Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules

Lab 4 Page 1 of 7

Protein Samples that you have prepared in lab 4 will be used in the subsequent labs.

Laboratory #4: Purification of Myoglobin by Ion-Exchange Chromatography

In the previous lab, we fractionated various protein samples based on their solubility in the presence of different
concentrations of ammonium sulfate. Also, we have utilized a preparative centrifuge to sequentially collect
protein samples isolated from our biological sample, hamburger meat. One of the fractionated samples has
been dialyzed to remove ammonium sulfate from the protein mixture or solution, which we will use as our
starting material in this laboratory.

In this laboratory, we will use ion exchange chromatography to further purify myoglobin from other undesirable
proteins (contaminants). The experiment is designed for students to practice fundamental techniques of protein
preparation. It is also for students to re-enforce the understanding of the concepts of protein ionization
properties along with the principle of ion exchange chromatography.

Background
Ion exchange chromatography is a form of adsorption chromatography in which ionic solutes interact with a
charged stationary phase using reversible electrostatic interactions. The stationary phase, or resin, consists of
two parts: (i) an insoluble, three-dimensional matrix and (ii) chemically bonded groups within or on the surface
of the matrix. Synthetic resins are made from different materials, such as cellulose, polysaccharides (dextran),
or polystyrene. The chemically bonded groups are either cationic or anionic groups depending on whether they
exchange cations or anions. A resin which has negatively charged functional groups will exchange positive ions,
therefore, is called a cation exchanger. Depending upon the ionic strength of the functional groups, the
exchange resins are classified into strong and weak.
Carboxymethyl Sepharose (CM-Sepharose) will be used as the stationary phase here. CM-Sepharose is
a cation exchanger and has negative charges that will exchange positive counter ions with solutes or proteins.
The ability of an ion exchanger to adsorb counter ions is defined quantitatively by capacity. The total capacity
of an ion exchanger is the quantity of charged groups per unit weight of dry exchanger (milliequivalents of
ionizable groups per milligram of dry weight).
Ionization properties of proteins: As we have learned from Lab 2, by titration we can identify an isoelectric
point (pI) of an amino acid. Pi is the pH at which a molecule carries no net electrical charge or net zero (0, plus
= minus) charge. The pI value of a small peptide can easily be calculated using the pK values of terminal carboxyl,
amino groups, and side-chain residues (please consult relevant lecture materials in Unit 2). During protein
purification, the amphoteric (acid-base) nature of amino acids and proteins can be exploited and used in an ion
exchange chromatography directed protein purification process. We can influence the ionization of amino acids
and proteins by exposing or incubating them in different pH solutions so that their net charge is predominantly
in negative or positive charges. At pH below pI, the peptides will predominantly be in their protonated forms or
have positive charge, and thus bind to a cation exchanger. At pH above pI, the peptides will predominantly be
in their deprotonated forms or have negative charge and bind to an anion exchanger.
Taken the ionization property of protein and the concept of ion-exchange chromatography together, any
target proteins can be enriched or purified from other protein contaminants based on their ability to
Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 2 of 7

differentially bind and/or release from an ion-exchange stationary phase (resin) using a buffer solution with an
appropriate pH and salt concentration(s). An appropriate pH is employed to influence the charge on the protein,
and salt concentration to affect the protein solubility to be either salted-in (solvated) or salted-out (de-solvated).
As the buffer solution or the mobile phase moves along the stational phase, the unbound (un-captured or
released) proteins are eluted out and can be collected by a fraction collector, which is a piece of laboratory
equipment commonly during chromatography. Using a fraction collector, we can program how much and how
often the samples will be taken for further analysis.
Ultraviolet-Visible (UV/VIS) spectrophotometer is another piece of laboratory equipment commonly used in
biochemistry laboratories. It allows researchers/scientists to follow the purification of a protein by measuring
the total amount of protein at every stage of purification. Most proteins are colorless and do not absorb the
energy in the visible region of the spectrum. However, due to the aromatic side chains (Phe, Trp, Tyr) of
polypeptides or proteins, most proteins strongly absorb the energy in the UV region of the spectrum ( = 200
to 400). Absorbance spectroscopy at 280 nm is a convenient way to monitor the concentration of protein eluted
from a chromatography column.
In this lab, we will exploit a unique characteristic of heme bearing nature of myoglobin to monitor its
purification. Heme-bearing proteins exhibit distinct absorption bands characteristic not only of the heme group,
but also of the oxidation state of the heme group. One of the signature bands of the heme group is the Soret
band which is seen as an absorption maximum around 410- 420 nm. Heme groups also bear additional
absorption bands in the 500 – 600 nm regions known as Q bands.

In practical terms, we require to do:

1. Prepare the stationary phase/resin and the chromatography column (packing a chromatography
column). CM-Sepharose is resuspended in 20 mM phosphate buffer pH 6 to allow the resin to fully hydrate
and form its negative charge. The CM-Sepharose suspension is then packed into a column and kept until
use.
2. Apply a protein sample (capturing step). The sample in a buffer solution will be applied to the column.
Proteins with cation will be captured by CM-Sepharose in the column, while anion-bearing proteins are
not captured and leave the column as the buffer solution moves along the column. Since the binding or
capturing of proteins is based on their charge (ionization) nature, a large volume of samples can be applied
to the column and will not affect the purification step.
3. Remove unbound proteins (washing step) and elute the protein of interest (releasing step). There are
two ways of removing unwanted protein and eluting a protein of interest. One is to alter pH of the buffer.
Using a buffer with the same pH as that of sample application step (pH 6), unwanted or unbound proteins
will move out from the column as the mobile phase moves along the column, while the protein of interest
stays bound. Then an elution buffer (with a higher pH or a pH matching the protein’s pI) is used to release
the protein from the stationary phase. Another way to wash unwanted proteins and to release wanted
proteins is to use buffer solutions with gradual changes of salt concentrations so that salt ions essentially
compete with the protein for binding sites. Proteins with different binding affinities to the stationary
phase will gradually release from the column as the mobile phase moves along the column (see more
information in the recommended textbook Chapter 5, section 2C).
Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 3 of 7

4. Regenerate the column for the next use. The ion-exchange resin or stationary phase is generally costly
and can be used multiple times. Therefore, a proper cleaning (or removing any materials left in the
column) is required. Here we use a high salt concentration of 1 M sodium chloride to remove all proteins
that may be left in the column. Water is then applied to the column to remove salt and any other
contaminants, and the cleaned ion-exchange resin is kept in the column with 20 mM phosphate buffer pH
6.
Chemicals & Reagents
• 20 mM phosphate buffer pH 6 • 20 mM phosphate buffer pH 8
• 1 M Sodium chloride

Glassware & Equipment

• Beakers: 50 and 100 mL • P1000 micropipette and tips
• Centrifuge • Retort stand and clamp
• CM-Sepharose column (prepacked) • Spectrophotometer (Cary)
• Cuvette with rack: 1.5 mL • Test tubes and rack: 20 (13 x 100 mm)
• Fraction collector • Vortex mixer
• Microcentrifuge tubes: 3

Procedure
Part A – Setting up
A1. Equilibrate the column: Use a retort stand and clamp to hold the prepacked column. Run 20 mL of 20
mM phosphate buffer pH 6 through the prepacked column. Be careful not to disturb the top of the
column bed. Leave 1 cm of the buffer on the top of the resin (sorbent) before you cap it and put the
yellow tip on the bottom of the column. Make sure the column never runs out dry. If the volume recedes,
add more buffer.
A2. Sample preparation: From the dialysis bag/tube, transfer the content into a microcentrifuge tube.
Centrifuge for 5 min at 3500 rpm. Transfer the clarified solution/supernatant to a clean microcentrifuge
tube making sure not to pick up any solid debris. Label this Sample D and keep on ice.
A3. Setup the fraction collector as follows:

a. Rotate the drop arm to the rear position.

Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 4 of 7

b. Load the carousel with 20 test tubes starting in slot #1.

Position the carousel so that slot #1 is in the back, directly over
the gear drive.

c. Lift and rotate the drop arm into the collection position over
tube #1. Press the [MODE] key to select the DROP collection
mode. Use the arrow keys to set the collection interval to 25
drops (25 drops = 1 mL).

Part B- Chromatography procedure:

Obtain phosphate buffer solutions pH 6 and pH 8 approximately 20 mL each.
B1. Loading sample:
a. From the centrifuged Sample D, pipet about 250 µL into a new microcentrifuge tube labelled also “
Sample D” and keep on ice (this sample will not be use in this lab but will be analyzed in the following
labs).
b. With your column still attached to the retort stand, adjust the level of buffer over the column bed of
your pre-packed column to 1-2 mm by draining the column into a waste beaker.
c. Very carefully, load the remaining of the centrifuged Sample D (about 750 µL) to the top of the column
bed.
B2. Binding and washing steps:
a. As soon as the protein solution has entered the resin bed, add (dropwise) about 1 mL of phosphate
buffer-pH 6 from a disposable pipet to wash the protein mixture into the resin bed. Be careful not to
disturb the top of the column bed and remember to never allow the column to run dry.
b. Continue to add ~1mL of phosphate buffer (pH 6) from a disposable pipet at a time until the brown
“flow through” band nears the bottom of the column.
c. Collect this band in a test tube labelled “F” and set aside.
d. Save 1000 µL of the flow through in a microcentrifuge tube labeled “Sample F” and keep on ice.
B3. Eluting steps:
a. Attach the column to the white rubber drop former located on top of the drop arm of the fraction
collector.
b. Press the [RUN] button on the fraction collector.
c. Carefully add ~15mL of phosphate buffer pH 8 and collect at least 15 fractions.
Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 5 of 7

d. Stop the collection by pressing the [RUN] button when your fractions have been collected. Make sure
there is ~2mm of buffer above the resin.
Detach the column from the fraction collector and securely place the yellow cap to the column’s tip.
Clamp the column to a retort stand.
Note: Make sure to keep the small white rubber piece on the fraction collector arm.
Part C- Regenerating the chromatography column’s resin.
C1. Slowly add 20 mL 1M NaCl solution (provided in a dropper bottle).
C2. When the level of NaCl solution is ~2 mm above the sorbent bed, add ~10 mL deionized water through
the column bed. Use a disposable pipet to carefully re-suspend the resin in the column (3 to 4 cycles).
C3. Allow the column to pack. When the water level gets low on top of the column bed, carefully add another
10 mL of DI H2O without disturbing the column bed.
C4. Finally, when water level is about 2mm above the sorbent bed, run ~15-20 mL of PB pH 6.0 through the
column bed. At the end make sure to leave about 2 cm of the buffer on top of the sorbent bed and place
the yellow tip as well as the top cap.
C5. Hand the column to your GA.

The column should be checked by the GA before you leave the lab. If the resin is not regenerated
according to the above procedure, students will lose 50% of this lab mark.

Part D – Sample analysis by spectrophotometer (Cary-50 system)

D1. On the spectrophotometer (Cary-50 system), choose “Simple Reads” application and wait 30 seconds for
the instrument to initialize.
D2. Under the Setup menu, type in 410nm (optimal absorbance of myoglobin). Click OK.
D3. Place a cuvette ¾ full DI H2O in the sample compartment to serve as the blank. Click the ‘Zero’ button to
set the absorbance to zero.
D4. Remove the cuvette from the compartment and replace the water in the cuvette with first fraction
sample (test tube #1). Click on the read button at the top of the screen and record the results. Pour
cuvette contents back into test tube #1. Rinse the cuvette with DI water.
D5. Fill the cuvette ¾ full of the second fraction sample (test tube #2) and click “Read”. Continue this
procedure and make sure to record the absorbance and sample volume in the table below. MAKE SURE
TO SAVE ALL SAMPLES.
D6. Once you have found the sample with the highest absorbance (highest myoglobin concentration), repeat
D5 for the next two fractions only.
D7. Identify the fraction that contains the highest myoglobin concentration (highest absorbance). Transfer
the entire sample to a microcentrifuge tube labeled SAMPLE E.
D8. Freeze SAMPLES D, SAMPLE F , and SAMPLE E for your analysis in Labs 5 & 6.
Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 6 of 7

Data Analysis Questions

a. At which wavelength does your protein exhibit maximum absorbance? [0.5]

b. Plot an elution profile of absorbance versus elution volume (notice volume = fraction number). Indicate
at which point the pH change occurred, the fraction(s) which contain the highest myoglobin content, and
the maximum absorbance exhibited by the myoglobin sample. [1.5]

c. Theoretically, the PI of myoglobin is 7.2. In phosphate buffer pH 6.0: [2.0]

i. What is the overall charge of myoglobin?

ii. How would it interact with CM Sepharose?

iii. Why do we change to a buffer of pH 8? Explain.

d. What is expected in Samples ‘D’, ‘F’, and ‘E’? [1.0]

e. Why do we keep the fraction with high absorbance, and not all fractions? [1.0]

f. At this point do you think your protein is pure? Why or why not? (Give specific reasons; do not cite
apparatus or human error) [2.0]

Assignment Requirements: Answers to Data Analysis Questions (typed) and the Data Sheet are due at the
beginning of the next lab session. [8.0 Marks]

• Upon completion of the experiment, make sure to set the micropipette to its maximum limit and
place vertically in the pipet holder.
• Wash and return test tubes upside-down in the test tube rack.
• Make sure the white rubber drop former located in the drop arm of the fraction collector remains
in place.
• Return orange dialysis clips to the front of the lab. Used dialysis membrane may be disposed to
the trash.
Winter 2025 BIOC-2010 Organic Chemistry of Biomolecules
Lab 4 Page 7 of 7

Lab 4 Data sheet

Name: Date:
Lab Partner: Section:

Record all data in PEN. Have your GA check and sign your data sheet before leaving the lab.

Abs
Fraction no. Elution Vol. (mL) (λ = ____)

Use the data above to plot an elution profile, in which the absorbance is on Y-axis, and elution volume (volume
= fraction number) is on X-axis. The elution profile must indicate at (1) which point the pH change occurred, (2)
the fraction(s) which contain the highest myoglobin content, and (3) the maximum absorbance detected.

GA signature: ___________________________________ Date: _____________