BIOCHEMISTRY LAB REPORT

AYU LAKSMI PUSPITASARI

BIOLOGY IUP
24020118190151

TOPIC IV
PROTEIN CHARACTERIZATION

I. Basic Competencies
Practitioners are able to identify protein based on their general characteristic that
includes precipitation, augmentation, denaturation, and protein hydrolysis with enzyme.

II. Literature Review

2.1. General Characteristics of Protein
Proteins are polymers of amino acids, with each amino acid residue joined to its
neighbor by a specific type of covalent bond. (The term “residue” reflects the loss of
the elements of water when one amino acid is joined to another.) Proteins can be
broken down (hydrolyzed) to their constituent amino acids by a variety of methods,
and the earliest studies of proteins naturally focused on the free amino acids derived
from them. Twenty different amino acids are commonly found in proteins. All the
amino acids have trivial or common names, in some cases derived from the source
from which they were first isolated. All 20 of the common amino acids are α-amino
acids. They have a carboxyl group and an amino group bonded to the same carbon
atom (α carbon). They differ from each other in their side chains, or R groups, which
vary in structure, size, and electric charge, and which influence the solubility of the
amino acids in water. For all the common amino acids except glycine, the α carbon is
bonded to four different groups: a carboxyl group, an amino group, an R group, and a
hydrogen atom. The α -carbon atom is thus a chiral center. Because of the tetrahedral
arrangement of the bonding orbitals around the α -carbon atom, the four different
groups can occupy two unique spatial arrangements, and thus amino acids have two
 possible stereoisomers. All molecules with a chiral center are also optically active—
that is, they rotate plane-polarized light. (Lehninger 7th edition, 2018).
2.2. Protein Denaturation
All proteins begin their existence on a ribosome as a linear sequence of amino
acid residues. This polypeptide must fold during and following synthesis to take up its
native conformation. Protein structures have evolved to function in particular cellular
environments. Conditions different from those in the cell can result in protein
structural changes, large and small. A loss of three-dimensional structure sufficient to
cause loss of function is called denaturation. The denatured state does not necessarily
equate with complete unfolding of the protein and randomization of conformation.
Under most conditions, denatured proteins exist in a set of partially folded states that
are poorly understood. (Lehninger 7th edition, 2018).
Most proteins can be denatured by heat, which affects the weak interactions in a
protein (primarily hydrogen bonds) in a complex manner. The effects of heat on
proteins are not readily predictable. Proteins can be denatured not only by heat but by
extremes of pH, by certain miscible organic solvents, such as alcohol or acetone, by
certain solutes such as urea and guanidine hydrochloride, or by detergents. Each of
these denaturing agents represents a relatively mild treatment in the sense that no
covalent bonds in the polypeptide chain are broken. Organic solvents, urea, and
detergents act primarily by disrupting the hydrophobic interactions that make up the
stable core of globular proteins; extremes of pH alter the net charge on the protein,
causing electrostatic repulsion and the disruption of some hydrogen bonding.
(Lehninger 7th edition, 2018).
2.3. Protein Hydrolysis
Hydrolysis is a chemical process that uses H2O as a breaker of compounds
including inversion of sugar saponification fats and protein breaking esters and
Grignard reactions. H2O as a reagent in the broad sense including acidic and basic
solutions (in organic compounds, neutralization hydrolysis). Protein hydrolysis is the
process of breaking the polymer into monomers with the help of enzymes as
biocatalysts. Hydrolysis in peptide bonds will cause some changes in the properties of
proteins, namely increasing solubility due to increased content of NH3 + and COO-
and reduced molecular weight of proteins or polypeptides, and damage to globular
structures of proteins. (Freskya, 2013).
2.3.1. Enzyme
Protease enzymes are enzymes that catalyze the breakdown of prote
molecules by hydrolysis. Protease enzymes can be divided into two types,
namely endopeptidase and exopeptidase. Endopeptidase only breaks down
proteins in certain places in protein molecules and the base does not affect the
group at the end of the molecule. While exopeptidase breaks down proteins at
both ends of protein molecules. Carboxypeptidase can release amino acids
that have a free -COOH group on protein molecules, while aminopeptidase
can release amino acids at the other end that have a free -NH2 group.
(Freskya, 2013).
III. Methods
3.1. Tools
1. Test tube
2. Test tube rack
3. Drop pipette
4. Water bath
5. Tube holder
6. Tissue
7. Camera
8. Laboratory manual book
9. Laboratory Temporary Report book
10. Stationary
3.2. Materials
1. Albumin solution
2. Ammonium Sulfate (10, 20, 30, 40, and 50%)
3. ZnSO4
4. Alcohol
5. Red Chlorophenol Indicator
6. 2% of Acetate Acid
7. Pepsin
8. Karmyn fibrin
9. 0,45% of HCl
10. Water
3.3. Procedure
3.3.1. Precipitation test
3.3.1.1.With salt
1. The test tube was prepared
2. 10 ml of protein solution was poured
3. Then, ammonium sulfate was added little by little
4. The mixture was stirred until dissolve
5. Then, ammonium sulfate was added again until little salt was left
 3.3.1.2.With hard metal
1. The test tubes were prepared
2. 2 cc of protein solution was dropped into test tube
3. 1 drop of ZnSO4 solution was added
4. Then, the emulsion that formed was divided again into 2 tubes
5. ZnSO4 solution was added again into one of the emulsion tubes
6. The changes were observed
3.3.1.3.With alcohol
1. The test tube was prepared
2. 2 cc of alcohol was dropped into test tube
3. 1 or 2 drops of protein solution were added into the tube
4. The changes was observed
3.3.2. Coagulation test
1. 2 cc of protein solution was dropped into test tube
2. 1 drop of Red Chlorphenol Indicator was added into the tube
3. 2 % of acetate acid was added into this solution until the “pink” color was
gone
3.3.3. Digestion test
1. test tubes were prepared
2. First tube, was added by 1 cc of pepsin, 1 cc of 0,45% HCl, and 2 slices of
Karmyn Fibrin
3. Second tube, was added by 2 cc of pepsin, 1 cc of water, and 2 slices of
Karmyn Fibrin
4. Third tube, was added by 1 cc of pepsin. Then, the tube was heated for 1
minute and after that, was cooled by water. Next, 1 cc of 0,45% HCl and
2 slices of Karmyn Fibrin were added into the tube
5. All tubes were entered into water bath at 37°c
6. The changes were observed
 IV. Result
Test Before After +/- Notes
Precipitation by + From clear (or
salt transparent)
solution to
white cloudy
solution and
precipitation
formed starting
from 40-50%
(Personal documentation, (Personal documentation,
2019) 2019)
Precipitation by + White
heavy metals precipitate
formed. White
Precipitate
turns back to
liquid upon
more addition
of ZnSO4

(Personal documentation, (Personal documentation,

2019) 2019)
Precipitation by (Personal documentation, (Personal documentation, + precipitation
alcohol 2019) 2019) occurred, with
substance
appeared hazy
 Protein + coagulation
coagulation happens after
the third drop
of acetic acid,
indicated by
the change of
color

(Personal documentation, (Personal documentation,

2019) 2019)
Protein + From orange-
digestion ish brown
solution with
camerin fibrin
to some visible
camerin fibrin
with clear (or
transparant)
(Personal documentation,
orange-ish
2019)
brown solution
Pepsin + HCl + Karmyn
Fibrin

(Personal documentation,
2019)
 - From orange-
ish brown
solution with
camerin fibrin
to whole
visible camerin
fibrin with
slightly
orange-ish
(Personal documentation, (Personal documentation,
brown solution
2019) 2019)
Pepsin + Water + Karmyn
Fibrin
+ From orange-
ish brown
solution with
camerin fibrin
to no visible
camerin fibrin
with slightly
orange-ish

(Personal documentation, brown cloudy

2019) solution
(Personal documentation,
2019)
Pepsin + HCL + Karmyn
Fibrin (Was heated and
cooled down)
V. Discussion
Biochemistry laboratory practice topic IV titled “Protein Characterization” on
Thursday, May 9th, 2019 in Biochemistry Laboratory of Science and Mathematics
Faculty, Diponegoro University, was held on the purpose to identify protein based on
their general characteristic that includes precipitation, augmentation, denaturation, and
protein hydrolysis with enzyme. The tools used were test tube, test tube rack, drop
pipette, water bath, tube holder, tissue, camera, laboratory manual book, laboratory
Temporary Report book, and stationary. The materials used were albumin solution,
ammonium sulfate (10, 20, 30, 40, and 50%), ZnSO4, alcohol, red chlorophenol
indicator, 2% of acetate acid, pepsin, karmyn fibrin, water and 0,45% of HCl.
5.1. Protein Precipitation
Protein precipitation is a process of separating protein and its solution, decreasing
its solubility. This aligned with Effendi (2003) that protein can be precipitated
because it have several characteristics, like being an amphoteric, which is having two
different charges in one molecule, also known as Zwitter ions. These characteristics
make proteins have different charges on different pH level, resulting in the protein
being soluble in a certain pH level, where it is charged. On a time, the protein will
reach isoelectric point, which the pH where the total charges of protein equals to zero
(positive ions equals to negative ions), and this will affect the protein solubility. In
isoelectric point, the solubility of protein is low thus the protein can precipitate.
5.1.1. Precipitation by Salt
This test works by mixing 2 ml of albumin samples with 3 drops of
ammonium sulfate [(NH2)4SO4] on different concentrations, which are 10%,
20%, 30%, 40%, and 50%. The mixtures were then shaken well until
precipitation occur, and then added again with ammonium sulfate until it
turns back to liquid or less salt is precipitate.
The principal of this test is the solubility of protein on a solvent. Protein
with the addition of salt on high concentration make the water molecules that
at first bonded with hydrophobic surface protein become bonded with salt and
resulted in the creation of precipitate. This is as stated by Triana (2013) that
the principal of protein precipitation is protein solubility on solvent. The more
 water molecules that bonded with salt ions affecting the protein to interact
with each other, segregated and precipitated (salting out).
The usage of ammonium sulfate [(NH2)4SO4] functions to decrease the
solubility of protein to create precipitation. This aligned with Poedjiadi
(2008) that explained that the albumin solution on water could be precipitated
by the addition of ammonium sulfate until saturated. If protein is added with
inorganic salt solutions in high concentration, then the protein solubility will
decrease until precipitation is created. This process happens because of the
competition between protein molecules and inorganic ions in bonding with
water molecules. The treatment done on the sample was strong shaking. This
shaking functions to accelerate the reaction to dissolve the sample. This is as
stated by Sakinah (2011) that the functions of shaking is to dissolve sample.
A positive result will show that the protein sample precipitate and un-
precipitate by ammonium sulfate. According to Hart (2007), the protein
precipitation with salt depends on its concentration and amount of ion charges
in the solution. On our practicum, we observed that the easiest sample to
precipitate was the tube with less concentration of ammonium sulfate, and the
easiest one to go back to liquid was also the less concentrated. Thus the
results were positive.
5.1.2. Precipitation by Heavy Metal
This test works by adding ZnSO4 to albumin sample until precipitation
occurs. Then the precipitate is transferred to a different tube and added with
ZnSO4 until it turns back to liquid. The principal of this test is that metal
compound will cut off the salt bridge and bonded with protein. The COOH
and NH2 group on protein react with heavy metal and creates chelation. This
is as explained by Wirahidakusumah (2010) that metal compound added to
albumin will break the saline bridge and bonded with protein, creating
proteinase metal precipitate that’s not dissolved. At the same time, the COOH
and NH2 groups in the protein will react with heavy metal ions, resulting in
chelate compounds. The amount of precipitate produced on each sample
depends on the activeness of the heavy metal added.
 The usage of ZnSO4 functions to neutralize protein charges with positive
ions from metal to produce precipitation. This aligned with Budiman (2009)
that ZnSO4 will neutralize the protein charges with positive ions possessed
by metal solution. The treatment done on the sample was strong shaking. This
shaking functions to accelerate the reaction to dissolve the sample. This is as
stated by Sakinah (2011) that the functions of shaking is to dissolve sample.
A positive result will show the addition of ZnSO4 resulted in precipitation,
and the precipitate given ZnSO4 will turns back into liquid. According to
Hamid (2005), the sample that contains protein will denaturize and produce
many precipitate if the heavy metal molecules are bigger. On our practicum,
we observed that the ZnSO4 could precipitate and un-precipitate in the
protein sample. Thus the result was positive.
5.1.3. Precipitation by High-Concentrated Alcohol
The test works by mixing high-concentrated alcohol with albumin sample.
The principal of this test is the functional alcohol group will bond water, thus
the decrease of protein solubility. This is as stated by Rismaka (2009) that
this method of precipitation is the competition between protein-water and
alcohol-water creation. Alcohol could precipitate protein because its
functional group is stronger on bonding water, resulting in the protein’s
decreasing solubility. The open C edge of amino acid on protein could react
with alcohol in acidic atmosphere, creating protein ester.
The usage of alcohol functions to decrease the solubility of protein. This is
as explained by Ariwulan (2011) that the addition of alcohol which is an
organic solvent will decrease protein solubility, because the solubility of
protein depends on the position and distribution of hydrophilic and
hydrophobic polar group on molecules. The treatment done on the sample
was strong shaking. This shaking functions to accelerate the reaction to
dissolve the sample. This is as stated by Sakinah (2011) that the functions of
shaking is to dissolve sample.
A positive result will show that precipitate is created. This is as according
to Effendi (2003) that protein can be precipitated by the addition of alcohol.
 On our practicum, we observed that precipitation occurred, with substance
appeared hazy. Thus the result was positive.
5.2. Protein Coagulation
Protein coagulation is the creation of protein lump because of water absorption
from heating. This is as stated by Makfoeld (2008) that coagulation is a state where
protein is no longer dispersed as a colloid because of the increasing amount of bonded
units. Coagulation can also be defined as the damage on protein because of heat that
creates lumps and hardens the protein in process.
This test works mixing a drop of chlorophenol to protein sample which resulted in
a pink change of color, and then adding 2% acetic acid carefully until the color is
unsaturated. The principal of this test is that protein can be turned from liquid to solid
to semi-solid by extracting water from it. This is as explained by Vacklavik (2008)
that coagulation is a continuous process that happens if a denaturized protein
molecules create a solid mass, by which water escaped from the structure, creating
open spirals that bonded with each other.
The usage of chlorophenol indicator functions to achieve pH level on isoelectric
point of protein. This is as explained by Nuryati (2001) that protein with red
cholorophenol will change the isoelectric point into alkalis. The protein on isoelectric
pH will decrease in solubility. The usage of 2% acetic acid functions to agglutinate
the protein in isoelectric point. This is as explained by Shurleff and Aoyagi (2007) the
acid generally used to agglutinate protein is acetic acid.
On our practicum, we observed that coagulation happens after the third drop of
acetic acid, indicated by the change of color. It is aligned with Makfoeld (2008) that
stated the addition of chlorophenol indicator resulted in a change of color into red,
indicating the pH is base. The acid will jeopardize the salt bridge in protein as the
positive and negative ion on protein switched partners, or could be said that it is
denaturized. Thus the result was positive.
5.3.Protein Digestion
This test works by creating three samples of pepsin. The first pepsin sample is
given HCl. The second tube is given water. The thirst tube is heated and then added
with HCl. All samples then is given karmyn fibrin, and then heated together in 37C.
The principal of this test is that protein is hydrolyzed by pepsin enzyme that active on
acidic environment. This hydrolysis produces amino acid where its carbocsyl group
releases H+ ions, while the base amino group receives H+ ions. This is as explained
by Poedjiadi (2008) that protein hydrolysis produces amino acids. The protein
digestion on pepsin works in acidic environment, and is damaged on base
environment or if heating occurs.
The usage of Pepsin is for the enzyme to hydrolyze protein. This is as explained
by Ridwan (2010) that pepsin could hydrolyze karmyn fibrin as protein source. HCL
functions to create acidic environment to activate pepsin. This is aligned with
Poedjiadi (2008) that stated digestion by pepsin works in acidic environment.
On our practicum, we observed on the first tube (pepsin+HCl) that the color of
karmyn fibrin was less saturated. On the second tube (pepsin+water), the color was
saturated and size is smaller. On the third tube (pepsin heated+HCl), the color was
saturated and the size was bigger. According to Ridwan (2010), the karymn fibrin
added with HCl and then heated will increase in size and turns light orange because
pepsin as enzyme could hydrolyze karmyn fibrin as protein source. The test with
water didn’t change in color thus it is not hydrolyzed. The addition of water didn’t
help with hydrolysis because water didn’t activate pepsin. It is as explained by
Suwandi (2009) that karmyn fibrin as substrate can’t hydrolyze because pepsin could
not activate without HCl. Thus all results were positive
VI. Conclusion
The general characteristics of protein include reactions of precipitation,
coagulation, and denaturation, and hydrolysis by enzyme. The precipitation reaction is by
salt, heavy metal, and high-concentrated alcohol. Precipitation by salt (NH4SO4) was
positive, indicated by the creation of white precipitate. In precipitation by heavy metal,
the more metal added, the more precipitate created. Precipitation by alcohol has a
positive result indicated by the creation of precipitate. Protein coagulation by acetic acid
has a positive result indicated by the creation of lump, because acetic acid breaks the salt
bridge so the amount of ion is equal and resulted in coagulation. Protein digestion used
pepsin enzyme and the result was positive. Pepsin hydrolyzes protein into peptone in
acidic environment.
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Bandung: ITB