CHM160L Biochemistry 1 Laboratory

4th Quarter SY 2018-2019

Protein Characterization and Enzyme Activity

Nanette D. Santos1, Amarrador, Jelsea, Cortez Mariene-syne Edisa P., Sardome, Rachelle, 2Victoria, Jane Xuan B.
1Professor, 2Student, CHM160L/E01, School of Chemical, Biological, and Materials Engineering and Sciences, Mapua University

ABSTRACT

Thin layer chromatography (TLC) is an easy, convenient and inexpensive way to determine how many components are in a
mixture and, in many instances, can be used to identify the components as well. The objective of this experiment is to separate
amino acids using thin layer chromatography. A spot of the sample is placed on a sheet of glass treated with an absorbent
substance. The glass is then placed in a solvent that will travel up the absorbent surface and cause the solid to move out of the
liquid with it. Different solids will move different distances on the sheet, but the distance will remain constant no matter how
many times chromatography is done. This distance is calculated into an amount called the Rf value, which can be used to
determine the identity of the substance.

Keywords: Thin layer chromatography, amino acids, Rf value, polarity, solublity, distance, molecular weight

INTRODUCTION them to aggregate and diminishing their interactions with

Proteins are biopolymers of the 20 common amino acids.
They have a lot of functions and are very important to any
organism. The amino acids are connected through the
peptide bonds formed between their α-amino and α-
carboxyl groups:
water molecules and rendering them insoluble in water.
Some of the amino acids in proteins are aromatic, such as
tyrosine and tryptophan. The nitration of these amino acids
using nitric acid will also be characterized, while it will be
investigated whether these reactions can also occur when
these amino acids are part of a protein chain. The reaction
of sodium nitrite with the phenol group in tyrosine to
produce a pink or red product will also be observed. Other
chemical reactions used to characterize proteins are due to
interactions with the peptide bond, such as the
complexation of copper (Cu2+) ions in the presence of
strong base, which is the basis of the biuret test for protein.

One biologically important class of proteins are the

FIGURE 3.1. An illustration showing how amino acids serve
as building blocks for proteins
The proteins to be studied in this experiment are casein
(milk protein) and albumin (in this case, from egg whites).
Casein and egg albumin are soluble in water in their native
or natural state. However, they may precipitate from
solution in the presence of acids, alkali, heavy metals and
other denaturing agents. Many of the chemical properties of
proteins are due to the characteristics of the R groups of
the amino acids that make up the protein. For example,
acidification of ionized groups in proteins may neutralize
their ionic charges resulting in decreased solubility in water
or alterations in their interactions with other ionic and polar
groups, causing them to precipitate. Heavy metals may bind
to ionic or polar groups on the surface of proteins, causing
enzymes. They are basically biological catalysts. Enzymes
are the workhorses of any living organism. If a cell is
represented by a city, the enzymes would be everyone that
works. Enzymes catalyze metabolic reactions to process
the food that we eat. They assemble various monomers into
structural polymers needed in the body. They send signals
between cells. They act as the defenders of the body.
Needless to say, it would not be possible to survive if there
were no enzymes. Thousands of enzymes are found in
living cells where they act as catalysts for the thousands of
chemical reactions which occur. In addition to making life
possible, many enzymes have numerous applications that
affect our daily lives in other ways such as food processing,
clinical diagnoses, sewage treatment, and the textile
industry. Enzyme experiments are ideal for “hands on”
opportunities and since several factors affect the rate at

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which enzymatic reactions proceed, an enzyme experiment
presents many opportunities in the biology laboratory. The
following experiments are based on the enzyme catalase.
The objective of the experiment is to characterize
the chemical and physical properties of proteins from
natural sources (egg and milk) and some of the chemical
reactions that the amino acid residues in these proteins
undergo, as well as the effects of denaturing agents on
these proteins. The experiment also aims for the students
to know what enzymes are and their importance in living
systems, as well as to observe the actual effects of
enzymes.
MATERIALS AND METHODOLOGY
The reagents used for the experimentation proper are skim
milk, egg, 0.5% glycine, 0.5% L-tyrosine, 0.5% L-
tryptophan, vinegar or 5% acetic acid solution, 10% sodium
hydroxide (NaOH), concentrated nitric acid (HNO3), 1.0 M
perchloric acid (HClO4), 1 M ammonium chloride (NH4Cl),
5% sodium nitrite (NaNO2), 95% ethanol, 0.1 M copper (II)
sulfate, 0.1 M mercury (II) chloride, and 0.1 M lead (II)
nitrate.
The materials and equipment used to perform this
experiment are as follows: micro test tubes, test tubes, test
tube rack, dropper, test tube holder, thermometer,
evaporating dish, hot plate, beaker, pipette, iron stand,
funnel, funnel support, and suction bulb.
Protein Characterization
Procedure. A 400 mL beaker was filled to about half full
with water and heated for a warm (40°C) water bath for the
beginning of part A. This was later heated to higher
temperatures for the other parts of the experiment. The egg
white was separated from the egg yolk and a solution of
one egg white in 500 mL deionized water was prepared to
serve as the egg albumin solution. About 20 mL of egg
white solution and about 20 mL of milk was used for the
tests performed. Approximately this amount of each
solution (egg white solution and skim milk) was added to
two separate beakers. None of the solutions were poured
back into their containers.
Part A: Precipitation of Casein from Milk and Albumin
from Egg White. Proteins are denatured (lose their natural
structure and solubility) by a variety of denaturing agents,
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such as organic solvents, acids, bases, heavy metals (e.g.,
mercury or lead) and high salt concentration. 2 mL of egg
white solution was placed in each of six clean large test
tubes and 2 mL of skim milk in each of six clean small test
tubes. One of the test tubes containing egg white solution
and one of the test tubes containing milk was placed in a
warm water bath at about 40°C. After the solution in the
test tubes had warmed for a few minutes, about 1 mL of
vinegar was added to each of the test tubes and it was
noted whether any precipitate formed in the test tubes.
Observations were recorded. The water bath was then
heated to boiling in preparation for the next tests. To a
second tube containing egg white solution, 1 mL of 1.0 M
HClO4 (perchloric acid) was added and the same was done
to a second tube containing milk. Each tube was mixed well
and observations were recorded. 3 mL of 95% ethanol was
added to the third tube of egg white solution and 3 mL of
95% ethanol to the third tube of milk. After mixing each tube
well it was noted whether any precipitate formed.
Observations were recorded.
Another test tube containing egg white solution and one
tube containing milk was placed in the by-then boiling water
bath for 5 minutes and observations were recorded noting
whether there was any precipitation or clumping of protein
in either tube. The contents of all the former test tubes were
disposed of in the sink making sure not to mix them with the
next ones.
The next tubes contained HgCl2 (mercuric chloride) and
Pb(NO3)2 (lead nitrate or plumbous nitrate) which are
poisonous. It was not allowed to come into contact with skin
and if it did, the areas of contact would have had to be
immediately washed off with water. To one test tube
containing egg white solution and one test tube containing
milk, 10 drops of mercury (II) chloride (HgCl2) solution was
carefully added, mixed well and observations were
recorded. To the sixth tube containing egg white solution
and the final tube containing milk, 10 drops of lead nitrate
(Pb(NO3)2) solution was carefully added, mixed well and
observations were recorded. NOTE: The contents of these
last 4 test tubes containing lead and mercury was disposed
of in the waste bottle. The test tubes that contained these
chemicals were then immediately washed.
Part B: The Xanthoproteic Test. Aromatic rings in the
amino acids react with nitric acid, as do all aromatic
compounds, forming yellow nitrated products, which often
turn deeper yellow in strong base. CAUTION: Since nitric
acid is very corrosive to skin and clothing it was important
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to work with care in the fume hood in performing this test,
immediately washing with plenty of water if any did get on
the skin or clothing.1 mL of egg white solution was added to
a clean test tube; 1 mL of skim milk to a second test tube; 1
mL of 0.5% tyrosine solution to a third test tube; 1 mL of
0.5% tryptophan solution to a fourth test tube; and 1 mL of
0.5% glycine solution to a fifth test tube. 1 mL of
concentrated nitric acid (in the hood) was then added to
each test tube and mixed carefully. After mixing, the test
tubes were warmed in the hot water bath. Any changes in
the color of the solutions were noted and recorded. Each
tube was then allowed to cool and 10% NaOH solution was
carefully added until each was alkaline to litmus paper
(about 4 to 6 mL of 10% NaOH was needed to achieve
basicity). Observations were recorded.
Part C: Biuret Test for Proteins. Biuret is obtained by
heating urea. Biuret contains amide bonds similar to those
in proteins. Biuret reacts with copper (II) ions (blue) in basic
solution to form a purple complex ion.1 mL of egg white
solution was added to a clean test tube and 1 mL of skim
milk to another clean test tube. Next, 1 mL of 10% NaOH
and 3 drops of copper (II) sulfate (CuSO4), also called
cupric sulfate, solution was added to each test tube and
mixed well. These were allowed to stand for about 10
minutes and then observations regarding the color of the
solutions were recorded. 1 mL of glycine solution was
added to another clean test tube. And then 1 mL of 10%
NaOH and 3 drops of copper (II) sulfate solution were
added to this test tube, mixed well and results were
compared with the results from the previous test solutions
containing protein. Results were recorded.
Part D: Millon's Test. Tyrosine is the only amino acid
found in proteins having a phenolic group. On the addition
of Millon's reagent, a pink color forms. The presence of Cl-
or NH4+ ions interfere with this reaction. Hence, the test
tube ought to be rinsed with deionized water before
performing this test. 1 mL of egg white solution was added
to each of two clean test tubes; 1 mL of skim milk to
separate clean test tubes; 1 mL of 0.5% tyrosine solution to
another, and 1 mL of 0.5% glycine solution to a fifth clean
test tube. Each tube was labelled. 5 drops of 1.0 M
ammonium chloride solution was added to one of the tubes
containing egg white solution to show the effect of NH4+
and Cl- ions on this test. Then to each tube, 10 drops of
Millon’s reagent was added and then the test tubes were
placed in a hot water bath for 10 minutes. The test tubes
were then cooled in an ice bath. When cooled, 10 drops of
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freshly prepared 5% NaNO2 solution was added to each
tube and the color formed was taken note of.
Part E. Test for Sulfur. Cysteine and methionine are the
sulfur containing amino acids, but different in that cysteine
can be oxidized in alkaline solution to form a disulfide bond
linking two molecules to form the dimer cystine, which
reacts with lead. On the other hand, methionine has a
methyl group on the sulfur making it less reactive toward
lead. 1 mL of egg white solution was added to a clean test
tube; 1 mL of 0.5% cysteine to another clean test tub; and 1
mL of 0.5% methionine to a third clean test tube. To each
test tube, 2 mL of 10% NaOH solution was added and then
they were heated in a boiling water bath for a few minutes.
5 drops of lead nitrate solution was then added to each tube
and again heated for a few minutes. Observations were
recorded.
Enzyme Activity
Catalase. Test tubes (one for each material to be tested
plus extra for control), hydrogen peroxide (H2O2) (3%
solution), assorted living tissue: sliced raw potato, ground
meat, liver, yeast cells, ground young leaves, and non-living
material: piece of bread were the materials used for the
following procedure. Each labelled test tube was filled to
approximately 1/3 full with fresh hydrogen peroxide. Then a
small amount of material to be tested was added. Whether
or not bubbles were produced was noted.
RESULTS AND DISCUSSIONS
Part A. Precipitation of Casein from Milk and Albumin from
Egg White
Proteins are denatured by a variety of denaturing agents,
such as organic solvents, acids, bases, heavy metals and
high salt concentration. Milk and egg white are both mostly
composed of water and each made up of several proteins.
Casein is simply the name for a family of related
phosphoproteins (αS1, αS2, β, κ) commonly found in
mammalian milk, whereas albumin is a class of several
hundred proteins and egg white happens to contain a
couple of these albumin proteins. However, the term
albumin is also used to refer to the most abundant protein
in egg white.
In Table 3.1 are found the observations made regarding
clumping or precipitation of proteins and whether the
clumps settled quickly to the bottom of the tube or remained
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suspended in the tube for a long time. It has also been

indicated whether the solution (especially egg white
solution, which is clear to begin) became cloudy (i.e., small
white aggregates) or whether there are large aggregates or
clumps.

Substance Milk Egg white

added solution
Vinegar Clumped and Formation of
remained at the precipitate
bottom observed
Mercuric Small aggregates Cloudy, some
Chloride whites settled at
(HgCl2) the bottom
Lead Nitrate Small clumps Clear solution
(Pb(NO3)2) formed Figure 3.3 Addition of Perchloric Acid to Egg and Skimmed
Boiling Water No noticeable Solution became Milk
Bath changes cloudy
Perchloric Clumped and Solution became
acid (HClO4) settled at the cloudy and
bottom clumped
Alcohol Clumped and Clear solution
some settled at
the top, others at
the bottom
Table 3.1 Data observed in Part A

Figure 3.4 Addition of Alcohol to Egg and Skimmed Milk

Figure 3.2 Addition of Vinegar to Egg and Skimmed Milk

Figure 3.5 Boiling Water Bath of Egg and Skimmed Milk

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Figure 3.6 Addition of Mercuric Chloride to Egg and
Skimmed Milk
Figure 3.7 Addition of Lead Nitrate to Egg and Skimmed
Milk
The differences between the behavior of egg albumin and
casein with respect to precipitation by vinegar and
precipitation by heating in a boiling water bath can be
explained by the difference in the way an acid causes
denaturation in a protein versus the way heat causes
denaturation in a protein. Changes in pH affect the
chemistry of amino acid residues and can lead to
denaturation by disrupting the salt bridges held together by
ionic charges. A type of double displacement reaction
occurs wherein the positive and negative ions in the salt
change partners with the positive and negative ions in the
new acid or base added. Salt bridges are one of the four
types of bonding interactions between amino acid side
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chains which are responsible for the secondary and tertiary
structures of proteins. Hydrogen bonding is another of the
four types of bonding interactions between amino acid side
chains. Protonation of the amino acid residues (when an
acidic proton H + attaches to a lone pair of electrons on a
nitrogen) changes whether or not they participate in
hydrogen bonding, so a change in the pH can denature a
protein. On the other hand, 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.
Casein has phosphate groups attached to the hydroxyl
groups of some of the amino acids side-chains. It exists in
milk as a calcium salt, calcium caseinate. It has an
isoelectric point of pH 4.6. This means it is insoluble in
solutions with a pH less than 4.6. The pH of milk is 6.6;
therefore, casein has a negative charge at this pH and is
solubilized as a salt. When vinegar or acetic acid was
added to the milk, the casein was neutralized due to the
addition of protons and the pH decreased to a point at
which the casein became insoluble thus causing its
precipitation. Only a little precipitation was observable since
vinegar is only a weak acid and the white color of milk tends
to make seeing the precipitate challenging. In the case of
the egg white solution, very little change was observed
even though it is expected that the acid will similarly
denature the albumin in the egg white. This can be because
either the egg white used was already a very dilute solution
which contained mostly water and so less of the protein
from the egg white may have been available to react with
the acid to cause a more observable result or because
acetic acid is a relatively weak acid.
When egg white solution is placed in water bath above
room temperature, denaturation is expected to occur
followed by coagulation given enough time. Heat disrupts
hydrogen bonds inside the protein, as a result of unfolding
of specific tertiary conformation of proteins. Polypeptides
are reformed in different ways by interacting with other
polypeptides to form coagulum. This can explain the
formation of white precipitate and lump at the bottom of the
boiling tubes.
If a solution containing a protein is heated, it will reach a
temperature at which properties such as viscosity or the
absorption of ultraviolet (UV) light will change abruptly. This
temperature is called the melting temperature of the protein
(because the measurement is analogous to that made for
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the melting of a solid). The melting temperature varies for

different proteins, but temperatures above 41°C (105.8°F)
will break the interactions in many proteins and denature
them. This temperature is not that much higher than normal
body temperature (37°C or 98.6°F), so this fact
demonstrates how dangerous a high fever can be.

Part B. Xanthoproteic Test for Aromatic Groups

The color of the solution after heating with concentrated

nitric acid (HNO3) was recorded as well as the changes
after making the solution alkaline with sodium hydroxide
(NaOH). Differences in the shade or intensity of color are
described in Table 3.2.
Figure 3.9 Test samples after heating
Test sample Color with HNO3 Change after
adding NaOH
Egg White Light yellow Yellow
Solution
Milk Yellow Light yellow
Tyrosine (T) Light yellow Light yellow
Tryptophan Orange yellow Red orange
(W)
Glycine (G) Clear Clear
Table 3.2 Data observed in Part B

Glycine showed no color change. This is probably because

the side chain of glycine is only a hydrogen atom which is
not reactive towards this test.

Figure 3.10 Test samples with HNO3

Figure 3.8 Test samples before heating

Figure 3.11 Test samples after addition of NaOH

Part C. Biuret Test

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G T Egg White Egg White Milk

The color changes and other observations for each of the w/ NH4Cl
test solutions and the glycine amino acid solution when Initial clear clear clear Egg white white
Cu2+ was added is indicated in Table 3.3. The +M.R No orange Small Few brow
purple/mauve color indicates a positive test. . rxn precipitate precipitate n
preci
pitate
+HW No No rxn Clear Clear brow
B rxn precipitate precipitate n
separated preci
pitate
separ
ated
+Na No No rxn Forms light Forms Sol’n
NO2 rxn yellow yellow turns
Table 3.3 Data observed in Part C precipitate precipitate white
with
brow
n
preci
piate
Table 3.4 Data observed in Part D

Millon’s Test test is a test for detection of the amino acid

Figure 3.12 Right after addition of CuSO4
tyrosine. Glycine is negative for the test since it has no
tyrosine. Tyrosine is positive since it has shown the positive
result which is brown precipitate. Egg white with NH4Cl
shows yellow precipitate which is a positive for brown (not
enough heating). Egg white with no NH4Cl where a pink
precipitate formed is positive for showing almost reddish
color (not enough heating). Milk also showed positive for
the test where a reddish-brown or orange clumping is
found.

Part E. Sulfur Test

Egg White Cysteine Methionine

Initial Egg white color colorless colorless

+NaOH No reaction No reaction No reaction
+Pb(NO Turns amber Turns brown No reaction
3)2 color
+HWB Darker amber brown No reaction
color
Table 3.5 Data observed in Part E
Figure 3.13 10 minutes after the addition of CuSO4
Part D. Millon's Test for Phenols Sulfur-containing amino acids include cysteine and
methionine. If a sample that contains one or both of these

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amino acids is acidified, the gas H2S is produced, which
smells like rotten eggs. When a piece of moistened lead (II)
acetate paper is held over the solution as the H2S is being
produced, the H2S reacts with the lead ion, forming a black
or gray coating of PbS (lead (II) sulfide). Appearance of this
black colour is taken as a positive test for a sulfur-
containing amino acid. In this experiment, It can be taken
that the egg white contains cysteine, since the outcome of
the solution is a dark amber color which is almost similar to
black.
Enzyme Activity
Living Tissue
Sample Observation
Sliced raw potato Bubbles appeared
Ground meat More bubbles appeared
Liver More bubbles appeared
Yeast Bubbles appeared
Ground young leaves Few bubbles appeared
Non living Tissue
Boiled potato Very few bubbles appeared
Cooked liver Very few bubbles appeared
Table 3.6 Enzyme Activity Assessment for both Living and
Non living tissues.
Sample Observation
Gelatin Semi-solid/gelatin-like
Table 3.7 Data observed as to the heating of gelatin
solution
Proteins are held in their native conformations by a
combination of forces: hydrogen bonds, ionic interactions,
disulfide bridges, and hydrophobic interactions.Changing
the conformation of a protein either temporarily or
permanently by disrupting these forces is called
denaturation. Denaturation results in a loss of protein
activity. It involves the disruption and possible destruction of
both the secondary and tertiary structures. Since
denaturation reactions are not strong enough to break the
peptide bonds, the primary structure (sequence of amino
acids) remains the same after a denaturation process.
Denaturation disrupts the normal alpha-helix and beta
sheets in a protein and uncoils it into a random shape.
Since the native conformation is usually the most water
soluble, disrupting the secondary and tertiary structures
causes changes in solubility and frequently results in the
formation of a solid in the solution. Reagents or conditions
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that can cause denaturation are called denaturing agents;
these include heat, pH changes, alcohol, and heavy metal
salts. In the experiment, the effect of several denaturing
agents on the protein albumin which is a simple globular
protein was observed.
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. Heat is also used in sterilization to
denature and hence destroy the enzymes in bacteria. A
slight precipitation was observed in the experiment since
the egg albumin was broken down by the heat.
Extremes of pH can cause a protein to denature. Addition of
strong acids such as nitric acid can cause extreme pH. The
R-groups in the amino acid chain are often charged and
can form ionic bonds with a group of opposite charge.
Extremes of pH can change the charges on these positive
and negative groups, disrupting ionic bonds interactions.
This can be observed by an appreciable white precipitate
as seen in the experiment.
Some reagents, such as alcohol, are capable of forming
hydrogen bonds with protein molecules which will disrupt
the hydrogen bonding present within the molecule. This
make the egg albumin denatured giving a white
precipitation as observed in the experiment.
CONCLUSIONS AND RECOMMENDATIONS
Therefore, protein plays a very vital role in our body by
having different function in depending upon the direction of
each. Protein, which are basically a polymers of amino
acids give us an insight on how the different R groups in
each amino acids dictate the protein structure into primary,
secondary, tertiary and quaternary structure of proteins.
This experiment also provided a better understanding on
the different qualitative test conducted in which either or
both proteins and amino acids can be tested for its
presence in a given sample. Moreover, the experiment
gives a closer look to what is the effect of the different
denaturing agents to a protein.
REFERENCES
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1. Reachdevices.com. (2019). TLC of aminoacids

and short peptides. [online] Available at:
http://www.reachdevices.com/TLC_aminoacids.ht
ml [Accessed 24 Jun. 2019].

2. https://www.promega.com/~/media/files/resource
s/technical%20references/amino%20acid%20abb
reviations%20and%20molecular%20weights.pdf
[Accessed 24 Jun. 2019]

3. Voet, Donald., Fundamentals of Biochemistry., “Amino

Acids”., John Wiley & Sons, Inc. 2nd Ed, 2006

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