University of Santo Tomas

Faculty of Pharmacy – Department of Pharmacy

Espana, Manila

Isolation and Characterization of Proteins

Revita, J.A., Singh, S.P., Soriano, J.C., Timtiman, D.R., Valeros IV, R., & Yutuc, P.N.
2H - Pharmacy

Abstract​: Proteins are biologically important molecules made up of amino acids

bonded by peptide bonds. Milk, wheat flour, and meat are examples of sources of
different types of proteins. The experiment aims to isolate selected proteins from
skimmed milk, wheat flour, and minced beef from their commercially available sources
using different isolation techniques for each; perform qualitative tests on the amino
acids in intact and hydrolyzed proteins; execute acid, alkaline, and enzymatic hydrolysis
on the isolated proteins; determine the amino acid components of proteins using
thin-layer chromatography (TLC), and to analyze chemical groups responsible for color
reactions and explain the principle involved in each test.

I. Introduction development of blood and tissue,

Proteins are biochemical
molecules consisting of polypeptides
linked by peptide bonds between the
amino and carboxyl groups of amino
acid residues, which are organic
compounds made of carbon, hydrogen,
nitrogen, oxygen or sulfur. They are the
main structural constituent of living
beings and are responsible for most of
the complex functions involved in
biological processes (U.S. National
Library of Medicine, 2019).
One example of a substance
containing different types of proteins in
milk. Milk is composed of three kinds of
“complete proteins” essential for the
namely, (1) casein, (2) lactalbumins,
and (3) lactoglobulins. Its principal
protein component is the protein group
casein, which is the common term for a
family of associated phosphoproteins
(Augustin & Margetts , 2003). It
involves a certain amino acid
arrangement vital for one’s growth and
development making milk as one of the
most essential components of a healthy
human diet. One of the significant
properties of casein is that it precipitates
or coagulates at pH 4.6 which is its
isoelectric point. Hence, a solution less
than pH 4.6 renders casein insoluble in
that solution (Fox, 2003).

Albumin, specifically lactalbumin,
is said to be the second most abundant
protein in milk, casein being the first.
This protein is categorized as a globular
protein and is generally soluble in water
as well as in diluted salt solutions. They
are easily denatured and coagulated by
heat, and weigh about 40,000 daltons.
After the casein is separated from the
milk and the solution has a lower ph
(acidic), the lactalbumin can be obtained
through the continuous heating of
mixture until precipitation has occurred.
Gluten is a protein compound of
glutenin and gliadin present in foods
processed from wheat and related grain
species (Flambeau , Redl, & Respondek,
2017). It is characterized by a
yellowish-white, tough, elastic, and
sticky protein that provides elasticity to
the dough which makes it useful in
bread-making. This is because it can
confine the carbon dioxide (CO​2​) from
the reaction caused by the mixture of
flour and yeast that causes it to rise and
keep its shape thus, giving the final
product a distinctive chewy texture (Hill,
2012). Aside from that, gluten can also
be found in cosmetics products and
dermatological preparations.
Myoglobin is considered as one of
the members of the globin superfamily
protein, it is located primarily within
vertebrates’ striated muscles, and low
concentrations in smooth muscle,
endothelial, and even tumor cells​. It is a
cytoplasmic hemoprotein, expressed
solely in cardiac myocytes and oxidative
skeletal muscle fibers, that reversibly
binds oxygen by its heme residue, a
porphyrin ring: iron ion complex. Its
primary function, as a result, is to
supply oxygen to myocytes. Myoglobin
also functions in the hemostasis of nitric
oxide, plays a role in the detoxification
of reactive oxygen species, and is the
reason for the red color of the muscle of
most vertebrates. ​Myoglobin has the
capability to release oxygen supply
when needed, specifically when the
body experiences hypoxia or anoxia.
Myoglobin is also thought to buffer
intracellular oxygen concentration when
muscle activity increases and to
facilitate intracellular oxygen diffusion
by providing a parallel path that
augments simple diffusion of dissolved
oxygen.
The objectives of this experiment
are to isolate casein and albumin from
skimmed milk by isoelectric
precipitation, gluten from wheat flour by
difference solubility, and myoglobin
from beef by salt-induced precipitation;
perform qualitative tests on the amino
acids in intact and hydrolyzed proteins;
execute acid, alkaline, and enzymatic
hydrolysis on the isolated proteins;
determine the amino acid components
of proteins using thin-layer
chromatography (TLC), and to analyze
chemical groups responsible for color
reactions and explain the principle
involved in each test.

II. Methodology
A. Isolation of Proteins
1. Isolation of
​ Casein from
skimmed milk
In a 100-mL beaker, 20.0 grams of
powdered Milk Magic nonfat dried milk
was placed and was mixed with 50.0 mL
of water. The mixture was heated up to
40˚C then 10% of acetic acid was
added dropwise and the solution was
stirred every 5 drops. Continuous
addition of acetic acid was done until
the pH reaches 4.6 and when the casein
has congealed. The casein was filtered,
and the filtrate was set aside for the
isolation and quantitative analysis of
albumin. The residue on the other hand
was dried and the weight % was
calculated.
2. I​ solation of Albumin from
skimmed milk
In a small beaker, half of the filtrate
from the isolation of casein was placed
and the remaining half was set aside for
the quantitative protein analysis. The
filtrate was heated up to 75 ˚C for 5
minutes and after that, the liquid was
decanted from the precipitated albumin
while the filtrate was discarded.
3. Isolation of gluten from wheat
flour
In a beaker, 128.0 grams of wheat flour
was weighed and was transferred to a
big evaporating dish and enough water
was added to make a thick dough. The
dough was then wrapped with
cheesecloth and was placed under
running water until all the starch was
removed. The dough washings were
tested by adding enough amount of
iodine solution until a clear yellow
solution is obtained. The insoluble
material which is the gluten was
collected for hydrolysis and qualitative
protein analysis
4. I​ solation of myoglobin from
muscle
In a small beaker, 6.0 grams of minced
beef was weighed, and 6 mL of 70%
ammonium sulfate solution was added.
The mixture was gently stirred for about
a minute to release the myoglobin. The
dark-red extract was expressed in a new
beaker using cheesecloth then the
extract was centrifuged at 4,000 rpm for
5 minutes. In another empty centrifuge
tube, 1.5 mL of the supernatant was
transferred, and 3 mL of saturated
ammonium sulfate solution was added.
The sample was then centrifuged again
for about 5 minutes and after that, the
supernatant was decanted and the
appearance of the isolated myoglobin
residue was described.
B.​ ​Hydrolysis of intact protein
1. Acid hydrolysis of intact
proteins
In a hard glass test tube, 0.5 g of the
isolated protein was placed and 5 mL of
6 M HCl was added. The tube was
labeled and was covered with cotton
then was submitted to the instructor for
autoclaving at 15 psi for about 5 hours.
After autoclaving, the appearance of the
mixture was noted. Ten mL of distilled
water was then added to the mixture
and was transferred into a 250-mL
beaker. To neutralize the mixture, 1 M
NaOH was added and this mixture was
used for the characterization tests and
chromatography.
2. ​ lkaline hydrolysis of intact
A
protein
In a hard glass test tube, 0.5 g of
isolated protein was placed and 10 mL
of 4 M NaOH was added. The tube was
labeled and was covered with cotton
then was submitted to the instructor for
autoclaving at 15 psi for about 5 hours.
After autoclaving, the appearance of the
mixture was noted. Ten mL of distilled
water was then added to the mixture
and was transferred into a 250-mL
beaker. To neutralize the mixture, 1 M
HCl was added and this mixture was
used for the characterization tests and
chromatography.
3. ​ nzymatic hydrolysis of intact
E
protein
In a 250-mL beaker, 1 g of protein was
placed and 100 mL of distilled water
was added. Ten mL of protein mixture
and ten mL of saturated proteases
solution was mixed. The mixture was
added 10 mL of 0.1 M phosphate buffer
until the pH is 7.5 and after, it was
incubated in a water bath with a
temperature ranging from 35 ̊C-40 ̊C for
60 minutes. The mixture was then
allowed to cool and it was set aside for
qualitative color reactions and for the
separation and identification of amino
acids by thin-layer chromatography.
C.​ ​Qualitative color reactions
Two sets of 10 test tubes were
prepared for the qualitative color
reactions. The first set contains the
intact protein solution which is, 0.5g of
intact casein in 1 mL distilled water and
the second set of test tube contains
0.5mL of the acid hydrolyzed sample.
The following tests were performed on
each sample:
Biuret test​: twenty drops of 2.5 M
NaOH was added to the samples and
was mixed well. After, 2-3 drops of 0.1
M CuSO​4 solution
​ was added.
Ninhydrin test​: In the diluted
samples, 6-10 drops of 0.1% ninhydrin
solution was placed. Then the test was
heated in a boiling water bath
Xanthoproteic test​: ten drops of
conc. HNO​3 was added slowly to the
diluted samples and was mixed. After,
10 drops of conc. NaOH was also added
slowly and was mixed.
 Millon’s test​: 5 drops of Millon’s
reagent was added to the samples.
Hopkin’s-Cole test​: twenty drops
of Hopkin’s-Cole reagent was added
slowly to the samples then was mixed
well. Then each test tube was inclined
and 20 drops of concentrated H​2​SO​4 was
slowly added.
Sakaguchi test​: ten drops of 10%
NaOH and 10 drops of the 0.02%
naphthol solution was added to the
samples then was mixed well and stand
for 3 minutes.
Nitroprusside test​: In the 0.5 mL
of sample, 0.5 mL of 3 M NaOH was
added then after which, 0.25 mL of 2%
nitroprusside solution was also added.
Fohl’s test​: five drops of 30%
NaOH and 2 drops of 5% Pb (CH​3​COO)​2
were added to the samples and was
placed in a boiling water bath.
Test for amides​: one mL of 20%
NaOH was added to the 10 drops of
sample and was placed in a boiling
water bath. A red litmus paper was
placed and moistened over the mouth of
the tube to test the evolution of gas.
Pauly’s test​: diazo reagent was
prepared by mixing 3-5 drops of 1%
sulfanilic acid with 3 drops of 5% NaNO​2
solution. Then five drops of the sample
and 3-5 drops of 10% Na​2​CO​3 was
added to the diazo reagent.
D.​ ​Thin-Layer Chromatography
Using a 12x15 cm TLC plate, a line
was drawn across the plate with 1.5-cm
margin from the bottom of the longer
edge of the plate and was marked with
13 equidistant points on the line for the
spotting of the amino acid standards
and 3 hydrolysate samples. The
standards were applied 5 times while
the samples were applied 10 times using
the capillary tubes and was dried in
between applications. The plate was
placed inside the pre-equilibrated
chamber and was then covered to allow
the solvent to ascend undistributed. The
plate was removed when the solvent
front was approximately 0.5 cm from
the top edge of the plate and was
immediately marked with a pencil line.
The chromatogram was air-dried and
after which, it was lightly sprayed with
1% ninhydrin reagent. After, the
chromatogram was placed on a hot
plate until all the color of the samples
appeared.
E.​ ​Quantitative protein analysis
Thirteen centrifuge tubes were
prepared, 1 for the stock solution which
is bovine serum albumin (BSA), 6 tubes
for the biuret total protein assay and the
other 6 for Bradford total protein assay.
For the stock solution, 0.01g/mL of BSA
was prepared. For the biuret test, test
tube #1 which has a concentration of
1000mcg/mL contains 600mcg/mL of
the stock solution and was diluted with
400mcg/mL distilled water. The other 5
test tubes were contained with Table 1: Biuret assay well
500mcg/mL of distilled water each. assignment
From test tube #1, 500mcg/mL was
pipetted out and was added to the next
test tube. From test tube #2, BSA PROTEIN
500mcg/mL was pipetted out and was SAMPLE
added to test tube #3. This successive
process was repeated until test tube #5. A1-A3 A4-A6 (G1 Sx)
While for the bradford test, 100mcg/mL (1000mch/mL)
of the stock solution was diluted with
900mcg/mL of distilled water and was
placed in the first test tube for Bradford. B1-B3 B4-B6 (G2 Sx)
500mcg/mL of distilled water was added (500mcg/mL)
to the other 5 test tubes. From the test
tube #1, 500mcg/mL was pipetted and C1-C4 C4-C6 (G3 Sx)
was mixed with test tube #2. Like in the (250mcg/mL)
biuret test, this process was repeated
until test tube #5. Then, 500mcg/mL of
D1-D3 D4-D6 (G4 Sx)
biuret and Bradford reagent were added
(125mcg/mL)
to their respective test tubes. After
which, 200mcg/mL of each test tube
was placed in a microwell plate E1-E3 E4-E6 (G5 Sx)
following the well assignment which is (62.5mcg/mL)
shown in table 1 and table 2. The
microplate was then brought to the F1-F3 F4-F6 (G6 Sx)
multiskan go microplate (31.25mcg/mL)
spectrophotometer in order to measure
the absorbance of each sample with a
wavelength of 540nm for Biuret and BLANK (D.W.) G4-G6 (G7 Sx)
595nm for Bradford. The result was
read and was graphed using excel.
Table 2: Bradford assay well changes in pH. It has an isoelectric
assignment point of approximately 4.6. It is a
slow-digesting dairy protein which
means it releases amino acids slowly. It
BSA PROTEIN can also help cells synthesize protein,
even during times when the body might
SAMPLE normally be breaking down its own
muscle mass to feed itself (eg.
A7-A9 A10-A12 (G1 starvation). Because of this, it is called
an “anti-catabolic” and helps reduce
(1000mch/mL) Sx)
muscle breakdown. Caseins are defined
as proteins that become coagulated and
B7-B9 B10-B12 (G2 precipitated from skim milk when the pH
of the milk is adjusted to pH 4.6 at
(500mcg/mL) Sx)
20​o​C, resulting in an insoluble,
noncrystalline white curd.
C7-C9 C10-C12 (G3
(250mcg/mL) Sx) Albumin is a water-soluble,
globular protein produced by the liver
and is the body’s predominant
D7-D9 D10-D12 (G4 serum-binding protein, and has several
(125mcg/mL) Sx) important functions: (1) keeps fluid
from leaking out of blood vessels, (2)
nourishes tissues, and (3) transports
E7-E9 E10-E12 (G5 hormones, vitamins, drugs, and
(62.5mcg/mL) Sx) substances like calcium throughout the
body. It takes up around 60% of the
total protein in the blood. It is possible
F7-F9 F10-F12 (G6 to denature and coagulate albumin by
(31.25mcg/mL) Sx) heat. The heating of milk to 75°C for 5
minutes results in the denaturation and
precipitation of albumin
BLANK (D.W.) G10-G12 (G7
Sx) Wheat gluten is a typical
water-insoluble protein, consisting of
more than 60 different polymorphic
polypeptides with a molar mass of
30,000-100,000 g/mol. Gluten is a
III. Results and Discussion mixture of two proteins: (1) glutenin,
and (2) gliadin, it is also a strong and
A. Isolation of Proteins flexible molecule. It is a commodity food
ingredient and its applications are
Casein is a major protein found in predominantly in baked goods and
milk which is very susceptible to processed meat products. It is a natural
protein derived from wheat or wheat
flour. Once dried, it has a creamy color,
neutral taste and is free-flowing. Dried
gluten is also able to recover its
viscoelastic structure when rehydrated.
Its typical composition would be:
Protein 75-80%
Carbohydrates 15-17%
Fat 5-8%
Ash 0.6-1.2%
In bread-making, gluten traps the
carbon dioxide produced by the reaction
between the yeast and flour which gives
flour its chewy characteristic.
It is possible to isolate gluten
with the use of the principle of
difference in solubility due to the fact
that starch is soluble in water but gluten
is insoluble in water. In order to confirm
that the starch is completely removed,
iodine solution is used. It is a
yellowish-white solid, tough, elastic, and
sticky.
Myoglobin is a monomer made
up of 153 amino acid residues and is a
member of a family of proteins
collectively called as globins, along with
hemoglobin. It is a globular protein that
serves as an oxygen storage protein and
transport in vertebrates, primarily in the
heart and the skeletal muscles.
Myoglobin is an iron and oxygen binding
protein found in large concentrations in
muscles, and this characteristic is
responsible for the color of mammalian
red muscle tissue.
In meat, with the increase in
ionic strength by the addition of
(NH​4​)​2​SO​4 results in a decrease in the
solubility of proteins, resulting in the
formation of a white precipitate which is
the myoglobin isolated from the muscle,
this is due to water preferring to
dissolve added salt ions instead of the
proteins in the muscle.
B. Hydrolysis of Intact Proteins
In hydrolysis, the protein is
subjected to extreme conditions usually
at high temperatures by prolonged
boiling in a strong acid or strong base
or using an enzyme to stimulate the
naturally occurring hydrolytic process.
This will cause denaturation of the
protein meaning that the protein’s
conformation is altered by the breaking
of the peptide bonds. This results to a
solution containing amino acid
fragments which is then called the
“hydrolysate.” Denaturation alters
protein function, demonstrating a
relationship between structure and
function. Hydrolysis of protein and
analysis of products are done to obtain
information about their compositions.
a. Acid Hydrolysis
Acid hydrolysis implies a chemical
mechanism of hydrolysis catalyzed by a
Bronsted or Arrhenius acid. This
promotes little to no racemization of
amino acids. However, there are
disadvantages with it like the
destruction of tryptophan and its
conversion to a black precipitate,
partial destruction of serine, threonine,
and cysteine, and hydrolysis of
asparagine and glutamine to aspartic
acid and glutamic acid, respectively.
 b. Alkaline Hydrolysis
Alkaline hydrolysis covers types
of reactions under nucleophilic
substitution that use a hydroxide ion as
the attacking nucleophile. In this,
tryptophan is stable even after being
treated with concentrated sodium
hydroxide (NaOH) solution and
autoclaved for five hours. However, it
causes racemization of most amino
acids such as the decomposition of
arginine to ornithine and urea, partial
destruction of cysteine, serine, and
threonine, and the hydrolysis of
asparagine and glutamine to aspartic
acid and glutamic acid, respectively.
A possible source of error for the
results obtained in the experiment is an
error in pipetting and the mixture not
being properly mixed. The reagents
used was drawn only from the surface
of the solution instead of the middle
part.
c. Enzymatic Hydrolysis
If protein samples are to be
subjected into identification, enzymatic
hydrolysis is the most ideal of all the
hydrolyzation process. It is the addition
of specific enzymes called “proteolytic
enzymes” or proteases, which is a group
of enzymes that hydrolyse specific
peptide bonds in proteins. A more
accurate results would be obtained
because these enzymes do not damage
or cause unnecessary reactions to the
amino acids that would be subjected in
the experiment. However, this
hydrolyzation process requires varying
temperature and pH conditions because
enzymes function better in these
conditions.
Most of the results obtained for
casein enzymatic hydrolysate yielded
positive results as seen in Table _,
except for Ninhydrin, Millon’s, and
Hopkin-Cole tests. The results for these
tests are considered false negative. The
test for Ninhydrin should yield a positive
result for all proteins since they contain
free amino groups. Casein also contains
varying percentages of tyrosine (5.7%)
and tryptophan (1.1%), which are the
target amino acids of the Millon’s and
Hopkin-Cole tests, respectively
(Rasmussen, Greenwood, Kalman, &
Antonio, 2008). A possible reason for
this is the fact that the intensity of
reaction in qualitative color is directly
proportional to the concentration of the
individual constituents of the sample
and the amount of samples and
reagents used. An increase in the
amount of samples and reagents might
give the desired results of the
experiment.
C. Qualitative Color Reactions
 Colorless
Nin No color solution No color
hydrin change with light change
purple ppt

Xantho No color
Yellow Yellow
proteic change

Colorless
Solution
solution No color
Millon’s with white
with white change
precipitate
ppt

Light
purple; No color
Hopkins- No color
turbid change at
Figure 1​. Visualization of Intact Casein Cole change
upper interface
portion

Salmon
pink
Saka Salmon
Light red solution
guchi Pink
with
bubbles
Figure 1​. Visualization of Intact
Myoglobin Yellow
interface;
Nitro Yellow-ora
colorless Yellow
prusside nge
lower
portion

Light
brown
Fohl’s Brown Black
turbid
solution

Red-blue Red-blue
Figure 1.​ Visualization of Intact Gluten Test for
Negative litmus litmus
Amide
paper paper
Table 1. ​Results of Qualitative Color
Red
Reactions of Intact protein of Casein, Light Red
Pauly’s orange
Myoglobin, and Gluten orange
solution
orange

Intact Intact Intact

Color Protein Protein Protein
Reactions (Casein) (Myoglobin) (Gluten)
Initial Color: Initial Color: Initial Color:
Colorless Light Pink Colorless

Purple
Biuret Blue Violet Purple
Solution
Figure 1.​ Visualization of Gluten Acidic Saka Salmon Colorless Light Red
Hydrolysate guchi Pink Solution

Nitro Yellow Clear Yellow

prusside Yellow
Solution

Fohl’s Brown | No Colorless Brown |

Sediments Solution No
Formed Sediments

Figure 1.​ Visualization of Myoglobin Test for No Color Colorless Yellow |

Basic Hydrolysate Amide Change | Solution Red to Blue
Red to Litmus
Blue Paper
Litmus
Paper

Pauly’s Red Clear Red

Orange
Solution

Biuret test, or also known as

Figure 1.​ Visualization of Casein Piotrowski’s test, uses the reagent 2.5 M
Enzymatic Hydrolysate NaOH and 0.1 M CuSO​4​. It is used to
detect the presence of peptide bonds in
Table 1​. Results of the Qualitative Color a sample; it relies on a color change in
Reactions Acid Hydrolysate of Gluten, order to confirm the presence of
Basic Hydrolysate of Myoglobin, and proteins, a positive result shows a
Enzymatic Hydrolysate of Casein pink-violet to blue color. The proteins
detected must have at least three amino
Color Gluten Acid Myoglobin Casein acids (two peptide bonds). The Biuret
Reaction Basic Enzymatic
test is based on the ability of Copper
Biuret Blue Colorless Light (II) ions to form a violet-colored chelate
Solution Purple complex with peptide bonds in alkaline
conditions. All test samples used in the
Nin Blue Violet Colorless No Color experiment showed a positive result
hydrin Solution Change with the exception of basic hydrolysate.
Possible sources of error would come
Xantho Orange Colorless Yellow
proteic Solution
from improper pipetting, unthorough
mixing of solution and improper rinsing
Millon’s No Color Colorless No Color of colorimeter tubes.
Change Solution Change
The Ninhydrin test is the test for
Hopkins- No Color Colorless No Color
amino acids and proteins with a
Cole Change at Solution Change at
Interface Interface
⍺-amino (-NH) group with the use of
1,2,3-Indanetrione Monohydrate or
Triketohydrindene Hydrate and Ethanol
as reagents. This test is due to a
reaction between an amino group of
free amino acid and ninhydrin.
Ninhydrin is a strong oxidizing agent
and with its presence it is able to cause
the amino acid to undergo oxidative
decarboxylation and deamination which
liberates the ammonia, CO​2​, aldehyde, a
reduced form of ninhydrin, hydrindantin.
Only the acidic hydrolysate of gluten
showed a positive result of blue to
blue-violet solution.
The Xanthoproteic Test is used to
detect amino acids containing an
aromatic nucleus such as tyrosine,
tryptophan and phenylalanine.
Phenylalanine gives a weakly positive or
even a negative reaction even though
this amino acid does contain an
aromatic ring, this is due to the fact that
it is challenging to nitrate under normal
conditions. With the addition of
concentrated nitric acid (HNO​3​) and
sodium hydroxide (NaOH), a yellow
solution or precipitate is formed with
concentrated nitric acid (HNO​3​) and
orange with the excess sodium
hydroxide (NaOH). The intact protein of
casein and gluten and the enzymatic
hydrolysate of casein yielded a positive
result of yellow solution while the acidic
hydrolysate of gluten also produced a
positive result of an orange solution.
The Millon’s test is used to detect
an amino acid containing phenol group
(hydroxyl attached to a benzene ring)
such as Tyrosine. Tyrosine when
reacted with acidified mercuric sulfate
solution gives yellow precipitate of
mercury-amino acid complex. With the
addition of sodium nitrate solution and
heating, the yellow complex of
mercury-amino acid complex converts
to mercury phenolate which is red in
color. None of the test samples used in
the experiment yielded to a positive
result.
The Hopkins-Cole Test is used to
detect the presence of indole group
containing amino acid, tryptophan, in
proteins with the use of glyoxylic acid
(Magnesium powder, Oxalic acid and
Acetic acid) and sulfuric acid as its
reagents. The indole group of
tryptophan reacts with glyoxylic acid in
the presence of concentrated sulfuric
acid, resulting in a pink to violet
interface. None of the test samples
used in the experiment yielded to a
positive result.
The Sakaguchi test is for the
detection of an amino acid guanidinium
group such as, arginine using
⍺-Napthol, Sodium hypobromite
(NaOBr), sodium hydroxide (NaOH),
and Urea, to stabilize the color and
destroy the excess -OBr ions. There is a
complexation- base-catalyzed
condensation of ⍺-Napthol with the
guanido group of arginine. The arginine
reacts with ⍺-Napthol and an oxidizing
agent such as bromine water or sodium
hypochlorite/sodium hypobromite to
give a red to red-orange colored
product. The intact protein of casein
and enzymatic hydrolysate of casein
yielded a positive result which showed
as light red solution, while the intact
protein of myoglobin and gluten and
the acidic hydrolysate of gluten yielded
a positive result which showed a
salmon pink solution.
The Fohl’s test is performed to
test for the detection of amino acids
containing sulfur, such as cysteine and
methionine, using lead acetate
(Pb(CH​3​COO)​2​) and sodium hydroxide
(NaOH), with the assistance of heat
(boiling). Once performed a positive
result shows a brown to black
precipitate which is the lead sulfide,
this is due to the degradation and
substitution. All the test samples used
with the exception of the basic
hydrolysate and intact gluten produced
a positive result of a light brown to
brown precipitate with no sediments.
The test sample intact glute produced a
black solution. ​The reason for the basic
hydrolysate not giving a positive result
or erroneous results in several tests
would be an error in pipetting (only
drawn liquid from the surface of the
solution instead of the middle part) and
not mixing the solution thoroughly.
Another source of error would be that
the students did not make use the
mixture of hydrolysed and intact
protein but instead only used the
former.
The nitroprusside test is specific
for cysteine, the only amino acid
containing a sulfhydryl group (-SH)
which reacts with nitroprusside in the
presence of excess ammonia (NH​3​)
using sodium nitroprusside
(Na​2​Fe(CN)​5​NO in diluted ammonia
(NH​3​) and sodium hydroxide (NaOH)
resulting to a complexation reaction
that shows a red coloration as a
positive result. None of the test
samples used in the experiment yielded
a positive result, most of the test
samples showed a yellow solution.
The test for amide is to detect
primary, secondary and tertiary amides,
and nitriles using sodium hydroxide
(NaOH) as a reagent resulting to a
basic hydrolysis. A red litmus paper is
used and a positive result shows a
change in color from red to blue litmus
paper. All of the test samples used in
the experiment with the exception of
the intact casein produced a positive
result.
The Pauly’s Test is used to detect
an aromatic acid Tyrosine or Histidine
using ehrlich’s diazo reagent consisting
of 1% sulfanilic acid with 5% sodium
nitrite (NaNO​2​) and 10% sodium
carbonate (Na​2​CO​3​) resulting in a
formation of an azo dye, the color being
red to orange for histidine while it is
light orange for tyrosine. The sulfanilic
acid will be diazotized with the addition
of sodium nitrite and sodium carbonate.
The diazotized sulfanilic acid will then
form a diazonium component which will
react with the imidazole ring of histidine
and the phenol group of tyrosine.
Diazotization is the reaction between an
aromatic amine, sulfanilic acid, with
sodium nitrite and sodium carbonate to
form a diazonium component. A positive
result for the presence of histidine was
produced from intact myoglobin, intact
gluten, acidic hydrolysate of gluten and
enzymatic hydrolysate of casein. A light
orange solution was produced from
intact casein and a clear orange solution
from the basic hydrolysate.
 D. Total Protein Assay
E. Thin Layer Chromatography
components’ affinity to the mobile and
stationary phase (Bheem, 2019). The
affinity of the components, which
include their solubility and adsorption to
the phases, allows them to rise up in
the plate.

Polar components tend to travel

slowly in the plate compared to
non-polar components. The main reason
for this is the base component of the
Figure 1​. Visualization of the Thin stationary and mobile phase used. The
Layer Chromatography stationary phase is made up of silicon
dioxide, a very polar compound in
Table 1​. Thin Layer Chromatography nature, which makes polar components
Amino Acids and Proteins’ Rf Values adhere to the plate tightly and attain a
Amino Standard Rf Values of the Spot lower Rf value. On the other hand, the
Acid mobile phase used – a mixture of
Acid Base Enzyme
butanol, acetic acid, and water – is very
non-polar in nature, thus making
Trp. 0.50 0.475 0.325 0.20
non-polar components move with it as it
Arg. 0.20 rose up in the plate and attain a higher
Rf value.
Pro. 0.25

Cys. 0.25 The principle of TLC explained

the results acquired. Tryptophan, which
Ser. 0.25
obtained the highest Rf value is a
Asp. 0.25 non-polar amino acid. Other non-polar
amino acids used in the experiment are
Tyr. 0.40
proline, glycine, and alanine. On the
His. 0.175 other hand, histidine, which obtained
the lowest Rf value, is a polar basic
Gly. 0.225
amino acid. Other polar amino acids
Ala. 0.25 used in the experiment are arginine,
cysteine, serine, aspartic acid, and
Thin Layer Chromatography tyrosine. In the case of hydrolyzed
(TLC) is an analytical technique that proteins, the acid hydrolysate, which is
mainly revolves around the principle of gluten, is more non-polar than
separation under the influence of the myoglobin (base hydrolysate) and
casein (enzyme hydrolysate). This result
is possible since gluten has prolamin,
which have a high proline content, and
proline is one of the non-polar amino
acids.
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