Isolation and Characterization of Egg White Proteins

Paulo T. Carpio
HUB32

De La Salle University-Dasmariñas
Dasmariñas, Cavite, Philippines

ABSTRACT

An egg white was isolated to separate the Ovoglobulin from Ovalbumin by salting out.
The proteins were then characterized based on different qualitative tests and denatured
by some denaturating conditions. The isolated egg white weighed 37.6314g and the
separated dried ovoglobulin weighed 4.9701g while the dried Ovalbumin weighed
3.9479g. The percent yield was calculated with 18.55% and 14.74% that were quite low
because of some errors. The other isolated egg white was diluted with phosphate buffer
and used to test the behavior of proteins from the addition of various reagents and then
denatured from different condition that altered the structure of proteins as manifested by
the precipitation or coagulation from the solution.

INTRODUCTION

Proteins are essential constituents of all organisms. It can be isolated from their native
source and then purified by different methods. These techniques depends on the differences in
the molecular weight, sizes, charges, solubility and their acid-base behavior.(1)
Egg white is the common name for the clear liquid contained within an egg. It is formed from
the layers of secretions of the anterior section of the hen's oviduct during the passage of the
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.(3)

MATERIALS AND METHODS

The first procedure of the experiment was the isolation of ovoglobulin and ovalbumin from the
egg white. The egg white was carefully separated from the yolk of one medium-sized chicken
egg. The volume of the egg white was measured in a pre-weighed graduated cylinder and then it
was weighed on the analytical balance. The isolated egg white was divided, and three-fourth was
used for the isolation and the remaining was for the characterization of egg white proteins. A
3.06g of NaCl powder was added for every 10ml of the divided egg white sample. Stirred gently,
until there were no precipitation of ovoglobulin was observed. Then it was filtered with a cheese
cloth to separate the filtrate. The residue was transferred to pre-weighed filter paper to dry. Then
the dried sample was weighed and the percent yield was calculated for ovoglobulin. The filtrate
placed on a beaker was added 2-3 drops of 0.200M acetic acid and subjected to heat to boil until
ovalbumin precipitates. Then the residue was transferred to pre-weighed filter paper to dry. Then
the dried sample was weighed and the percent yield was calculated for the ovalbumin.
The remaining isolated egg white sample was used for the qualitative test and denaturation
of protein. It was diluted with equal volume of phosphate buffer and then filtered with cheesecloth.
For the qualitative test of protein, a 1.00ml of egg white solution was transferred to four separate
test tubes and the color change in every test was observed. The table for the reagents added is
shown in Table 1.1

Table 1.1 Reagents for Qualitative test for Proteins

Test Tube Reagents added and Procedure

1 2.00ml of ninhydrin solution; heat gently in water bath
2 5 drops of conc. nitric acid (Xanthroproteic test); heat gently in water bath
3 5 drops of α-naphthol reagent,2.00ml 0.200M NaOH & 5drops of bromine
water (Sakaguchi test)
4 5 drops of glyoxylic acid (Hopkins-Cole test), 2.00ml conc. sulfuric acid

The diluted egg white was also used for denaturation of protein. 1.00ml of the solution was
transferred to eight separate test tubes and labeled properly. The added reagents and procedure
to the respective tubes were shown in the Table 1.2 and all the reagents were added drop by
drop until observable changes occur.

Table 1.2 Reagents for Denaturation of Proteins

Test Tube Reagents added and Procedure

1 0.200 M HCl
2 0.200 M NaOH
3 0.200 M lead acetate
4 0.200 M tannic acid
5 70% ethanol
6 Heat in water bath
7 Agitate vigorously

RESULTS AND DISCUSSIONS

The isolated egg white placed in a pre-weighed cylinder weighed 37.6314g. From the
isolation of ovoglobulin from ovalbumin in the egg white with the used of salting out, the dried
sample of ovoglobulin in the filter paper weighed 4.9701g and the filtrate which was the
ovalbumin weighed 3.9479g. The percent yield was calculated from the weight of the egg white
minus the weight of the added salt which was 10.8397g as the theoretical yield and the weight of
the dried ovoglobulin and ovalbumin as the actual yield. The Table for islotaion was shown in
Table 2.1

Table 2.1 Isolation of Ovoglobulin and Ovalbumin from Egg White

Ovoglobulin Ovalbumin
Weight of dried 4.9701g 3.9479g
sample
% Yield 18.55% 14.74%
 The calculations for the percent yield were shown below:

Actual Yield X 100%

Theoretical Yield

Theoretical value = 37.6314g - 10.8397 = 26.7917g

Ovoglobulin

4.9701g X 100% = 18.55%

26.7917g
Ovalbumin

3.9479g X 100% = 14.74%

26.7917g

The percent yields were low because of some error like not filtering the samples properly and
while transferring the egg white or the filtrate, there may be some spill so the volume changed.
Ovoglobulin and Ovalbumin are both proteins found in egg white. However, they have certain
differences like Ovoglobulin can be precipitated from egg white in salting out using NaCl or
ammonium sulfate. The ovalbumin is the one that serve as nourishment and the main protein
found in the egg white which is approximately 60-65%. The globulin is the one that serves as
antibodies in the immune system and bind to certain compounds in the body. The complex
protein structure of the typical egg white is illustrated below:

Table 2.0 Protein Structure in Egg White

(%) In Typical Egg

Protein Type Function of Protein
White
Nourishment, blocks digestive
Ovalbumin 54%
enzymes
Ovotransferrin 12% Binds iron
Ovomucoid 11% Blocks digestive enzymes
Globulins 8% Plugs defects in membranes, shell
Enzyme that digests bacterial cell
Lysozyme 3.5%
walls
Ovomucin 1.5% Thickens egg white; inhibits viruses
Avidin .06% Binds vitamin (biotin)
Bind vitamins, block digestive
Others 10%
enzymes

Proteins showed different color changes from the addition of several reagents. The test used
in the experiment to observe these characteristics are the Ninhydrin, Xanthroproteic, Sakaguchi
and Hopkins-Cole test. The observation from these tests were shown in Table 2.2

Table 2.2 Qualitative Test for Proteins

 Test Tube Added Reagent Color Observations
1 Ninhydrin solution Yellow
2 Conc. nitric acid Yellow to Orange
3 α-naphthol reagent, NaOH & Bromine water Red
4 Glyoxylic acid, conc. sulfuric acid Violet (exothermic)

The sample that was added with ninhydrin solution showed a color yellow but it supposes to
show a violet color. The reason of this might be the ninhydrin reagent was contaminated so the
test showed a wrong result. The other sample showed the write color based on the theoretical
results of the tests done.
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary
and secondary amines. When reacting with these free amines, a deep blue or purple color known
as Ruhemann's purple is evolved.(4) The Xanthroproteic test is a test for proteins in which a
yellow color forms on addition of conc. nitric acid and the color turns orange when made alkaline.
The Sakaguchi is a test for guanidines, in alkaline solution they give a red color with the
sakaguchi reagent.
The natural or native structures of proteins may be altered, and their biological activity
changed or destroyed by treatment that does not disrupt the primary structure.
This denaturation is often done deliberately in the course of separating and purifying proteins.
The denaturation of protein and the agents or condition done were shown in Table 2.3

Table 2.3 Denaturation of Protein

Test Tube Denaturating Agent or Observations

Condition
1 0.200M HCl Coagulate
2 0.200M NaOH Clear with white precipitate
3 0.200M lead acetate Homogenous white
4 0.200M tannic acid Brown with white residue
5 70% ethanol Coagulate
6 Heat in water bath Coagulate
7 Agitate vigorously Form bubbles

Following denaturation, some proteins will return to their native structures under proper
conditions; but extreme conditions, such as strong heating, usually cause irreversible change.
Some stabilizing forces or bond were disrupted when subjected to denaturating conditions. In the
heat, hydrogen bonds are broken by increased translational and vibrational energy like
coagulation of egg white albumin in frying. By addition of strong acids and base, there were salt
formation and disruption of hydrogen bonds. On some organic solvents such as ethanol, there
were change in dielectric constant and hydration of ionic groups. And on agitation, there were
shearing of hydrogen bonds. The disrupted forces or bonds in denaturating agents that were
used in the experiment were shown in the table below:

Table 3.1 Disrupted Stabilizing Forces on Denaturation

Denaturating Agent or Disrupted Stabilizing Force or Bond

Condition
0.200M HCl Salt bridges
 0.200M NaOH Salt bridges
0.200M lead acetate Disulfide bonds
0.200M tannic acid Salt bridges
70% ethanol Hydrogen bonding
Heat in water bath Hydrogen bonding & non-polar
hydrophobic interaction
Agitate vigorously Hydrogen bonding

REFERENCES

(1) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory

(2) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.
McGraw-Hill Companies, Inc
(3) Retrieved 15 July 2010 from http://en.wikipedia.org/wiki/Egg_white
(4) Retrieved 15 July 2010 from http://www. Ninhydrin%20-%20Wikipedia,%20the%20free
%20encyclopedia.htm