Experiment 2: Proteins
Crystal Gale M. Resurreccion, Crizzia Belle B. Reyes, Markus Gerard O. Reyes,
Catherine Joy B. Rodriguez, Ann S. San Juan
Group 7 2E Medical Technology Biochemistry Laboratory
ABSTRACT
Proteins are essential parts of all cells, and they are known as polymers of amino acids.
Different proteins such as casein, albumin, myoglobin and gluten are isolated through
different methods like isoelectric precipitation and difference in solubility. The different
proteins are then analyzed through qualitative color reactions, thin layer chromatography
and quantitative analysis.
INTRODUCTION curd-like substance was formed. Using
Separation and identification of a funnel and a filter paper, the formed
amino acids can be done using thin curd-like substance was filtered and
layer chromatography. This technique dried then the filtrate was set aside for
uses a TLC plate made up of a thin the isolation and quantitative analysis
layer of silica gel adhered to an of albumin.
aluminum for support. The separation
effects are based from the degree of
solubility and size of each amino acid,
and the interaction of the amino acids
(mobile phase) to the stationary phase
(silica gel).
EXPERIMENTAL
A. Samples used
● Flour
● Skimmed Milk
● Ground Beef Figure 1. Isolated casein
B. Procedure
Isolation of Albumin from
Skimmed Milk
Isolation of Casein from Skimmed
Milk
Using the filtrate from the
process of isolating casein, half of it
A mixture of 20 grams of
was transferred to a small beaker and
powdered nonfat milk and 50 ml of
was heated on a hot plate for five
water in a 100-ml beaker was heated
minutes until it reached 75𝇈c. After
up to 40𝇈c. As it reached the said
heating, a white precipitate was formed
temperature, 10% acetic acid was
and was separated from the liquid.
added in a dropwise manner until a
the supernatant was decanted off after
Isolation of Gluten from Wheat centrifugation.
Flour
A thick dough was prepared by
adding water to one cup of wheat flour.
The dough was then wrapped with
cheesecloth and washed under running
water to remove the starch. Then,
iodine solution was used to test if all
starch content was removed.
Figure 3. Isolated myoglobin
Acid Hydrolysis of Intact Protein
Once all proteins - gluten,
casein, albumin, and myoglobin - have
been isolated, 0.5 g of each had been
Figure 2. Isolated gluten procured in separate test tubes, which
are then labelled. 5 mL of 6 M HCl had
Isolation of Myoglobin from Muscle been subsequently added to them. The
In a small beaker, six grams of test tubes had been covered with
minced beef and six milliliter of 70% cotton and were then submitted to the
(NH₄)₂SO₄ solution was mixed professors for autoclaving. After about
together and was gently mixed for a 5 hours, the mixture will be removed
minute releasing the myoglobin from the autoclave. 10 mL of distilled
content of the meat. The dark-red water was added and then was
extract is then expressed to a new transferred into a 250 mL beaker. The
beaker using a cheesecloth. This mixture was then neutralized with 1 M
extract was centrifuged for five NaOH. The neutralized mixture was
minutes at 13,000 x g. Then, 1.5ml of used as a sample for the
the supernatant was transferred to characterization tests and
another conical tube where 0.33 grams chromatography.
of (NH₄)₂SO₄ crystals was mixed by
stirring it gently and avoiding formation Alkaline Hydrolysis of Intact
of small bubbles until the solid Protein
substances dissolve. The sample was
centrifuged again for five minutes then Another set of the intact proteins
- gluten, casein, albumin, and
myoglobin - are put into separate drops of concentrated NaOH was slowly
labelled test tubes. 10 mL of 4 M NaOH added.
are added to 0.5 g of each isolated Millon’s test: 5 drops of Millon’s
protein. The test tubes were covered reagent was added to the samples.
with a cotton and submitted to the Hopkins-Cole test: 20 drops of
professors for autoclaving. After about Hopkins-Cole reagent was mixed to the
5 hours, 10 mL of distilled water was samples. Then each test tube was
added to the mixture. This was inclined and 20 drops of concentrated
transferred into a 250 mL beaker. It H2SO4 was slowly added.
was neutralized with 1 M HCl. This Sakaguchi test: 10 drops of
neutralized solution had become the 10% NaOH and 10 drops of 0.02%
sample for the characterization tests napthol solution was added to the
and chromatography. samples. After 3 minutes, 3 drops of
2% NaOBr was mixed to the samples.
Qualitative Color Reactions Nitroprusside test: 0.5mL of
3M NaOH was added to the samples.
Five (5) sets of 10 test tubes Then, 0.25mL of 2% nitroprusside
were prepared for the qualitative color solution was added.
reactions. The first set of test tubes Fohl’s test: 5 drops of 30%
contains intact protein solution (0.5g of NaOH and 2 drops of 5% (CH3COO)2Pb
intact casein in 1mL distilled water). was added to the samples then the rest
The second one contains 0.5mL of acid were placed in a boiling water bath.
hydrolyzed sample of casein while the Pauly’s test: Diazo reagent was
third set contains 0.5mL of basic prepared by mixing 3-5 drops 1%
hydrolyzed sample of casein. The sulfanilic acid with 3 drops 5% NaNO2
fourth set of test tubes containing solution. Five drops of the sample and
0.5mL of acid hydrolyzed sample of 3-5 drops 10% Na2CO3 was added to
gluten and lastly, the fifth one contains the diazo reagent.
0.5mL of basic hydrolyzed sample of Test for amides: 1mL of 20%
gluten. NaOH was added to 10 drops of the
Biuret test: 20 drops of 2.5M sample. The samples were then placed
NaOH were added and mixed well to in a boiling water bath and red litmus
the samples. Then, 2-3 drops of 0.1M paper was placed and moistened over
CuSO4 was added. the mouth of the tube.
Ninhydrin test: 6-10 drops of
0.1% ninhydrin solution was placed
into the sample then heated in a boiling Separation and Identification of
water bath. Amino Acids by Thin Layer
Xanthoproteic test: 10 drops Chromatography
of concentrated HNO3 was slowly
added to the samples. After which, 10 On a 12x15 cm TLC plate, the
origin was drawn, using a pencil, with
a margin of 1.5 cm from the bottom of
the longer edge of the plate. At equal
Standard BSA Distilled
distances, the standards of tryptophan, (uL) Standard Water
arginine, proline, cysteine, serine, Stock
aspartic acid, tyrosine, histidine, Solution
glycine, and alanine were applied 5 (uL)
times and the acid and basic
Blank 0 1000
hydrolysed casein and albumin were
spotted 10 times using a capillary tube Standard 10 990
and were dried in between application. tube 1
The plate was then placed inside the
Standard 20 980
pre-equilibrated chamber wherein the
tube 2
solvent was leveled below the line of
origin. The chamber was covered and Standard 50 950
the solvent was allowed to rise until it tube 3
reached approximately 0.5cm of the
Standard 70 930
other end of the TLC plate. The tube 4
chromatogram was then dried and
sprayed with 1% ninhydrin reagent. Standard 100 900
tube 5
Quantitative Protein Analysis
95 uL of distilled water were placed in
MicroBradford Total Protein each plate of the 96-microwell plate
Assay was used to measure the which was marked based on the
concentration of the total protein in the instructions in the laboratory manual. 5
samples. There are standard protein uL of the standard protein solution
concentrations prepared in a 1.5mL- were added using a micropipette. The
microcentrifuge (Eppendorf) tubes. samples were diluted 1:1000 in the
These concentrations are prepared wells. The 1.0 mL Bradford reagent was
using the basis given. diluted with 4.0 mL of water and the
100 uL of the diluted Bradford reagent
Table 1. Standards and Blank for was dispensed in the wells which are
Quantitative Protein Analysis marked and placed with the blank,
standards and the sample proteins.
RESULTS AND DISCUSSION Table 1. Qualitative Color Reactions
Hydrolysis of Intact Proteins
Acid Hydrolysis
This was catalyzed by a strong
acid, 6 M HCl. The reaction mechanism
took place via Sn acyl mechanism,
hydrolysis of amide. Its effects include
complete hydrolysis producing L-amino
acids, and little or no racemization. Not
all amino acids were recovered
quantitatively. Trp was destroyed and
there were some loss of Ser and Thr.
The color after hydrolysis was brown-
black or black amorphous solid called
melanin or humin. This was due to the
condensation of Trp with an unknown
aldehyde which may had been a Separation and Identification of Amino
carbohydrate. Acids by Thin Layer Chromatography
Alkaline Hydrolysis
This was catalyzed by a strong
base, 6 M NaOH. Its effects include
complete hydrolysis producing L-amino
acids, and the occurrence of
racemization, which entailed its optical
inactivity. Not all amino acids were
recovered quantitatively: Cys, cystine,
Ser, and Thr. Trp was stable.
Figure. Thin Layer Chromatography
(Standard samples of Cysteine, Proline,
Tryptophan, Arginine, Glycine, Alanine;
Acid and Basic Hydrolyzed Casein and Quantitative Protein Analysis
Albumin)
Due to time constraints, the group was
not able to do this part of the
experiment.
REFERENCES
Crisostomo A. C., Daya, M. L., et al
(2010) Laboratory Manual in General
Biochemistry Quezon City: C & E
Publishing, Inc.
Figure. Thin Layer Chromatography
(Standard samples of Aspartic acid,
Serine, Tyrosine, Histidine; Acid and
Basic Hydrolyzed Casein and Albumin)
Amino Acids contain an acidic -COOH
group and a basic -NH2 group. The
most prevalent ionic form of an amino
acid in a solution, therefore, depends
on the pH of the solution. The rate
and distance of migration of an amino
acid is dependent on the pH of the
mobile phase and the presence of an
acidic and basic R- group further
complicates the pH dependence.
