Ology Science
Research Article
Journal of Research Analytica
Affinity Capillary Electrophoresis for the
Estimation of Binding Constants and
Comparison of Binding Interactions of
Heparin-Derived Disaccharides to Histidine
Abstract
Heparin is a linear polysaccharide of the glycosaminoglycan (GAG) family of carbohydrates. Heparin and
other structurally related GAGs are characterized by the presence of carboxylate and sulfate groups on their
polysaccharide chains giving rise to an overall negative charge. GAGs are known to mediate a host of many
biological mechanisms such as cell differentiation, proliferation, metastasis and inflammation. The mechanisms
of these interactions are still poorly understood hence the continued interest in their study. Due to the structural
complexity and variability of functional groups in the disaccharide units that make up GAGs, their interactions
with proteins continue to be a major and challenging topic in analytical, biochemical and biological studies. We
present an affinity capillary electrophoresis (ACE) method to determine and compare the binding constants of
heparin-derived disaccharides to histidine.
Keywords: Affinity capillary electrophoresis, Heparin; Glycosaminoglycans
Alex Schrader, McKenna Feltes, Minh Duong and Albert K. Korir*
Department of Chemistry and Physics, Drury University, Springfield Missouri, USA
Corresponding author: Albert K. Korir
*
Received : October 10, 2016; Accepted: November 14, 2016; Published: November 21, 2016
E-mail: [email protected] (A.K.R)
Copyright: ©2016 OLOGY Group.
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
1
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
Introduction
The characterization of specific interactions and the
determination of corresponding binding constants are of
substantial interest in analytical and biochemical research [1].
These interactions are critical in understanding the functions
and molecular mechanisms of biological systems as well as
the roles the interactions play in health and human disease. A
number of analytical techniques have been employed in the
study of interactions between ligands and biomolecules [2]. In
many cases these techniques often require the separation and
quantification of either the complexed or free (uncomplexed)
molecule in an equilibrium mixture. However, it is not always
possible to distinguish complexed and uncomplexed molecules
thus complicating the binding assay [1].
In this study, we present the use of affinity capillary
electrophoresis (ACE) which offers several advantages including
low sample consumption (nanogram amounts), speedy analysis,
ease of automation and high-resolution separation. Using ACE,
weak binding constants (102-104 M-1) can easily be estimated
[3]. Since measurements are based on shifts in migration
times, the amounts of free or complexed molecule need not be
known. In addition, ligand or receptor under study needs not
be pure provided that the ACE method can distinguish it from
impurities. Unlike other methods used for studying ligandreceptor interactions, radiolabeling of molecules is not required
in ACE. An additional advantage of ACE is that the binding
interactions are studied in free solution without the need to
immobilize the ligand or receptor, a process that may possibly
denature or alter conformation. Therefore the binding assay can
be set up in such a way that the interacting molecules are freely
mobile at pH and ionic strength relevant to biological conditions
if desired.
The ACE method has become a well-established method
in the determination of binding constants particularly in 1:1
interactions [4]. The two major factors that influence the
migration behavior of an analyte are molecular interactions and
the effect of electric field on charged molecules. By monitoring
the migration time or mobility of an analyte, we can obtain
information on the binding affinity of the analyte under study
to an additive ligand mixed with the run buffer. There are many
examples on the successful application of ACE in a wide array
of interactions including protein-drug, protein-DNA, peptidecarbohydrate, peptide-peptide, and carbohydrate-drug [5-10].
There are a several reports on the use of ACE to study heparinprotein interactions. [11-13]. However, to our knowledge,
the differential binding of the various heparin-derived
oligosaccharides to biomolecules has not been studied. In this
study we chose two model molecules (histidine and histamine
structures of which are shown in Figure 1) to study their binding
interactions to heparin and heparin-derived disaccharides using
the ACE method. Histidine residues on proteins play important
role in mediating heparin interactions with certain proteins
[14,15]. For example, studies have shown histidine residues are
involved in heparin binding, tetramerization, and activation of
the tryptase mouse mast cell protease 6 [16]. When mast cells
are activated, they release the contents of their secretory granules,
2
ISSN : 2473-2230
including histamine, heparin proteoglycan, cytokines and proteases,
into the extracellular space.
Because of the significance of histidine residues in heparin
binding to proteins, we developed an ACE method to study its
interactions with heparin-derived disaccharide standards. The
success of binding studies using ACE is dependent on proper
experimental design and mathematical interpretation of data [17].
Materials and Methods
Chemicals
All chemicals were of analytical grade and were used without
further purification. All solutions were prepared in deionized water.
Apparatus
All experiments were performed on a Beckman P/ACE MDQ
system (Beckman Coulter, Fullerton, CA, USA) equipped with
a photodiode array detector. Data were collected with 32Karat
software (Beckman). Electropherograms were extracted at a
wavelength of 214 nm. Bare fused silica capillary was used with
an internal diameter of 50 µm (360 µm outer diameter) , a length
from the inlet to the detector of 55.3 cm, and a length from the
detector to the outlet of 16.2 cm. Samples were introduced by
hydrodynamic injection.
ACE procedure
The ACE injection protocol is summarized in Table 1. Briefly,
the neutral marker (benzyl alcohol) was first injected at 1.5 psi
pressure for 15.0 s followed by the running buffer at 1.0 psi for
10.0 s. The sample analyte was then injected at 1.5 psi for 15.0 s
and again followed by the running buffer at 2.5 psi pressure for
5.0 s. The running buffer was 20.0 mM sodium phosphate buffer
at pH 6.00 containing the GAG. The concentration of the GAG
in the buffer was gradually increased in a series of experiments
from zero to about 1mM. Between measurements, the capillary
was successively flushed with 1.0 M NaOH, water, and running
buffer at 50.0 psi each for 1 min. All separations were performed
in triplicate to check for precision of data. The temperature of the
cartridge was kept at 25.0 ± 0.1 ºC. The CE unit was operated at
a positive voltage of 18 kV in the normal polarity mode.
Table 1: ACE injection protocol and calculated zone lengths.
Injection
step
1
2
3
4
Sample
Pressure, time
Zone length (cm)
Benzyl alcohol
Buffer
Sample analyte
Buffer
1.5 psi, 15.0 s
1.0 psi, 10.0 s
1.5 psi, 15.0 s
2.5 psi, 5.0 s
1.9 cm
0.9 cm
1.9 cm
1.1 cm
N
O
HN
OH
A
B
H2 N
OH
N
HN
C
NH2
Figure 1: Chemical structures of (A) benzyl alcohol, (B) histidine and (C)
histamine used in the affinity capillary electrophoresis experiments.
J Res Anal. (2016) Volume 2 • Issue 4
Ology Science
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
Results and Discussions
affinities to histidine (~105) allowing qualitative comparison
based on magnitude of the binding constant.
Theory
In the ACE set up, determination of binding constants is
based on measuring changes in the migration time of the sample
analyte, R in the running buffer containing different concentration
levels of the ligand, L [3]. Throughout our discussion, L will
represent heparin or heparin-derived disaccharide (GAG) while
R will represent the sample analyte studied (e.g. histamine or
histidine). As the interaction occurs, a complex C is formed and
an equilibrium is established. For a 1:1 molar interaction, the
binding constant can be determined based on equations below.
L + R  C Kf =
C
[ L ][ R ]
lR =l − ( l2 + l3 + l4 ) − 0.5l1 (5)
(2)
l NM =l − l4 − 0.5l3 (6)
The basic principle of ACE is that complexation of the
ligand and the receptor induces a change in the electrophoretic
mobility. The effective mobility of analyte R ( µ Reff ) is influenced
by the mole fraction of the free molecule and that of the complex
C and is related by the equation 3 below:
R
C
µ RO +
µ RO C+R
C+R
(3)
Equation 3 can be expressed in terms of the binding constant
(Kf) to yield equation 4 below.
=
µ Reff
K f [ L] O
1
µ RO +
µC 1+ K f
1 + K f [ L]
(4)
Different linearized mathematical equations, summarized in
Table 2, can then be used deduce the interaction stoichiometry
and to calculate binding constants based on measurement of the
effective mobility as a function of GAG concentration [L] in the
buffer [18,19]
Studies have shown that nonlinear regressions perform better
in minimizing both error and bias in the estimates of binding
constants [20,21]. Bias particularly becomes significant in the
three linearized methods (x-, y- and double-reciprocals) when
the binding constant is low and therefore it is often necessary to
weight the data. Despite these limitations, linearized equations
are still useful in ascertaining the binding stoichiometry and
comparison of the relative binding affinities of structurallyrelated ligands such as the heparin-derived disaccharides used in
this study. In addition, these disaccharides exhibited high binding
Table 2: Linearized transformation equations used for the determination of
binding constants.
Linearized
transformation
Equation
µ PO − µ Peff
f
= K=
f [ L]
µ Peff − µCO
Linear Isotherm
X-reciprocal
µ Peff − µ PO
Y-reciprocal
Double-reciprocal
Ology Science
[ L]
([ L ])
=
− K f ( µ Peff − µ PO ) + K f ( µCO − µ PO ) =
f ( µ Peff − µ PO )
[ L ]=
µ Peff − µ PO
1
µCO − µ PO
× [ L] +
1
= f
K f ( µCO − µ PO )
([ L ])
 1 
1
1
1
1
=
×
+ = f 

µ Peff − µCO K f ( µCO − µ PO ) [ L ] µCO − µ PO
 [ L] 
Because of the successive hydrodynamic injection steps
involved in the ACE protocol (Table 1), it is necessary to
accurately determine the migration distance of the neutral marker
(NM) and the sample analyte (R) to the detector. These distances
will be denoted l NM and l R , respectively. If l is the length of the
capillary length from the injection inlet to the detector, then the
corrected distances will be as follows:
(1)
Where Kf is the binding constant or often referred to as
formation constant.
=
µ Reff
Corrected migration distances to detector and
calculated electrophoretic mobilities
The length of each of the successive zone li ( l1 through l4 ) was
calculated according to equation 7 derived from the HagenPoiseuille law that governs pressure-injection into capillaries
[22].
li =
(d
2
c
.P.t )
32η L
(7)
where dc=inner capillary diameter,P=Hydrodynamic Injection
Pressure,t=injection time in seconds, η=buffer viscosity.
Using equations 5-7, where l=55.3 cm, L=71.5 cm, dc=50
µm, and η=0.88×10-3 Pa, the corrected migration lengths for
the neutral marker ( l NM ) and the sample analyte ( l R ) were
calculated as 53.25 and 50.45 cm respectively.The corrected
migration lengths are used to calculate electrophoretic mobilities
using classical equations [3,11].
Reproducibility of ACE runs
The electrostatic attraction between analyte or additive
molecules and the capillary wall can be detrimental in the
ACE experiments. Although the interaction can be reduced by
increasing the ionic strength of running buffer, this can influence
the maximum voltage that can be applied which in turn will
decrease the efficiency. A 20 mM sodium phosphate buffer at
pH 6.00 produced the best ACE runs in terms of reproducibility
of migration times and separation efficiency. Since bare fused
silica capillary was used in our study, we conducted control
experiments to determine whether the ligand additives were
adsorbed in the capillary. To do this, the mobility of the
analyte in the running buffer without additive was measured
after each ligand concentration. Figure 2 is a representative
electropherogram showing triplicate ACE runs to measure the
mobility of 2.5 mM histidine in the presence of 0.008 mM
heparin added to the running buffer. Benzyl alcohol, used as
the noninteracting neutral marker and to aid in the accurate
measurement of migration shifts, had a migration time of about
5.52 s while the histidine peak was detected at around 3.00 s.
During the runs, there were minimal shifts in the migration
time of the neutral marker and the migration time of histidine
was reproducible with a relative standard deviation of 1.16%
indicating that the ligand was not adsorbed in the capillary wall.
Binding of histamine to heparin and heparan
sulfate by the ACE method
J Res Anal. (2016) Volume 2 • Issue 4
The glycosaminoglycans (GAGs) heparin and heparan
ISSN : 2473-2230
3
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
of the histamine-heparin complex peaks degraded at higher
concentrations of heparin in the running buffer. There are several
possible causes of peak-shape asymmetry in CE including the
initial asymmetry of the injected sample plug itself and physical
processes, such as capillary wall adsorption. The various causes
of peak asymmetry and possible ways of peak-shape correction
have been discussed in the literature [33,34]. To avoid the
complications in measurements associated with asymmetric
peaks, we used data from ACE electropherograms obtained using
lower concentrations of heparin for our ACE binding assays.
Figure 4 shows representative electropherograms obtained using
lower heparin concentrations ranging from (0 to 0.0094 mM).
sulfate are known to interact with many proteins [23-25] and
are structurally related differing only in the variability of the
substitution patterns in the disaccharide constituents [2628]. Table 3 shows the repeat disaccharide unit found in both
heparin and HS. Up to 50% of heparin (depending on its source)
consists of the trisulfated disaccharide unit with α (1→4)-linked
L-iduronic acid that is 2-O-sulfated and D-glucosamine which is
N- and 6-O-sulfated [27].
The formation constants (Kf) for the binding of heparin to
histamine have previously been determined from chemical shiftpH titration data obtained using proton NMR spectroscopy
[30]. In addition, equilibrium dialysis studies have shown a
1:1 histamine-heparin stoichiometry based on the disaccharide
building block as the combining unit [31,32]. Therefore, the
histamine-heparin model system was chosen to optimize our
ACE method and to compare the determined binding constants.
An added advantage of the histamine-heparin system is that both
compounds are relatively inexpensive.
The difference in the mobility between the free and the
complexed forms of the analyte is small and can cause errors
in the measured migration times leading to large discrepancies
in the determination of binding constants. To overcome this
problem, we conducted replicate experiments to determine the
effective mobility of the heparin-histamine complex at each level
of concentration and the values obtained from these replicate
measurements were then averaged.
Figure 3 shows a portion of representative overlay of ACE
electropherograms for 2.0 mM histamine in buffer containing of
increasing concentrations of heparin in the running buffer (20.0
mM sodium phosphate at pH 6.00). At pH 5.2-6.0, histamine
is a diprotonated cation that binds to heparin. The histamine
peak was detected at 3.0 s while the benzyl alcohol peak is
detected at around 9.5 s. As seen in Figure 3, the symmetry
The effective mobility of histamine-heparin complex was
calculated at each concentration level from classical equations and
using the corrected migration lengths (Table 1) [3,11]. A plot of
effective mobilities of histamine-heparin complex versus heparin
concentration yielded the binding isotherm shown in Figure 5A.
10.0
histidine
7.5
5.0
benzyl alcohol
2.5
0.0
-2.5
-5.0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
Minutes
Figure 2: ACE electropherogram of 2.0 mM histidine and 2.0 mM benzyl alcohol using a phosphate buffer containing 0.078 mM heparin.
Table 3: Structure of the repeat disaccharide unit of heparin and nomenclature of the commercially available heparin-derived disaccharides.
6 CH
5
5
O
O
CO 2H
4
6
1
OH
3
4
2
OR2
ISSN : 2473-2230
4
2OR1
O
1
OH
3
O
2
NH Y
Disaccharide
IS
IIS
IIIS
IVS
IA
IIA
IIIA
IVA
IH
IIH
IIIH
IVH
R1
SO3H
SO3H
SO3H
SO3H
SO3H
SO3H
J Res Anal. (2016) Volume 2 • Issue 4
R2
SO3SO3H
H
SO3SO3H
H
SO3SO3H
H
Y
SO3SO3SO3SO3Ac
Ac
Ac
Ac
H
H
H
H
Ology Science
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
histamine
P/ACE MDQ-214 nm
ACE-pH5Hep0p03125mM-09-18-13-run1
16
PDA - 214nm
ACE-pH5Hep0p0625mM-09-18-13-run1
PDA - 214nm
ACE-pH5Hep0p125mM-09-18-13-run1
PDA - 214nm
ACE-pH5Hep0p25mM-09-18-13-run1
benzyl alcohol
14
12
10
8
D
6
C
4
B
2
A
0
2
3
4
5
6
7
8
9
10
11
Minutes
Figure 3: Representative high-concentration region ACE electropherogram of 2.0 mM histamine and 2.0 mM benzyl alcohol using a phosphate buffer containing
(A) 0.0031 mM (B) 0.0063 mM (C) 0.013 mM and (D) 0.025 mM heparin.
22.5
P/ACE MDQ-214 nm
0.0078125mM
histamine
PDA - 214nm
0.015625mM
PDA - 214nm
0.03125mM
PDA - 214nm
0.0625mM
PDA - 214nm
0.09375mM
22.5
20.0
20.0
17.5
17.5
benzyl alcohol
15.0
15.0
12.5
12.5
10.0
10.0
E
7.5
7.5
D
5.0
5.0
C
B
2.5
2.5
A
0.0
0.0
-2.5
2.0
-2.5
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
Minutes
Figure 4: Representative low-concentration region ACE electropherograms of 2.0 mM histamine and 2.0 mM benzyl alcohol using a phosphate buffer containing
(A) 0.00078 mM (B) 0.0016 mM (C) 0.0031 mM (D) 0.0063 mM and (E) 0.0094 mM heparin.
A binding isotherm plot is a graphical representation of changes
in net electrophoretic mobility of an analyte as a function of the
concentration of the additive in the running buffer.
Nonlinear regressions are increasingly being recommended
for accurate and precise determination of binding constants
Ology Science
because of some of the advantages these methods offer [20,21].
However, in a recent review article, it was reported that binding
data obtained by the different linearization approaches and
the nonlinear regression method have been compared in
several studies and found that the values may or may not differ
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
5
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
A: Binding isotherm
significantly [34]. It is anticipated that, with the contemporary
computational power and statistical tools, it is becoming more
convenient to automate estimation of the binding parameters
by using both nonlinear and linear regressions. For ease of
comparing the binding data for the structurally-related heparinderived disaccharides in this study, we chose to use the linearized
equations shown in Table 2.
-0.365
0
0.001
0.002
0.003
0.004
0.005
-0.37
µeff
-0.375
-0.38
The slope of the linear portion of the plot is used to determine
the equilibrium constant (K). It is observed from the graph
that there is generally good linearity at lower concentrations of
heparin (0-0.0010 mM) and therefore this concentration range
was used to generate linearly transformed plots (Figures 5B,
5C and 5D) per the equations shown in Table 2 thus allowing
the determination of the binding constants. Binding constants
were only obtained for linear plots that produced correlation
coefficients (R2) greater than 0.98. The binding constant
calculated from the ACE method (~Kf = 6×105) was in the same
order of magnitude as that obtained from chemical shift-pH
analysis in NMR spectroscopy averaged at ~ 2×105 over values
obtained at different ionic strengths and pH conditions [28]. The
ACE method was therefore adopted to measure and compare the
binding constants for the interaction of histidine binding with
heparin-derived disaccharides.
-0.385
-0.39
[L] (mM)
B: X-reciprocal
-0.022
-0.02
-0.018
-0.016
-0.014
0
-0.012 -0.01
-2000
(µeff-µ0)/[L]
y = -992757x - 24787
R² = 0.9838
-4000
-6000
-8000
-10000
Binding of heparin-derived disaccharides to
histidine
-12000
µeff-µ0
-14000
Table 3 shows the nomenclature and structures of the
twelve commercially available heparin/HS disaccharides. Six
of these disaccharides (IS, IIS, IIIS, IA, IIIA and IH) were
selected for ACE analysis to study binding to histidine. Figure
6 is a representative electropherogram following the injection
of 2.0 mM histidine in a running buffer containing 0.001 mM
disaccharide IIS. The commercially available disaccharides are
obtained by enzymatic depolymerization during which a double
bond is formed with a strong UV-absorption at 232 nm [31].
In our ACE runs, the electrophoretic peaks are monitored at
214 nm and therefore the disaccharides added into the running
buffer do not interfere with detection of neither histidine nor
benzylalcohol. The binding constants were calculated similarly
to the histamine-heparin model and are summarized in Table
4. It should be noted that although the linear equations are not
all equivalent [36,37], the results obtained in this study as still
useful in comparing the relative binding affinities of the heparinderived disaccharides to histidine.
C: Y-reciprocal
0
0
0.000001 0.000002 0.000003 0.000004 0.000005
[L]/(µeff-µ0)
-0.00005
y = -40.999x - 4E-05
R² = 0.9986
-0.0001
-0.00015
-0.0002
-0.00025
[L] (M)
D: Double reciprocal
-40
100000 300000 500000 700000 900000 1100000
-45
1/(µeff-µ0)
-50
-55
y = -4E-05x - 39.835
R² = 0.9949
-60
-65
-70
-75
-80
-85
1/[L]
Figure 5: Graphical plots based on (A) binding isotherm, (B) X-reciprocal, and
(C) Y-reciprocal and (D) double-reciprocal equations.
6
ISSN : 2473-2230
The disaccharide IS is the most sulfated and therefore the
most negatively charged. It makes up almost 50% of heparin
[27] and its binding affinity to histidine was determined to be
approximately 5×104. Whereas the disaccharide IS is trisulfated,
four other disaccharides (IIS, IIIS, IA and IH) each have two
sulfates while the disaccharide IIIA has one sulfate. Compared
to the disaccharide IS, the disaccharide IIS is desulfated at the
C2-O position of the iduronic acid residue and was found to
have a binding constant of ~3×104 which is not significantly
different from that of disaccharide IS. However, the disaccharide
IIIS which is instead desulfated in the C5-O position of the
glucosamine residue and is isomeric with disaccharide IIS
was found to have a binding constant of ~1×105, an order of
magnitude greater than Kf for both disaccharides IS and IIS. The
J Res Anal. (2016) Volume 2 • Issue 4
Ology Science
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
Table 4: Summary of the binding constants for the interaction of heparin-derived disaccharides with histidine obtained using different linearized plots.
Disaccharide
I-S
II-S
III-S
I-A
III-A
I-H
Linear Isotherm
3 × 104
3 × 104
1 × 105
3 × 102
3 × 104
1 × 105
X Reciprocal
5 × 104
5 × 104
1 × 105
N/A
1 × 104
1 × 105
Y Reciprocal
5 × 104
5 × 104
1 × 105
N/A
1 × 104
1 × 105
Double reciprocal
5 × 104
3 × 104
1 × 105
3 × 102
3 × 104
1 × 105
Average value
5 ± 1 × 104
4 ± 1 × 104
1 ± 0 × 105
3 ± 0 × 102
2 ± 1 × 104
1 ± 0 × 105
benzyl alcohol
3.0
2.5
2.0
histidine
1.5
1.0
0.5
0.0
-0.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
3.1
Minutes
Figure 6: ACE electropherogram of 2.0 mM histidine and 2.0 mM benzyl alcohol using a phosphate buffer containing 0.0010 mM disaccharide IIS.
disaccharide IH is desulfated at the C2-N of the glucosamine
residue and was also found to have a Kf of ~1×105.
The data suggest that replacement of the sulfonate group in
the glucosamine with hydrogen resulted in a ten-fold increase
of the binding of the disaccharide unit to histidine. For the
disaccharide IA, however, the sulfonate group of the glucosamine
is replaced with an acetyl group instead of hydrogen at the
C2-N position. This significantly decreased the binding of IA to
histidine (Kf of ~3×102). The disaccharide IIIA is also acetylated
at the C2-N position of the glucosamine but, in addition, it is
desulfated in the C2-O position of the iduronic acid residue.
Surprisingly, this did not seem to change its binding to histidine
(Kf of ~2×104) compared to disaccharide IS. Knowledge of these
binding interactions at the disaccharide level can provide insight
into specific influence of iduronate and glycosamine substitution
in protein binding [32].
Conclusion
The results presented demonstrate the influence of
substitution patterns at the disaccharide level has on heparin
binding to specific compounds and can find relevance in studying
interactions of GAGs to proteins. A few studies have reported
the use of ACE for the interaction of GAGs with proteins of
interest [11]. In many cases, there are limited amounts of protein
Ology Science
due to difficulties in purification from biological matrices or
prohibitive costs of the purified protein. The ACE method offers
the advantage of low sample consumption allowing studies of
interactions of GAGs with such proteins. We have undertaken
preliminary studies for the binding interactions of GAGs to the
chemokine CXCL14 using ACE.
Although the linear transformations in ACE generally permit
the determination of binding data for 1:1 stochiometry, there
are differential equations that can be used to describe the analyte
migration behavior where a different stoichiometry is involved.
Affinity interaction involving 2:1 stochiometry and the analytical
solutions of corresponding binding constants have been reported
[5]. The authors also offered suggestions that can be adapted
for cases where a different stoichiometries are involved thus
expanding the potential of ACE in studying binding interactions
of GAGs to proteins. The utility of ACE to estimate accurate
binding constants can be enhanced by using computational tools
to automate determination of the binding parameters using both
nonlinear and linear regressions.
Acknowledgement:
The author gratefully acknowledges the Research Corporation
for Science Advancement for the initial funding that facilitated
this research work.
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
7
Citation: Schrader A, Feltes M, Duong M, Korir AK. Affinity capillary electrophoresis for the estimation of binding constants and comparison of binding
interactions of heparin-derived disaccharides to histidine. J Res Anal. 2016; 2(4): 113-121.
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J Res Anal. (2016) Volume 2 • Issue 4
Ology Science