Protein separation via ion-exchange chromatography and
identification via SDS-PAGE.
Introduction
Proteins are a sequence of aminoacids that have functional groups such as carboxylic
acid, amines, sulfhydryl, guanidiino, etc. These functional groups can either donate or attract
protons at varying pH conditions, leading to a negative, positive, or a neutral charge. It is the
sum of the charges in the functional groups that will determine the net charge of the protein.
The net charge determines physical properties of the protein such as its attraction to a
resin with charged functional groups (known as ion-exchange resins). By manipulating the
charge in proteins, attractive forces between the protein and the ion-exchange resin can be either
reversibly strengthened or weakened. Since proteins tend to have different functional groups, the
rate at which different protein will elute in an ion-exchange column will differ from protein to
protein. The greater the electrostatic potential is, the greater the attractive forces will be, and the
slower the elution will take place. A salt or pH is then used to desorb those proteins with the
highest electrostatic attraction between the protein and the ion-exchange resin. On one hand, the
salt will competitively bind to the resin and remove the protein; pH, on the other hand, ionizes
functional groups to enhance or weaken electrostatic forces.
Another physical property of proteins useful for their analysis is the molecular weight.
Actually, it is possible to separate proteins based on their molecular weight. First, heat is used to
disrupt the attractive forces in proteins to partially linearize the molecule. Second, Bmercaptoethanol is used as a reducing agent to disrupt disulfide bonds to linearize the protein.
Finally, sodium dodecyl sulfate (SDS), an anionic detergent, binds to proteins in a ratio of 1.4:1
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(mass of SDS:mass of protein). By doing so, the proteins will be rod-like structures surrounded
by the negatively charged SDS molecules. Since the ratio of SDS to protein is constant, the
negative charges in the SDS-surrounded protein has a positive linear dependence to the
molecular weight and negative linear dependence to the displacement of protein in an electric
field; therefore, the molecular weight and displacement’s negative dependence can be found.
Based on aforementioned principles, the purpose of this experiment is to separate three
proteins based on their overall isoelectric points and estimate their recovery with the use of mass
balances. This will be done via ion-exchange chromatography using a step-change in NaCl
concentration. Additionally, identification of proteins will be done using SDS-Polyacrylamide
Gel Electrophoresis (SDS-PAGE).
Theory
Electrostatic interactions
Electrostatic interactions govern ion-exchange chromatography. The attractive forces
between two oppositely point-charges are described by Coulomb’s Law:
F = -q1·q2/d·r2 Eq. 1
Where q1 and q2 are the charge of point charges, d is the dielectric constant of the
medium and r is the distance between q1 and q2. For this reason, the attractive force will repel if
the charges are the same, and vice versa.
Protein Charge
Handerson-Hasselbach equation describes ionization behavior upon changes pH changes.
pH = pKa + log ([A-]/[HA]) Eq. 2
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where pH is a measure of hydrogen activity, pKa is the negative log of the acid dissociation
constant, A- is the negatively charged species, and HA is the conjugate acid. Since pKa is
constant, the log ([A-]/[HA]) will increase if the pH of the solution increases; thus, the negative
charge (A-) will increase. It follows from this simple analysis that proteins will have negative
charges if they are above their isoelectric point (or pH at which they have no charge), and vice
versa.
Mass balances
Beer-Lambert is an empirical relationship that provides a positive linear dependence of
absorption of light and concentration. Typically, Beer-Lambert law is expressed as
A=lec Eq. 3
where A is absorbance, l is the path length traveled by the light, e is the extinction coefficient
and c is the protein concentration.
On the other hand, the mass of the protein in column can be expressed as:
m = ∫ C· F·dt Eq. 4
where m is the mass of protein, c is the concentration of protein, f is flow rate and dt is
differential time.
By combining equation equation 3 and 4, a new relationship of the mass of protein
flowing out of the column with absorbance is obtained:
m = kR Eq. 5
where m is the mass of the protein that flows out of the column, k is a constant dependant on
flow rate (F), extinction coefficient (e), and light path length (l), and R is the area under the
curve in absorbance (A) versus time (t) plot.
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Since the initial mass of protein can be determined, and the mass of the exit stream can be
determined, the governing equation is.
m0-m=0 Eq. 6
where m0 is the initial mass of protein.
Langmuir isotherm
A system in which there the following equilibrium exists:
Keq,1 = [SP] / ([P][S]) –Eq. 7
Keq,2 = [SCl] / ([Cl][S]) –Eq. 8
where P- is the protein anion, S+ is the unoccupied binding site, SP is the protein-binding
site complex, Cl is chloride ion, and SCl is the chloride-binding site complex, Keq are
equilibrium constants. Mass balances on the sites in the resin, indicates that:
Stot = [S] + [SP] + [SCl] –Eq. 9
where Stot is the total amount of binding sites. The following Langmuir isotherm can be
obtained from combining equations 7-9.
[S P] = (Keq,1 Stot [P])/(1+ [P] Keq,1+ [Cl] Keq,2 ) –Eq. 10
Experimental Methods
Reagents
All proteins were purchased from Sigma (St. Louis, MO, U.S.A.). Cytocrhrome C,
Myoglobin and Bovine Serum Albumin (BSA) were dissolved in 0.02 M Tris buffer (pH 8.5) to
0.15 mg/ml, 0.07 mg/ml and 0.3 mg/ml respectively. Previous to loading, the dissolved protein
was filtered in a 0.22 μm hydrophilic polyvinylidene (PVDF) to remove particulate matter.
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Extinction coefficient was previously determined to be 0.0610±0.002 for BSA,
0.21±0.002 for Cytocrhrome C, and 0.170±0.001 for Myoglobin using a Shimadzu UV-Vis
Spectrophotometer Model #UV160U.
Ion-exchange column
The resin used was Macro-Prep High Q Support (methylacrylate copolymer) from BioRad (Hercules, CA, U.SA.). All solutions (sterilization, regeneration, equilibration, protein,
wash, elute) were introduced into the column using a peristaltic pump at a flow rate of
approximately 1/6 (1mL/min) solution volume flow per unit of liquid volume per minute.
Ionic strength in the medium was step-changed using solutions of 0.02 M Tris buffer with
0 M, 0.3 M, 0.5 M and 1.0 M NaCl (pH 8.5).
Data acquisition and validation
Conductivity and UV readings were collected using LabView from National Instruments
(Austin, TX, U.S.A.). Fractions were 3 mL in volume and analyzed further in 15% Tris-HCl
ready gels (SDS-PAGE).
SDS-PAGE was ran on Mini-PROTEAN II and the low range molecular weight standard
was used; both manufactured by Bio-Rad.
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Figure 1. Schematic representation of the apparatus used.
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Results
Molecular weight estimation
The low range molecular weight standard
Gel #1
was Bio-Rad was ran along with the eluted
proteins. From this standard, a rough molecular
weight estimations can be made: cytochrom C is
14-21 KDa and Myoglobin about 21 KDa. BSA is
difficult to determine because the standard is
Gel #2
smeared. This compared to the theoretical values:
cytochrom should have 12KDa, Myoglobin 17.6
KDa, and BSA 66.5 KDa.
A positive control of the proteins used was
run in the gel. From the positive control it is clear
that the fractions with each protein were identified.
Figure 2. SDS-PAGE ready gels with labels, where
MW is molecular weight standard, Mix is the
mixture of the three proteins, and F is the fraction
number.
Protein separation
Two different SDS-Page gels were run because the first gel did not show the stain
corresponding Myoglobin.
Figure 2 shows that the fractions 9, 15, 17 and 23 contained cytochrome C; fraction 25
contained BSA. It also shows that fraction 18 contained myoglobin.
Thus, these gels suggest that the elution order was: cytochrome C, myoglobin and BSA.
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Protein mass balances
In figure 3, gels identified the peak 1 and 2 to be cytochrome C (fractions 9, 15, 17), peak
3 to be myoglobin (fraction 18), and peak 4 to be BSA (fraction 25).
Figure 3 shows 4 peaks. Using polymath, the area under the peaks (of the A.U. versus
time plot) were found to be, from left to right, 0.106±0.024, 0.036±0.007, 0.059±0.007, and
0.119±0.013 minutes.
By using equation 5, the mass that the peaks represent are, from left to right,
0.438±0.100, 0.149±0.029, 0.174±0.021, and 0.975±0.110 mg of protein. Which represent
percent recovery of 97.8±21.5% for cytochrome C, 62.0±7.7% for myoglobin, and 81.3±9.2%
for BSA.
Salt concentration effects
No step change was necessary to recover 73.0±16.7% of cytochrome C. A step change in
NaCl resulted in further cytochrome C desorption from column. The first step change,
corresponding to 0.1 M NaCl, desorbed 24.8±4.9% cytochrome C and myoglobin out of the
column; the second step change of 0.3 M NaCl detached BSA.
Normalized variable
Conductiviry and AU superimposition
1
0.8
0.6
0.4
0.2
0
-0.2 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Fraction
Normalized AU
Normalized conductivity
Figure 3. Plots of normalized AU (AU/0.06) and normalized conductivity (Conductivity/3) versus 3 mL fractions.
Note that, because of frequent sampling, error bars were not included because they obstruct the shape of the curves.
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Discussion
Protein elution order
Cytochrome B5
Because all the proteins were buffered at a
pH of 8.5, it is expected from the HendelsonHasselbach equation that the net charge for
cytochrome C will be positive. In other words,
Horse heart wild-type myoglobin
cytochrome C should be repelled from the column.
However, results show that only 73.0±16.7% is
repelled; the rest elutes after addition of 0.1 M NaCl.
Results suggest that part of the cytochrome C binds Human serum albumin monomer
as opposed to what is predicted by the net charge
model used. This behavior is not strange because net
charges do not take into account that the surface
charges might not be symmetrically distributed. In
fact, it has been suggested by retention mapping
studies that the net charge model is inadequate
(Kopaciewicz et al., 1983). The only bovine
Figure 4. Red represents positively charged
groups (Lys, Arg, His), blue represents
negatively charged groups (Asp, Glu).
cytochrome NMR shows that the negatively charged residues, indicated in figure 4, are on the
surface (Muskett and Whitford, 2003). An improved model, therefore, is that sometimes the
negatively charged surfaces have a probability to attach to the resin despite the protein’s positive
net charge. However, since these charges are weak, it is easy to desorb them –a 0.1 M increase in
NaCl detaches them immediately as indicated in figure 3.
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The order of elution of the other proteins followed expectations. At pH 8.5 myoglobin
should elute first because its pI is 7.2 and its electrostatic force, indicated by Coulomb’s Law, is
weaker –as opposed to 4.7 pI of BSA.
An approach to quantitatively assess the electrostatic attractions between a protein and
the resin is by treating each ionizable functional group as a force vector. The addition of these
vectors should give an idea of the total force that the protein is attracted to the resin. However,
the implementation is out of the scope of this lab report.
Protein recovery
Protein recovery was not constant. Percent recovery of 97.8±21.5 was observed for
cytochrome C, 62.0±7.7 for myoglobin, and 81.3±9.2 for BSA. It is interesting to observe that
the recovery of repelled proteins, such as cytochrome C, is higher than attracted proteins, such
myoglobin and BSA. Although this seems to indicate that proteins that are most strongly
attached to the resin will have less protein recovery, it is not always the case. BSA, the protein
that is most strongly bounded, has a recovery of 81.3±9.2 compared to the 62.0±7.7 for
myoglobin. Thus, even if the errors are considered, results indicate that some protein did not exit.
To address these considerations, we can use cytochrome C as a working example. As
seen earlier, the behavior of proteins binding to the column is a probabilistic phenomenon (recall
that some cytochrome C was even found in fraction 23). Proteins that bind to the column from
patches of negative charge in the surface will tend to attach stronger. Thus, a reasonable
speculation will be that myoglobin’s charges are distributed asymmetrically -as seen in figure 5.
BSA on the other hand, has charged distributed throughout its tertiary structure.
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Another factor that might have an effect on attractive forces is the size. Whereas it is
difficult for BSA to attach different sizes simultaneously to the ion-exchange resin, it is easy for
myoglobin to orient its negative charges towards the ion-exchange matrix.
Error analysis
The raw A.U. data from the UV unit was noisy. Even when buffer was running and the
apparatus was recalibrated to zero; the A.U. values fluctuated intermittently from -0.06 to 0.05.
Since the fluctuations were symmetrical about 0 A.U, it is reasonable to assume that the noise is
a random error; the average of all the values of the noise over time should be the set point. The
set-point was calculated to be -0.00027, the standard deviation due to noise was determined to be
0.00140.
The standard deviation was added to each data-point that corresponded to each peak in
figure 3. A new set of data-points with standard deviation was obtained. These data was then
inputted into Polymath to calculate the area under the curve using the Simpson’s area
approximation algorithm.
The area under the peak was subtracted from the area under the peak with error. The
difference should be the error of the area due to noise.
Molecular weight determination
Although the molecular weight estimations based on the low range molecular weight
standard were off by 2 to 4 KDa. It did follow the prediction that the proteins with lower
molecular weight would cover a longer distance. Despite these differences, it does not posses
significant relevance for the experiment. Positive controls were used to determine the identity of
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the proteins. Since the eluted protein aligned with the positive controls, it can be concluded that
the eluted proteins are indeed the proteins of interest –and not contamination.
Salt concentration effects
Decisions on steps of NaCl concentrations were made arbitrarily. However, the resolution
was good since no fraction contained more than one protein. A better approach to this is by
constructing Langmuir isotherms (see Figure 5). By doing so, it is possible to determine the
precision at which the protein of interest will detach from the ion-exchange resin. This is useful
in proteins with close isoelectric points require to be separated using ion-exchange
chromatography.
Another important aspect from figure 5 is that the detachment of proteins becomes
subsequently difficult. An increase of 0.5 mg/ml decreases the protein concentration on resin
from ~0.08 to 0.06, whereas an increase of 90 mg/ml decreased the protein concentration on
resin from ~0.01 to 0. This is because the Langmuir isotherm is represented as:
[S P] = (Keq,1 Stot [P])/(1+ [P] Keq,1+ [Cl] Keq,2 )
Fig 5. Langmuir isotherms tells the behavior of protein with increasing NaCl concentrations.
Source: Bioseparations science and engineering.
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Considering that figure 5 shows the effect of equilibrium binding of a monovalent protein, it is
even probable for a plurivalent (such as myoglobin) protein to attach strongly to the ion
exchange resin. However, as the Langmuir isotherm shows, it is difficult to detach the last
portion to obtain a 100% recovery.
Conclusions
Although the study of ion-exchange chromatography based on the net charge model is
inadequate, as it does not describe the reason why two peaks for the same protein was observed
or the reason why the recovery of different proteins was different, it is generally a reasonable
description of protein elution order.
The study of protein structure can shed light on how proteins will elute. Asymmetrical
distribution of charges will typically increase the ways in which proteins can attach; high
molecular weight, on the other hand, will decrease the ways in which protein can be absorbed
into the ion-exchange matrix. Since the protein structure is complex and variable, it is difficult to
develop a model that takes into account the structure of the protein that desires to be separated on
a case-per-case basis.
Despite the limitations of the existing net charge model, the resolution was good. None of
the fractions had more than one protein and the resolution is 5.26±0.1. Recovery of the protein
was fairly decent: 97.8±21.5% for cytochrome C, 62.0±7.7% for myoglobin, and 81.3±9.2% for
BSA. Despite a 100% protein recovery is desired, Langmuir isotherm shows us that doing so is
inefficient –since it will require high quantities of competitors for the binding site.
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Nomenclature
Variable
q1
q2
D
r2
pH
pKa
[A-]
[HA]
A
l
e
c
m
F
Dt
K
R
m0
Meaning
Charge of point charge 1
Charge of point charge 2
Dielectric constant
Distance between q1 and q2
Negative log of hydrogen concentration
Negative log of the acid dissociation constant
Negatively charged species
Conjugate acid
Absorbance
Path length traveled by the light
Extinction coefficient
Protein concentration
Mass
Flow rate
Differential time
Fudge constant
Area under the curve in absorbance (A) versus time
(t) plot.
Initial mass of protein
[S X]
Keq
Stot
[P]
[Cl]
[S]
X-binding site complex
Equilibrium constant
Total binding sites
Protein anion
Cloride ion
Free binding site
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References
Bhattacharya, A.A., Curry, S., Franks, N.P. 2000. Binding of the general anesthetics propofol
and halothane to human serum albumin. High resolution crystal structures. J.Biol.Chem.
275,38731-38738.
Harrison, R.G., Todd P., Rudge, S.R., Petrides D.P. 2003. Bioseparations science and
engineering. New York, 2003.
Maurus, R., Overall, C.M., Bogumil, R., Luo, Y., Mauk, A.G., Smith, M., Brayer, G.D.
1997. A myoglobin variant with a polar substitution in a conserved hydrophobic cluster
in the heme binding pocket. Biochim.Biophys.Acta.1341, 1-13.
Muskett, F.W., Whitford, D. Cytochrome B5NMR Structure of Bovine. To be published.
Kopaciewicz, W., Rounds, M.A., Fausnaugh, and Regnier, F.E. 1983. Retention model for highperformance ion-exchange chromatography, J. Chrom., 266, 3-21.
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Appendix A
Mass balance derivation
The Beer-Lambert Law can be expressed as
A = lec –Eq I
Differential mass can be expressed as
dm =c ·dV –Eq II
Which upon integration yields
m = ∫ c · dV –Eq III
Since dV=F·dt
m = ∫c· F·dt –Eq IV
Combining equation I and IV
m = ∫ FA/el · dt –Eq V
Which can also be expressed as
m = F/el ∫ A· dt –Eq VI
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Langmuir isotherm
A system that has the following equilibrium
Keq,1 = [SP] / ([P][S]) –Eq. A
Keq,2 = [SCl] / ([Cl][S]) –Eq. B
And where mass balances indicate
Stot = [S] + [SP] + [SCl] –Eq. C
Combining the equations A-C
Stot = [S] + Keq,1 [S] [P] + Keq,2 [S] [Cl]
Stot = [S] (1+ [P] Keq,1+ [Cl] Keq,2 )
Stot / (1+ [P] Keq,1+ [Cl] Keq,2 ) = [S] -Eq. D
Combining equation A with D
Stot / (1+ [P] Keq,1+ [Cl] Keq,2 ) = [SP] / (Keq,1 [P])
[S P] = (Keq,1 Stot [P]) / (1+ [P] Keq,1+ [Cl] Keq,2 ) –Langmuir isotherm
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Sample calculations
Area error propagation
1. Noise data over 20 minutes was obtained.
2. Average was -0.0002744
3. Standard deviation was 0.0014073
4. Each data point from a peak in raw data, “set A”, was added to the standard deviation,
namely “Set B”
5. “Set B” is integrated (Plots of A.U. versus time), Area B.
6. “Set A” is integrated (Plots of A.U. versus time), Area A.
7. Area B – Area A = Error
Recovery error propagation
Raw data and report
Please download from
www.zhuam.com/report.zip
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