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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.
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:
2
NH 2 OOC Ar
N H O C S S Ar
C O H N
Hydrophobic
hydrogen bonds salt bridge disulfide bridge Interaction
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
4
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:
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:
O O
protein C O + Ag protein C O Ag
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.
NO2
COO - COOH
H2 conc. -H2O H2
+H OH + Hg22+ + HNO
3N C C +H
3 3N C C OH + HgO
H H
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.
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.
9
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
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.
10
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.