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PROTEIN TESTING IN EGG’S ALBUMIN

Ni Putu Giyan Adnya Antari

Chemistry Education Department, MIPA Faculty,

GANESHA University of Education, Singaraja, Indonesia
E-mail: putu_giyan@yahoo.com

Abstract
Protein plays an important role in all biochemical and physiological body
processes. They act as enzymes, hormones, receptors, antibodies and are required
for the structural integrity of cells. Protein can be found in the egg’s albumin. The
structure of proteins can be classified into four levels, namely primary, secondary,
tertiary, and quaternary. The primary structure is made up with covalent bond
which is the strongest binding. Secondary, tertiary, quaternary structures were
made up of weaker bindings than covalent bonds. Therefore, the high plane
structures above primary structure are disrupted easily. This alternation results in
denaturation and loses of the biological activity. Because of those factors, it is
essential to conduct an analysis to identify the protein in egg’s albumin and also
its characteristics. The experiment was purposed to identify the protein in Egg’s
albumin by using Biuret, precipitation by metal, precipitation by salt, coagulation,
precipitation by alcohol, and denaturation test. Furthermore, the methods used
was experiment in qualitative aspect based on the tests mentioned above.
According to the experiment result, can be concluded that the protein in Egg’s
albumin can be identified by using Biuret, precipitation by metal, precipitation by
salt, coagulation, precipitation by alcohol, and denaturation test.

Key words: Egg’s Albumin, Denaturation, Identification, Protein.

Introduction
The word protein is derived from the Greek word “proteios”, which means
“of primary importance”. In fact, a protein plays an important role in all
biochemical and physiological body processes; they act as enzymes, hormones,
receptors, antibodies and are required for the structural integrity of cells.
Proteins are organic compounds made of “amino acids” joined together by
“peptide linkages”. These peptides linkages are obtained by condensation
reactions (removal of water) between carboxylic & amino groups of two adjacent
amino acids, as what the figure explain bellow:
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Figure 1. Protein Structure

Proteins are polymers of amino acids. A typical protein may be composed

of hundreds of amino acids. The side chains of the amino acid residues may
contain nonpolar, neutral polar, acidic, or basic groups. The primary structure of
a protein is determined by the sequence of amino acids, but the secondary and
tertiary structures of proteins define their natural or native states, which are often
folded. This is called the native conformation and is usually the state in which the
protein is most active and functional. Proteins are held in their native
conformations by a combination of forces: hydrogen bonds, salt bridges (also
called ionic interactions), disulfide bridges, and hydrophobic interactions. The
figure can be seen as bellow:

NH 2 OOC Ar
N H O C S S Ar

C O H N

Hydrophobic
hydrogen bonds salt bridge disulfide bridge Interaction

Figure 2. Combination Force in Protein

The structure of proteins can be classified into four levels: primary,

secondary, tertiary, and quaternary. The basic structure, primary structure, is made
up with covalent bond which is the strongest binding in the chemical bonds, so the
primary structure is hardly affected even if the protein changes its form. When the
conformation of proteins is altered, this process is called denaturation, whereas
the intact protein is referred to the native protein. Secondary, tertiary, quaternary
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structures were made up of weaker bindings than covalent bonds. Therefore, the
high plane structures above primary structure are disrupted easily. This alternation
results in denaturation and loses of the biological activity. The factors of
denaturation are heat, strong acid, organic compounds, and heavy metal ions.
Each sources of denaturation reacts on different bonds in the protein and makes
the protein visible as a precipitate or coagulation.
Heat can supply kinetic energy to protein molecules, causing their atoms
to vibrate more rapidly. This will disrupt relatively weak forces such as hydrogen
bonds and hydrophobic interactions. The most common example is observed in
cooking an egg. Heat is also used in sterilization to denature and hence destroy
the enzymes in bacteria.
Extremes of pH can cause a protein to denature. Although the backbone
of a protein chain is neutral, the amino acid residues that comprise the protein
often contain acidic and basic groups. These groups are usually charged and can
form salt bridges with a group of opposite charge. Extremes of pH can change the
charges on these acidic and basic groups, disrupting salt bridges.
Less drastic changes in pH can also affect the activity and solubility of a
protein. Like individual amino acids, proteins have an isoelectric point at which
the number of negative charges equals the number of positive charges. This is
frequently the point of minimum water solubility. At the isoelectric pH, there is
no net charge on the molecule. Individual molecules have a tendency to approach
one another, coagulate, and precipitate out of solution. At a pH above or below
the isoelectric pH, the molecules have a net negative or positive charge,
respectively. Thus when protein molecules approach each other, they have the
same overall charge and repulse each other. This prevents coalescence and
precipitation.
Reagents such as ethanol that are capable of forming intermolecular
hydrogen bonds with protein molecules will disrupt the intermolecular hydrogen
bonding within the molecule. A 70% solution of alcohol can be used as a
disinfectant, because the alcohol functions to denature the proteins in bacteria. A
70% solution is used because it will effectively penetrate the bacterial cell wall; a
95% solution coagulates proteins at the surface of the cell wall, forming a crust
that prevents the alcohol from penetrating into the cell.
Salts of metal ions such as mercury (II), lead (II), and silver can form
strong bonds with disulfide groups and with the carboxylate ions of the acidic
amino acids. Thus, they disrupt both disulfide bridges and salt linkages and cause
the protein to precipitate out of solution as an insoluble metal-protein salt.
Proteins are precipitated from aqueous solution by high concentration of
neutral salts. This is the “salting-out” effect. Commonly used salts are ammonium
sulfate, sodium sulfate, magnesium salts and phosphates. The most effective
region of “salting-out” is the isoelectric point of the protein. Mechanism of
“salting-out” is due to dehydration of the protein by added salt. Removal of water
from hydrophilic ionic groups of protein to other ions will decrease protein
solubility. If this “salting-out” is carried out at a cold temperature, the proteins
will precipitate without denaturation. Thus, the proteins can be collected by
centrifugation an then redissolved in solution using buffer with low salt contents.
“Salting-out” or ammonium sulfate precipitation is useful for concentrating dilute
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solutions of proteins. It is also use for fractionating a mixture of protein. Since

large proteins tend to precipitate first, smaller ones will stay in solution.
In this experiment, protein solution used was obtained from the albumin of
the egg. Albumin refers generally to any protein readily soluble in water, dilute
acids and alkalies which may be precipitated out from solution using high salt
concentration in a process called 'salting out'. Albumin experiences heat
coagulation (protein denaturation). Substances containing albumin, such as egg
white, are called albuminoids. Albumin is the main protein of plasma; it binds
water, cations (such as Ca2+, Na+ and K+), fatty acids, hormones, bilirubin and
drugs. Its main function is to regulate the colloidal osmotic pressure of blood.
Furthermore, the experiment was purposed to identify the protein in Egg’s
albumin by using Biuret, precipitation by metal, precipitation by salt, coagulation,
precipitation by alcohol, and denaturation test.

Materials and Method

In this experiment, there are severals equipments used such as, 1 racks of
test tube, drop ppipette, funnel, beaker glass 25 mL and 100 mL, volumetric
pippette, volumetric cylinder 10 mL, erlenmeyer 100 mL, spatulla, stirring rod,
spritus, watch glass, and heater. Meanwhile material needed namely, solution of
egg’s albumin, NaOH 0.1N, NaOH 2.5N, solution of CuSO4, HgCl2, Pb-acetate,
ammonium sulfate crystal, Millon’s reagent, Acetate buffer pH 4.7, HCl 0.1N,
aquades, BaCl2, ethyl alcohol 95%, and the solution of CH3COOH 1M.
Furthermore, the methos used was experiment in qualitative aspect. The
test was conducted to several test, namely Biuret test, precipitation by metal,
precipitation by salt, coagulation, precipitation by alcohol, and denaturation test.
The steps of the test were as follows:
a. Biuret’s Test: 3 mL of protein solution was added by 1 mL NaOH 2.5N, then
added drop by drop CuSO4 0.01N. All of the mixture then stirred. The color
was observed.
b. Precipitation by Metal Test: 3 mL of protein solution was added by 5 drops
HgCl2 0.2M. The solution was observed. Then continued by the addition of 5
drops Pb-accetate 0.2M and the solution was also observed.
c. Precipitation by Salt Test: 3 mL of protein solution was saturated by
ammonium sulfate. The solution then filtrated to obtain the precipitate. The
precipitate then tested by soluting it in water. Some of precipitation was tested
by Millon’s reagent. The filtrate was tested by Biuret. The color of solution
was observed.
d. Coagulation Test: 5 mL of protein solution was added by 2 drops of
CH3COOH 1M and boiled about 5 minutes. The precipitation formed was
taken by stirring rod. Then it dissolved in water and observed. Some of
precipitation was tested by Millon’s reagent. The color of solution was
observed.
e. Precipitation by Alcohol Test: about 3 test tubes were prepared. Test tube I
filled with 5 mL of protein solution, 1 mL of HCl 0.1 M, and 6 mL ethyl
alcohol 95%. It was mixed and then observed. Test tube II filled by 5 mL
protein solution, 1 mL NaOH 0.1M, and 6 mL ethyl alcohol 95%. It was mixed
and then observed. Meanwhile the test tube III was filled by 5 mL of protein
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solution, 1 mL of buffer accetate pH 4.7, and 6 mL ethyl alcohol 95%. It was

mixed and then observed.
f. Denaturation Test: about 3 test tubes were prepared. Test tube I filled with 9
mL of protein solution and 1 mL of HCl 0.1 M. It was put in the heat water and
then observed. Test tube II filled with 9 mL of protein solution and 1 mL of
NaOH 0.1 M. It was put in the heat water and then observed. Test tube III filled
with 9 mL of protein solution and 1 mL of buffer acetate. It was put in the heat
water and then observed.

Result and Discussion

Sample preparation
The protein solution was prepared from the egg albumin and dilluted in
aquades with proportion 1:5. The solution than tested by several test
identification, such as Biuret, precipitation by metal, precipitation by salt,
coagulation, precipitation by alcohol, and denaturation test. Based on the
observation, the result can be concluded as the table bellow:

Table 1. The Observation Result of Sample Test Identification

Test
Identifi- Observation Result Confirmation
cation
Positive
Biuret’s
- Producing violet color contained
Test
peptide bonds
Positive
Precipitatio
produced
n by Metal - Formation of precipitation
insoluble metal-
Test
protein salt
- Formation of precipitation
Precipitatio Positive
- The precipitaion soluble in water.
n by Salt contained
- The precipitation produced red-orange color with Millon.
Test tyrosin.
- The filtrate produced blue color with biuret.
- Formation of precipitation Positive
Coagulatio
- The precipitaion soluble in water. contained
n Test
- The precipitation produced red color with Millon. tyrosin.
Positive test for
Precipitatio the characteristic
- Formation of precipitation in acid condition (Tube I)
n by of protein with
- No formation of precipitation in base condition (Tube II)
Alcohol regard to the pH
- Formation of precipitation in buffer condition (Tube III)
Test and isoelectric
point.
- Formation of precipitation in acid condition through Positive test for
heating (Tube I). the characteristic
Denaturatio - No formation of precipitation in base condition through of protein with
n Test heating (Tube II). regard to heat,
- Formation of precipitation in buffer condition through pH, and
heating (Tube III). isoelectric point.
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Biuret’s Test
The name of the test comes from the compound biuret, which gives a
typically positive reaction. Biuret is obtained by heating urea to about 180 C°. In a
kind of chelation reaction a blue color is formed with Cu2+. The reaction can be
seen as bellow:

Figure 3. Biuret Preparation

The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in
proteins to form a blue to violet complex known as the “biuret complex”. as
bellow:

Figure 4. Structure of Biuret COmplex

Alkaline copper sulfate reacts with compounds containing two or more

peptide bonds to give the violet-colored complex. The depth of the color obtained
is a measure of the number of peptide bonds present in the protein. The reaction is
not absolutely specific for peptide bonds, since the compounds containing two
carbonyl groups linked through hydrogen or carbon atom will give a positive
result. According to this experiment, it was obtained the violet color which is
indicated that the solution contained peptide bonds.

Precipitation by Metal Test

Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high molecular weight cations. The
positive charge of these cations counteracts the negative charge of the carboxylate
group in proteins giving a precipitate. At pH 7 and above, proteins are usually
negatively charged so that addition of a positively charged metal ion neutralizes
this charge and the proteins come out of solution. Precipitation by heavy metals is
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therefore, most effective at neutral to slightly alkaline pH values, although the

solution must not be too alkaline otherwise there is a risk of metal hydroxides
being precipitated. The formed precipitate is frequently soluble in excess of the
heavy metal ion solution since the excess ions confer a stabilizing positive charge
on the particles. One of the reaction by using heavy metal, as follows:

O O
protein C O + Ag protein C O Ag

Figure 5. One of Reaction in Protein Precipitation by Metal

Salts of metal ions form strong bonds with disulfide groups and with the
carboxylate ions of the acidic amino acids. Thus, they disrupt both disulfide
bridges and salt linkages and cause the protein to precipitate out of solution as an
insoluble metal-protein salt. According to the result, it produced precipitation and
showed the positive test for protein.

Precipitation by Salt Test

Protein molecules contain both hydrophilic and hydrophobic amino acids.
In aqueous medium, hydrophobic amino acids form protected areas while
hydrophilic amino acids form hydrogen bonds with surrounding water molecules
(solvation layer).
When proteins are present in salt solutions (e.g. ammonium sulfate), some
of the water molecules in the solvation layer are attracted by salt ions. When salt
concentration gradually increases, the number of water molecules in the solvation
layer gradually decreases until protein molecules coagulate forming a precipitate;
this is known as “salting out”. As different proteins have different compositions of
amino acids, different proteins precipitate at different concentrations of salt
solution. The figure can be seen as follow:

Figure 6. Protein Precipitation by Salt

Based on this experiment, it resulted in formation of precipitation. The

precipitation was dissolved in water. And by the testing of Millon to the
precipitation, it produced red-orange color which indicate the positive test for the
presence of tyrosin in protein, as reaction bellow:
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NO2
COO - COOH
H2 conc. -H2O H2
+H OH + Hg22+ + HNO
3N C C +H
3 3N C C OH + HgO
H H

Tyrosine Nitrated Tyrosine

Figure 7. Tyrosin and Millon’s Test Reaction

The filtrate of also confirmed positive test for the protein. It indicated by the
formation of blue color with biuret. The number of peptide bonds present in the
filtrate might be not too significant due to the the bright blue color of solution (not
violet). But, it still a positive test for the protein presence.

Coagulation Test
Salt bridges result from the neutralization of an acid and amine on side
chains. The final interaction is ionic between the positive ammonium group and
the negative acid group. Any combination of the various acidic or amine amino
acid side chains will have this effect.
As might be expected, acids and bases disrupt salt bridges held together by
ionic charges. A type of double replacement reaction occurs where the positive
and negative ions in the salt change partners with the positive and negative ions in
the new acid or base added.
In this experiment, coagulant formed by addition of acid causes H+ ions
bound to the protein negative group (amine group). At the isoelectric point of the
protein will have positive and negative poles of the same ratio. When H+ ions
entered the acid solution into the system, the balance will be disturbed and the
damaged conformation of tertiary and quaternary protein structures. It caused the
protein undergo coagulation in the form of precipitation. The precipitation was
soluble in water and it produced red color with Millon indicated a positive test for
tyrosin.

Precipitation by Alcohol Test

According to the three test tube used, respectively from the left to the right, there
were acid, base, and buffer condition.

Figure 8. Test tube of Acid, Base, and Bufer Condition

The formation of precipitation due to the the weak of protein in competing with
alcohol to bond the water that caused it resulted in demaged conformation. It
caused the solubility of protein in water decrease and caused precipitation.
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In tube I, the addition of acid and alcohol causing proteins under

isoelectric point and lost to compete in binding water molecules that undergo
deposition. In tube II, the addition of base and alcohol do not cause precipitate
formation due to the addition of NaOH causes the pH is above the isoelectric
point so that increased protein solubility. In tube III, the addition of acetate buffer
and alcohol led to the formation of a white precipitate. Buffer solution serves to
maintain the pH of the solution while the alcohol function is to bind water
molecules because the hydroxy group is stronger in binding water molecules
compare to the protein, so that the protein precipitate.

Denaturation Test
As before, this experiment also used three test tube with different condition. Tube
I contained HCl and albumin, Tube II contained NaOH and albumin, and tube III
contained Buffer-acetate and albumin. All then heated in hot water. The
observation were as the figure bellow:

Figure 9. Test tube of Acid, Base, and Bufer Condition with Albumin After Heating

Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic

interactions. This occurs because heat increases the kinetic energy and causes the
molecules to vibrate so rapidly and violently that the bonds are disrupted. The
proteins in eggs denature and coagulate during cooking.
The precipitation of protein by hydrochloric acid through heating can be
caused the protein was in bellow of its isoelectric pH so that the solubility
decreases. Besides that, heating can disrupt the hydrogen bonds caused the
deposition of protein.
The precipitation of protein by NaOH through heating produced no
precipitate because NaOH caused the protein in above of the isoelectric pH, so
protein solubility in water was increase.
The precipitation of protein by buffer through heating caused protein
deposits. In general, with the help of heating up to 80-100oC, it can destroy the
hydrogen bonds and nonpolar hydrophobic interactions, so the protein coagulated.

Conclusion
According to the experiment result, can be concluded that the protein in
Egg’s albumin can be identified by using Biuret, precipitation by metal,
precipitation by salt, coagulation, precipitation by alcohol, and denaturation test.
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Acknowledgment
This practicum report would not have been possible without the guidance
and the help of several individuals who in one way or another contributed and
extended their valuable assistance in the preparation and completion of this study.
First and foremost, my utmost gratitude to Mr. Dr. I Nyoman Tika, M.Si., as the
biochemistry practicum lecturer of Chemistry Education Department UNDIKSHA
for his sincerity and encouragement. Mr. I Ketut Lasia, S.Pd., M.Pd. as the
laboratory assistant that help during the practicum. and also my teamwork,
Ningsih Handayani for the corporation during the practicum.

References
Anonymous. 2013. Identification of Unknown Amino Acid. www.google.com.
Redhana, I Wayan dan Siti Maryam. 2010. Penuntun Praktikum Biokimia.
Singaraja: IKIP Negeri Singaraja.
Stryjecka, Marta and Zimmer. 2013. Amino Acids and Proteins.
www.google.com.
Tika, I Nyoman. 2010. Penuntun Praktikum Biokimia. Singaraja: Universitas
Pendidikan Ganesha.
Willbrand, Ann. Proteins and Denaturing Agents .USC-Aiken. www.google.com.