ISOLATION, CHARACTERIZATION

AND QUANTITATIVE ANALYSIS

OF PROTEINS

OBJECTIVE
S
At the end of this chapter, the student must be able to:
• Isolate proteins using appropriate techniques.
• Perform qualitative and quantitative analysis of isolated proteins.

BACKGROUND OF THE EXPERIMENT

Proteins are naturally occurring large biomolecules that serve various functions in an organism.
They may have catalytic, structural, regulatory, or transport functions depending on their composition.
Plants can synthesize proteins from inorganic substances present in the atmosphere and in the soil.
Animals, on the other hand, can only synthesize proteins from amino acids that come from their diet.
Thus, they must obtain them from plant- or animal-based food. Other than being sources of essential
nutrients, there are also protein-based remedies known as therapeutic proteins (Dimitrov, 2012). These
proteins are represented mostly by antitoxins and antibodies. Insulin, a glucose-blood level regulatory
hormone, was the first non-antibody therapeutic protein that was sequenced. It was first administered
to patients with diabetes mellitus in 1922. It was further improved in the 1970s as the recombinant
protein therapeutic Humulin.

Proteins are polymers composed of an unbranched series of amino acids that are joined
together by peptide bonds. There are commonly 20 naturally occurring amino acids found in proteins.
These amino acids give them their distinct structures and shapes and contribute to their physico-
chemical properties. The isolation and characterization techniques used for proteins are largely based
on their physico-chemical properties.

Amino acids contain an amino (-NH2) group, an alpha-carbon

(-CH), and a carboxyl group (-COOH). The alpha-carbon
bears the side chain (R) that gives the protein its distinct
shape, function, and physico-chemical properties.

Proteins may be classified according to function, composition, and shape (Table 1). They are
generally odorless, tasteless, and colorless. They are amphoteric in nature owing to the amino and

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
carboxyl groups present in the amino acids that compose them. The amino acid composition of a
protein give rise to four levels of protein organization:

§ The primary structure shows the sequence of amino acids making up the protein.
§ The secondary structure represented by the alpha-helix and beta-pleats, arise from the
H-bonding interactions of the R groups within the peptide chain.
§ The tertiary structure is the overall three-dimensional shape of a protein and results
from the interaction of the R groups of amino acids that are far apart in the peptide
chain. This structure is maintained by four stabilizing interactions namely: disulfide bond,
H-bond, hydrophobic and electrostatic interactions.
§ The quarternary structure is the highest level of protein structure that can only be found
in multimeric proteins (a protein with 2 or more polypeptide chain).

A protein’s shape or native conformation defines its basic function. When proteins are exposed
to unfavorable conditions such as extreme pH or heat, or chemicals that disrupt their stabilizing
interactions they tend to lose their native conformation. Mechanical stress such as extreme agitation
can also cause destabilization in the protein structure. This is called protein denaturation. Denaturation
can take the form of coagulation just like when eggs are boiled where the albumin is transformed from
clear to opaque white and the protein is no longer fluid. This shows that denatured proteins are no
longer functional. Protein denaturation may be reversible or irreversible. Some proteins regain their
native conformation when favorable conditions are restored, others cannot and are permanently
denatured. Cooking is one form of irreversible denaturation of proteins in plants and animal tissues.

When proteins are denatured, its primary structure, i.e. the amino acid sequence, is not lost.
This means the peptide bonds are still intact and the protein backbone is not yet disrupted. When
proteins are heated in a solution of a strong acid or a strong base, these peptide bonds break, and
free amino acids are produced, this is called protein hydrolysis. Protein hydrolysis may be complete
or partial. Complete hydrolysis is achieved by heating the protein in strong acids and bases producing
free amino acids, while partial hydrolysis is achieved using proteolytic enzymes producing peptide
fragments. The freed amino acids from protein hydrolysis are the basis for the qualitative assessment
of a protein.

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 METHODOLOGY

A. ISOLATION OF PROTEINS

Reagents and Materials

50 mM Tris-Hcl Buffer, pH 7.5 Cheesecloth Ice bath

Ammonium sulfate crystals Centrifuge tubes
Distilled water Centrifuge
Ammonium sulfate crystals Osterizer

Protein Source: Bring 50 grams of each of the following (minced or osterized)

1. Beef muscles 6. Shrimp meat
2. Chicken meat (breast) 7. Tuna meat
3. Heart of pig/chicken 8. Egg white (50 mL and take note the
4. Liver of pig/chicken number of eggs)
5. Salmon

Procedure
1. To 25 grams or 25mL of protein source add 25 mL of Tris-HCl buffer.
2. Stir the mixture for 1 minute.
3. Express the extract in a beaker using a cheesecloth (except for egg white). *Centrifuge the
solution if not filtered well.
4. Measure the volume of extracted solution and add equal amount of 50% ammonium sulfate
solution.
5. Transfer all the mixture in a centrifuge tube.
6. Put the solution in an ice bath for 10 mins to facilitate precipitation of proteins.
7. Centrifuge the cold solution @4000 rpm for 5 minutes at 4C.
8. Pour off the supernatant liquid carefully. Take note of the white precipitate/pellet and describe
the appearance of isolated protein.
9. Divide the precipitate into three and transfer in an amber bottle and add 20 mL of 1M
Phosphate buffer, pH 7.5 and store in a refrigerator for further analysis.

B. HYDROLYSIS OF PROTEINS

Reagents and Materials

Protein isolates Centrifuge tube

6 M HCl Para film
Saturated protease solution Litmus paper
1 M NaOH 250-mL Erlenmeyer flask

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
B.1 Acid Hydrolysis

1. In a test tube place 0.5 g of the isolated protein add 5 mL of 6 M HCl.

2. Cover the test tube with cotton plug and autoclave for 5 hours at 15 psi.
3. Observe the change in the appearance of the protein sample after autoclaving.
4. Transfer to a 250-mL Erlenmeyer flask and add 10 mL of distilled water. Wash the test tube with
little distilled water and add the washing to the flask.
5. Neutralize the mixture with 1 M NaOH. Swirl the flask upon every addition of base and check
the pH using red and blue litmus paper.
6. Once neutralized, cover with para film, and label as “acid hydrolysate.”

B.2 Enzyme Hydrolysis

1. Transfer 10 mL of the protein isolate preserved in phosphate buffer (pH 7.5) in a 100-mL beaker.
2. Add 5 mL of saturated protease solution.
3. Incubate the mixture in a water bath, maintaining the temperature at 35-40°C.
4. Cool the mixture, cover with parafilm, and label as “enzyme hydrolysate.”

C. QUALITATIVE ANALYSIS OF PROTEINS

Amino acids are distinguished from one another by their R group. The R groups give
characteristic color reactions with reagents used to identify the amino acids components of the
proteins.

C.1 Protein Color Reactions

Reagents

Protein isolates Conc. NaOH 2% NaOBr

Protein hydrolysates Conc. H2SO4 2% Nitroprusside solution
2.5 M NaOH Millon’s reagent 5% Pb(CH3COO)2
0.1 M CuSO4 Hopkins-Cole reagent 1% Sulfanilic acid
0.1% Ninhydrin solution 10% NaOH 10% Na2CO3
Conc. HNO3 0.2% Alpha-naphthol solution 5% NaNO2

Materials

10-mL Test tubes Litmus paper Test tube holder

Dropper Water bath

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
Procedure

For all the tests, use 0.5 mL of the protein hydrolysates. Record the color of the solution and
take note of the formation of precipitate.

C.1.1 Biuret Test. To the sample, add 400 uL of 2.5M NaOH then add 20 uL of 0.1M CuSO4. Shake
the mixture and record the result.

C.1.2 Ninhydrin Test. To the sample, add 50 uL of 1% Ninhydrin solution. Shake the mixture then heat
in a boiling water bath.

C.1.3 Xanthoproteic Test. To the sample, add 10 drops of Conc. HNO3. Mix well and observe the
color change. Slowly add 10 drops of Conc. NaOH. Shake the mixture.

C.1.4 Millon’s Test. To the sample, add 50 uL of Millon’s reagent. Shake the mixture.

C.1.5 Hopkins-Cole Test. To the sample, add 100 uL of Hopkins-Cole reagent. Shake the mixture.
Incline the tube 45° and add 10 drops of conc. H2SO4 by running it down the side of the tube. Do not
shake the mixture but observe the interface.

C.1.6 Sakaguchi Test. To the sample, 50 uL of 10% NaOH and 50 uL of 0.2% alpha-naphthol solution.
Shake the mixture and let it stand for 3 minutes. Add 20 uL of 2% NaOBr. Shake to mix well.

C.1.7 Nitroprusside Test. To the samples, add 250 uL of 3M NaOH and 250 uL of 2% Nitroprusside
solution. Shake the mixture.

C.1.8 Fohl’s Test. To the samples, add 50 uL of 30% NaOH and 20 uL of Pb(CH3COO)2. Shake the
mixture. Heat the mixture in a boiling water bath.

C.1.9 Test for Amines. To the samples, add 50 uL of 20% NaOH. Shake the mixture then heat in a
boiling water bath and test the evolution of gas by placing a red litmus paper over the mouth of the
tube.

C.1.10 Pauly’s Test. To the samples, add 100 uL of Diazo reagent and add 20 uL of 10% Na2CO3.
Shake the mixture.

C.2 Thin Layer Chromatography (TLC)

TLC is a technique used to separate and identify the amino acid constituents of a hydrolyzed
protein isolates. The amino acids are separated based on their polarities. This technique utilizes a
stationary phase (TLC plate made of silica gel) and a mobile phase (solvent). The protein hydrolysates
spotted on the TLC plate migrates or “rises” on the plate as it is carried by the mobile phase. Different
amino acids will travel at varying distances depending on its affinity with the mobile phase. After the
amino acids are separated, the retention factor (Rf) is calculated. It has a formula:
Distance travelled by sample
Rf = Distance travelled by solvent

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 The Rf value can be used to identify the amino acid in reference to the Rf value of a given
standard. High Rf values would mean that the amino acid is less polar (or more non-polar) while lower
Rf values indicate a more polar amino acid.

Reagents

Amino acid standards 1-Butanol:Acetic acid: Water (4:1:1) *upper layer

Protein hydrolysates Ninhydrin solution

Materials Aspartame is the methyl ester of the dipeptide aspartylphenylalani

hydrolysis with HCl it yields aspartic acid, phenylalanine, and methyl al
artificial sweetener was approved by the Food and Drug Administration,
Paper stationary phase using a filter paper Capillary
aspartame tubesclaimed that itPencil
is a health hazard, because aspartame would
Chromatography chamber Ruler
would yield Filter
poisonous methyl paper
alcohol in soft drinks that are stored over
time. The Food and Drug Administration ruled, however, that aspartam
stable and fit for human consumption. Only a warning must be put on th
Procedure containing aspartame. This warning is for patients suffering from pheny
cannot tolerate phenylalanine.
To run a thin-layer chromatography experiment, we use silica gel in
1. Prepare developing chamber by lining it filter paper.
plasticPut a few
or glass mLWe
plate. of apply
BAWthe into the(aspartame
sample chamberorand amino acids) a
allow the solvent to saturate the chamber. of a thin-layer plate. The plate is dipped into a mixture of solvents. The s
the thin gel by capillary action and carries the sample with it. Each amin
2. Prepare the Stationary phase. Very lightly, mark awith a pencil
different a 0.25-cm
migration distance
rate depending from the
on the solubility topside chain in
of the
(solvent front) and 0.5-cm from the bottom (origin)Amino
edges respectively.
acids At the
with similar side origin,
chains mark ato0.5-cm
are expected move with similar,
identical, rates; those that have quite different side chains are expected
distance for each of the standard amino acid solutions and the samples. Properly label the markings
different velocities. Depending on the solvent system used, almost all am
with the one-letter symbol of the amino acids. dipeptides can be separated from each other by thin-layer chromatograp
3. Spot the markings on the plate with the amino acid standards
We actually do and
not the samples
measure the rateusing a capillary
of migration of an amino acid o
rather, how far a particular amino acid travels in the thin silica gel layer
tube. migration of the solvent. This ratio is called the Rf value. In order to calc
4. Place inside the developing chamber. Make surevalues, that the levelbeof
one must ablethe solventthe
to visualize in position
the chamber is acid or dip
of the amino
done by spraying the thin-layer silica gel plate with a ninhydrin solution
below the origin. Cover the chamber. the amino group of the amino acid. A purple color is produced when the p
5. Remove the TLC plate when the solvent reaches the (Thesolvent front.
proline not Leta it
having dry. amine gives a yellow color with ninhy
primary
6. Spray with visualization (staining) reagent. Then dry the plate using a hair an
example, if the purple spot of blowamino acid appears on the TLC plate 4.
dryer.
the origin and the solvent front migrates 9.0 cm (Fig. 43.1), the Rf value
7. Visualize the spots (using UV light) and mark lightly with pencil. Measure the distance of the spot
is calculated
from the origin (Refer to Figure 1). This is the distance travelled by the protein
distance hydrolysate.
traveled by the amino acid
Rf ! ! 4.5 cm ! 0.50
8. Compute for Rf values of the amino acid standards and the samples. distance traveled by the solvent front 9.0 cm
In the present experiment you will determine the Rf values of three
phenylalanine, aspartic acid, and leucine. You will also measure the Rf v
aspartame.

Solvent front à Figure 43.1

Solvent
(0.25 cm from TLC chromatogram.
front
top edge)
Amino
9.0 cm
acid
Markings for spotting of samples 4.5 cm

Origin ×
Origin à
(0.5 cm from Figure 1. TLC Chromatogram.
bottom edge) Measurement of distance from origin
to the spot.

438 Experiment 43
UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory
FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
D. QUANTITATIVE PROTEIN ANALYSIS

The most common method to determine protein concentration are Biuret Test, Bradford Assay,
Lowry Assay, and Bicinchoninic Acid (BCA) Test. The intact protein sample is made to react with a
compound producing a visible colored product which can be analyzed using spectrophotometer. A
standard calibration curve of known protein sample is utilized to compute the amount of protein in the
isolate using the absorbance with the application of Beer-Lamberts law.

Reagents

Biuret Reagent Bovine Serum Albumin (BSA)

Bradford Reagent Protein Isolate (per group)
0.1 g/mL BSA stock solution

Materials

96-well microplate Microplate reader Micropipettors and tips

D.1 Biuret Assay

Procedure

1. Use 0.1 g/mL stock solution to prepare a 6-point standard concentration using 2-fold dilution
starting at a concentration of 4000 ug/mL.

Concentration of standards for calibration curve in the Biuret Assay

4000 ug/mL 2000 ug/mL 1000 ug/mL 500 ug/mL 250 ug/mL 125 ug/mL

2. To 500 uL of each of the standard BSA solutions add 500 uL of Biuret reagent. Do the same with
the albumin isolate.
3. Incubate the treated standards and the albumin isolate at 60°C in a water batch for 10 minutes.
4. Transfer 200 uL of the mixture from the reaction mixture into the designated wells in the microplate.
5. Set the microplate reader at 540 nm and read.

D.2 Bradford Assay

Procedure

1. Use 0.1 g/mL stock solution to prepare a 6-point standard concentration using 2-point dilution
starting at a concentration 1000 ug/mL.

Concentration of standards for calibration curve in the Bradford Assay

1000 ug/mL 500 ug/mL 250 ug/mL 125 ug/mL 62.5 ug/mL 31.25 ug/mL

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
2. To 500 uL of each of the standard BSA solutions add 500 uL of Bradford reagent. Do the same with
the albumin isolate.
3. Transfer 200 uL of the mixture from the reaction mixture into the designated wells (see the figure
below)in the microplate.
4. Set the microplate reader at 595 nm and read.
Biuret Assay Bradford Assay

4000 ug/mL 1000 ug/mL

2000 ug/mL 500 ug/mL

1000 ug/mL 250 ug/mL

500 ug/mL 125 ug/mL

250 ug/mL 62.5 ug/mL

125 ug/mL 311.25 ug/mL

DW (Blank) DW (Blank)

SAMPLES SAMPLES

REFERENCES

Bathan, G., Crisostomo, A.B., Daya, M., De Guia, R., Farrow, F., Gabona, M., Guevarra Jr, L., Liu, M.I.,
Peña, G., Peña, L., Santiago, L., Santiago, M., Sarile, A., Torres, P., Vargas, A., & Ysrael, M. (2017).
Laboratory Manual in General Biochemistry. 2nd ed. C&E Publishing, Inc.

Stoker, H.S. (2017). Biochemistry. 3rd ed. C&E Publishing, Inc.

Bettelheim, F.A. & Landesberg, J.M. (2000). Laboratory experiments for general, organic, and
biochemistry. 4th ed. Brooks Cole

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 DATA SHEETS

NAME: GROUP NO.

A. Isolation of Intact Protein

Indicate the source of the protein. Describe the proteins isolated organoleptically. Insert/attach a
picture of the isolate.

Isolated intact protein

Source:
Characteristics:

Total Weight:
% Yield:

B. Hydrolysis of Protein
Describe the protein hydrolysate.
Acid Hydrolysate Enzyme Hydrolysate
Characteristics: Characteristics:

C. Qualitative Assay

1. Color Reactions
Indicate the visual positive result (VPR) for each of the tests. Indicate a positive result with a (+)
and a negative result with a (-).

Descriptive Analysis and Interpretation of the Result

Reference
Test
Acid Enzyme
Functionality/ Groups Positive
Description Interpretation Description Interpretation
detected Result

Biuret

Ninhydrin

Xanthoproteic

Millon’s

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 Hopkins-Cole

Sakaguchi

Nitroprusside

Test for
Amines

Pauly’s

2. Paper Chromatography
Attach a picture of the chromatogram below.

Fill out the table with the necessary information. Show your computations for the Rf values.

Acid: Enzyme:
Number of spots from the protein
hydrolysate revealed in the chromatogram
Distance travelled by the solvent (cm):

Rf values of the of the protein hydrolysate:

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 Sample Computation:

Interpretation of the chromatogram:

What are the amino acids present in the hydrolysate and compare the result of acid and
enzymatic hydrolysates?

Acid Hydrolysate Enzymatic hydrolysate

Interpretation:

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
D. Quantitative Assay (Total Protein Assay)
Attach a picture of the microplate and Show the graph and equation of the line.

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
1. Biuret Assay
Fill out the table below.

Absorbances
Samples
Trial 1 Trial 2 Trial 3 Average
Distilled water (Blank)
4000 ug/mL
2000 ug/mL
1000 ug/mL
500 ug/mL
250 ug/mL
125 ug/mL
Isolated Intact protein

R2 value of the standard

Equation of the Line
Concentration of isolate

2. Bradford Assay
Fill out the table below.

Absorbances
Samples
Trial 1 Trial 2 Trial 3 Average
Distilled water (Blank)
1000 ug/mL
500 ug/mL
250 ug/mL
125 ug/mL
62.5 ug/mL
31.25 ug/mL
Isolated Intact protein
R2 value of the standard
Equation of the Line
Concentration of isolate

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024
 LABORATORY PERFORMANCE

Name: Group No.

Section: Date Completed:

Rating Scale
Criteria
4 3 2 1
1. All required materials/samples are available and correct.

2. Instruments needed in the experiment are clean and

Pre- placed neatly on the working table.
laboratory
3. Calculations are correct.
Work
4. Schematic diagram is accurate and is easy to follow.

5. PPEs are worn appropriately.

Total /20
6. Proper decorum in the laboratory is observed.
7. Workspace is tidy and clean.
8. Safe and proper disposal of wastes are observed.
9. Procedures are followed correctly with little assistance
from the facilitator.
Laboratory 10. Proper techniques in weighing and pipetting are
Performance observed.
11. Containers are properly labeled and identifiable.
12. No repetition of procedures was done.
13. Reagent bottles, equipment, instruments are placed
back in their designated places after use.
14. Finished 15 minutes before the end of the class.
Total /36
Post- 15. Working table is clean.
laboratory 16. Instruments are clean and kept in the locker.
Work 17. Data sheets are checked and signed by the laboratory
facilitator.
18. Proper decorum in the laboratory is observed.
Total /14
Overall score /70

UNIVERSITY OF SANTO TOMAS Pharmaceutical Biochemistry Laboratory

FACULTY OF PHARMACY PHA 6112
Department of Pharmacy A.Y. 2023-2024