are changed to a non-
polar environment causing the globulins to be dissolved in the egg white.

Milk is the most nutritionally complete food found in nature. It is a globular protein, which
tend to fold back on themselves into compact, nearly spheroidal units and are more easily
solubilized in water as colloidal suspensions than fibrous proteins are. They are "complete
proteins", so-called because they contain all the amino acids essential for building blood and
tissue, and they can sustain life and provide normal growth even if they are the only proteins in
the diet. These proteins not only contain more amino acids than plant proteins, but they contain
greater amounts of amino acids than the proteins in eggs and meats.
Casein, the main protein in milk, is a phosphoprotein, meaning that phosphate groups
are attached to the hydroxyl groups of some of the amino acid side-chains.  Compared to other
proteins in milk, casein has a larger molecular weight and a larger structure. Several competing
theories regarding the precise structure of the micelles are present but they share one important
feature: the outermost layer consists of strands of one type of protein, k-casein, reaching out
from the body of the micelle into the surrounding fluid. All these protein molecules have negative
electrical charge and therefore repel each other, keeping the micelles separated under normal
conditions and in a stable colloidal suspension in the water-based surrounding fluid. Because of
the density of casein, which is denser than the other proteins, they get in the bottom layer of the
centrifuge after centrifuging.

METHODOLOGY, RESULTS AND DISCUSSION

Isolation of Casein

For the isolation of casein from milk, 25 ml of evaporated milk was measured and was
diluted using tap water. It was put to heat at 40 oC and slowly after a 10 minute period, 0.1 M of
HCl was added dropwise, with thorough stirring until flocculent precipitate was formed. If, on
pouring the liquid from the tube, a sediment remains at the bottom, it is an indication that
acid has been added too rapidly and has caused some coagulation of casein at the point
of contact of acid and milk (Van Slyke and Beaker, 2016).
To check for the amount of protein present in the supernatant, a spectrophotometric
protein determination was carried out. Calculation on concentration of protein in the isolation of
casein is shown below:
Abs at 280nm wavelength = 2.198
Abs at 260nm wavelength = 0.76

Protein Concentration=1.55 ( A 280 ) −0.76 ( A 260 )

 Protein Concentration=1.55 ( 2.198 )−0.76(0.192)
Protein Concentration=(3.4069−1.9477)
Protein Concentration=1.9477 mg/ml
Isolation of Egg Albumin
In isolating the albumin from an egg, 30 mL (26.6 grams) of egg white was measured
and then placed on a beaker. After stirring the egg white using a stirring rod, 3 ml of 1N HOAc
was added (this is one-tenth of the egg white’s volume). Resulting mixture was then filtered by
the use of cheese cloth and an equal amount of saturated ammonium sulfate solution was
added to break up the membranes while vigorously stirring the glass rod to facilitate its
passage. The mixture was left to stand for 30 minutes. It was then placed in the centrifuge and
the precipitate was discarded. The supernate in a centrifuge tube was immersed in an ice bath
for 15 about minutes. The buffered ammonium sulfate solution was added until the precipitate
ceases to dissolve. The mixture as allowed to stand for complete precipitation. It was
centrifuged again and the resulting supernate was discarded. 1% solution of the albumin in
0.9% NaCl on a beaker was prepared from this and was stored in the refrigerator for further
experiments.

Fig 1. Mixture of egg white & 1N Fig 2. Centrifugate of Filtrate + Fig 3. Ice Bath of supernatant
HoAc buffered saturated solution
 Upon addition of HOAc as seen in Fig. 1, the egg white turns turbid, hence presence of
precipitation. Proteins present in egg has
Fig 4.an isoelectric
Albumin Solutionpoint at an acidic pH. The addition of 3
mL of HOAc was enough to bring the pH down to less than the isoelectric point. Thus, this
resulted to precipitation of proteins which is present in the egg white. Addition of buffered
saturated ammonium sulfate was done to precipitate out contaminant proteins prior to
precipitation of albumin. The second addition of buffered saturated ammonium sulfate solution
precipitated the albumin. This was then centrifuged out of the mixture and dissolved in NaCl
solution for preservation. This was used for the analysis using a spectrophotometer. Beer-
Lambert law states that the absorbance of a given sample is directly proportional to its
concentration.
A = abc
where A is the absorbance of solution, a is the molar absorptivity, b is the path length and c is
concentration of the sample solution. This just means that as the concentration of the solution
increases, its absorbance also increases.
Maximum absorbance at 280 nm is characteristics of proteins like albumin and this was
used to verify the presence of protein in the solution. Fig. 5 presents the ideal concentration of
albumin in a solution.

Fig 5. Spectrograph of the Isolated Albumin Solution

To quantify the presence of protein, the following equation was used.

Concentration (mg/ml) = (1.55 x A280) – (0.76 x A260)
Concentration (mg/ml) = (1.55 x 0.483) – (0.76 x 0.132)
Concentration (mg of protein/ mL of solution) = 0.648 mg/mL
Calculation of mass percent of albumin in the egg white was not made possible from the
experiment. The maximum absorbance at 280 nm is not specific to albumin. Other proteins
present in the egg would contribute an absorbance and would give erroneous mass percent
calculation. Further purification and verification of the albumin in the mixture should be done.
Volumes of ammonium sulfate added in the isolation of albumin from the egg white were not
recorded. Also, prepared solution were not all centrifuged. Moreover, the volume of these
solutions were not documented.
To conclude with, albumin was probably isolated. However, to confirm presence of
albumin and its concentration in the sample, further purification and characterization should be
done. Also, volumes of reagents used should be noted carefully for quantification of isolated
protein.

Isolation of Crystalline Globulin from Vegetable Seeds

A 25 g fershly ground squash seeds was added in a beaker of 100 ml 10% NaCl. It was
heated at 50oC for an hour, with frequen stirring. The beaker was heated in a water bath at
75oC and was strained through a cheese cloth. The filtrate was squeezed and warmed at 60 oC
in a water bath. A large flutted filter paper in a funnel with warm 10% NaCl was moistened. It
was then filtered into a 300 ml flask returning the filtrate to the paper until a clear filtrate is
obtained. It was kept at 60oC for 30 minutes. The solution and the water bath was allowed to
cool spontaneously at room temperature. It was kept refrigerated until the next meeting.
Decantation of the supernatant fluid into the beaker was done. A drop of the heavy expediment
was examined under the microscope.
In an isoionic or salt-free state, proteins are least soluble and most precipitable. Protein
molecules when in their most compact, least-hydrated conformation. In this process, freshly
ground squash seeds were used as samples and 10% NaCl solution was added. Even a small
concentration of salt can have large effects on protein solubility [ CITATION Neh13 \l 1033 ].
Temperature was maintained as low as possible, thus minimized heat denaturation and effects
of degradative enzymes. Then after the decantation, a drop of heavy sediment was examined
under a light microscope.

Figure 5. Crystalline globulin from squash seeds (400X HPO).

Calculation on percentage recovery:
Weight of ground squash seeds = 3 grams
Weight obtained after centrifugation = 1.21 grams

Final Weight (g)

Percentage Recovery= x 100
Initial Weight (g)

1.21 grams
Percentage Recovery= x 100
3 grams

Percentage Recovery=40.33 %

CONCLUSION
It is therefore concluded that proteins can be extracted and purified in different methods. A
more accurate protocol as well as utilization of equipment and chemicals should be precisely
done to come up with accurate results.

REFERENCES
Nehete, Bhambar, Narkhede, & Gawali. (2013, July). Natural proteins: Sources, isolation,
characterization and applicaions. Retrieved December 18, 2016, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841988
Milio, F. R. and Loffredo, W. M. (n.d.). Qualitative Testing for Amino Acids and Proteins.
Retrieved
November 13, 2016 from https://labopslton.wikispaces.com/file/view/Qualitative+Testing+for+
Amino+Acids+%26+Proteins.pdf
Van Slyke, L. and Baker, J.. (n.d.) The preparation of pure casein.
Quantifying Protein Using Absorbance at 280 nm.
<http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html>