Faculty of Technology and Science Chemistry Karlstad University Studies 2006:43 Maria E. Bohlin Capillary electrophoresis of b 2 -glycoprotein I Method development and binding studies
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Karlstad University StudiesISSN 1403-8099
ISBN 91-7063-074-7
Faculty of Technology and Science Chemistry
Karlstad University Studies2006:43
Maria E. Bohlin
Capillary electrophoresis
of b2-glycoprotein I
Capillary electrophoresis of b2-glycoprotein I
Human b2-glycoprotein I (b2gpI) is a phospholipid- and heparin-binding plasma glyco-protein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against b2gpI. With the final goal of assessing autoantibody influence on binding interactions of b2gpI we have developed capillary electrophoresis (CE)-based assays for interactions of ligands with b2gpI. The analysis of peptides and proteins by CE is desirable due to low sample con-sumption and possibilities for non-denaturing yet highly effective separations. However, adsorption at the inner surfaces of fused silica capillaries is detrimental to such analyses. This phenomenon is especially pronounced in the analysis of basic proteins and pro-teins containing exposed positively charged domains. The problem with these analytes is that they stick to the wall, which is negatively charged at neutral pH. To avoid wall interactions numerous procedures have been devised. Here, some of these methods were evaluated. Capillaries permanently coated with acrylamide and dimethylacryl-amide did not permit recovery of this basic protein (pI about 8) at neutral pH, unless the negatively charged ligand heparin was added to mobilize the protein. However, we found the pH hysteresis behavior of fused silica surfaces useful in avoiding b2gpI adsorption problems. The protonated surface after an acid pretreatment counteracted protein adsorption efficiently. This simple approach made estimates of heparin-b2gpI interactions possible and the principle was shown also to work for detection of b2gpI binding to anionic phospholipids. We also investigated the effects of different pretreat-ment techniques on the electroosmotic flow and the rate of the deprotonation process and show the more general utility of this approach for CE of various basic proteins in plain silica capillaries at neutral pH. The realization of a successful generic approach to facilitate protein analysis by CE is an important foundation for carrying out functional studies on b2gpI and other basic proteins.
Method development and binding studies
Karlstad University Studies
2006:43
Maria E. Bohlin
Capillary electrophoresis
of b2-glycoprotein I
Method development and binding studies
Maria E. Bohlin. Capillary electrophoresis of b2-glycoprotein I: Method development and binding studies
Licentiate thesis
Karlstad University Studies 2006:43ISSN 1403-8099 ISBN 91-7063-074-7
© The author
Distribution:Karlstad UniversityFaculty of Technology and Science Chemistry SE-651 88 KARLSTADSWEDEN+46 54-700 10 00
www.kau.se
Printed at: Universitetstryckeriet, Karlstad 2006
Abstract Human β2-glycoprotein I (β2gpI) is a phospholipid- and heparin-binding plasma
glycoprotein involved in autoimmune diseases characterized by blood clotting
disturbances (thrombosis) together with the occurrence of autoantibodies against
β2gpI. With the final goal of assessing autoantibody influence on binding
interactions of β2gpI we have developed capillary electrophoresis (CE)-based
assays for interactions of ligands with β2gpI. The analysis of peptides and proteins
by CE is desirable due to low sample consumption and possibilities for non-
denaturing yet highly effective separations. However, adsorption at the inner
surfaces of fused silica capillaries is detrimental to such analyses. This phenomenon
is especially pronounced in the analysis of basic proteins and proteins containing
exposed positively charged domains. The problem with these analytes is that they
stick to the wall, which is negatively charged at neutral pH. To avoid wall
interactions numerous procedures have been devised. Here, some of these
methods were evaluated. Capillaries permanently coated with acrylamide and
dimethylacrylamide did not permit recovery of this basic protein (pI about 8) at
neutral pH, unless the negatively charged ligand heparin was added to mobilize the
protein. However, we found the pH hysteresis behavior of fused silica surfaces
useful in avoiding β2gpI adsorption problems. The protonated surface after an acid
pretreatment counteracted protein adsorption efficiently. This simple approach
made estimates of heparin-β2gpI interactions possible and the principle was shown
also to work for detection of β2gpI binding to anionic phospholipids. We also
investigated the effects of different pretreatment techniques on the electroosmotic
flow and the rate of the deprotonation process and show the more general utility
of this approach for CE of various basic proteins in plain silica capillaries at neutral
pH. The realization of a successful generic approach to facilitate protein analysis by
CE is an important foundation for carrying out functional studies on β2gpI and
other basic proteins.
2
List of abbreviations α-CTrg α-chymotrypsinogen AA acrylamide ACE affinity capillary electrophoresis
β2gpI β2-glycoprotein I CE capillary electrophoresis cyt c cytochrome c DMA dimethylacrylamide ELISA enzyme-linked immunosorbent assay EOF electroosmotic flow KD equilibrium dissociation constant (binding constant) koff dissociation rate constant kon association rate constant ld capillary length to detector LIF laser-induced fluorescence Lys lysozyme Mr molecular weight n number of measurements pI isoelectic point POPC phosphatidylcholine PS phosphatidylserine RnA ribonuclease A RSD relative standard deviation tm migration time tM migration time of marker molecule Trg trypsinogen
∆µ change in electrophoretic mobility
∆µmax change in electrophoretic mobility at saturating ligand concentration µEOF electrophoretic mobility of the electroosmotic flow
υEOF velocity of the electroosmotic flow µep electrophoretic mobility
υep electrophoretic velocity µapp apparent mobility
ζ zeta potential
ε dielectric constant
η viscosity E electric field strength
3
List of papers I. Capillary electrophoresis-based analysis of phospholipid and
glycosaminoglycan binding by human ββββ2-glycoprotein I M.E. Bohlin, E. Kogutowska, L.G. Blomberg, N.H.H. Heegaard, J. Chromatogr. A 1059 (2004) 215-222.
II. Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries M.E. Bohlin, L.G. Blomberg, N.H.H. Heegaard, Electrophoresis 26 (2005) 4043-4049.
4
Table of contents
1. Aim of study ................................................................................................. 5
2. β2-glycoprotein I........................................................................................... 6
3. Capillary electrophoresis.............................................................................. 7
4. Analytical recovery of β2gpI ....................................................................... 10
4.1. Coated capillaries (Paper I) .............................................................................................................10
4.2. Mobilization using ligand addition (Paper I)................................................................................11
5. The pH hysteresis effect ............................................................................ 13
5.1. The slow equilibrium .......................................................................................................................13
5.2. Dependency of the EOF on the pretreatment method (Paper II)...........................................14
5.3. Utilization of the pH hysteresis effect for separation of some basic proteins (Paper II).....15
6. Migration shift affinity capillary electrophoresis ...................................... 17
6.1. Binding experiments with heparin (Paper I) ................................................................................20
6.2. Binding experiments with phospholipids (Paper I) ....................................................................22
7. Future perspective...................................................................................... 24
References...................................................................................................... 26
5
1. Aim of study
The purpose of this study is to establish microscale, solution phase quantitative
binding assays for the characterization of interactions between the human plasma
protein β2-glycoprotein I (β2gpI) and anionic phospholipids and autoantibodies.
This will help elucidate the mechanisms involved in the increased thrombosis risk
associated with the presence of circulating autoantibodies against β2gpI. Capillary
electrophoresis (CE) is an analytical technique that provides simultaneous analysis
of low- and high molecular weight compounds in solution under non-denaturing
conditions, which is important for molecular interaction studies where active
species are required. CE also offers high efficiency, high resolution and it is easy to
automate with on-line detection. The sample consumption is low, the speed of
analysis is high and the equipment is simple. Therefore, CE is a promising
technique for studying molecular interactions. One problem though, is that protein
analytes often exhibit recovery problems due to interactions with the inner surface
of the fused-silica capillaries. Suppression of such interactions is therefore often a
requirement when performing protein studies with CE, especially at neutral and
basic pHs. For the establishment of a CE-based binding assay, methods for
suppression of solute-wall interactions had to be developed.
6
2. β2-glycoprotein I
Human β2gpI, Fig. 1, is a basic plasma protein (pI about 8) with a molecular weight
of approximately 55 kDa [1]. It consists of a single polypeptide chain folded in five
domains. Domains I and II provide binding sites for thrombosis-associated
antibodies [2,3], domains III and IV are heavily glycosylated [1,4] while domain V
shows affinity for negatively charged phospholipids [2,5-12].
Hydrophobic loop
Positive region
I
II
III
IV
V
Figure 1. A ribbon structure of human β2gpI based on crystallography [12].
The physiological role of β2gpI is not completely known, except that it is involved
in the function of the coagulation cascade and that anti-β2gpI autoantibodies are
associated with the thrombophilic events of the anti-phospholipid syndrome.
Different functions of β2gpI have been discussed in the literature. It has been
shown that β2gpI binds to DNA [13], mitochondria [14], lipoproteins [15], platelets
[16] and heparin [17]. β2gpI likely plays a primary role in mediating the clearance of
liposomes and foreign particles [18] as well as being an anticoagulant [19]. It is one
of the key antigens in the autoimmune disease antiphospholipid syndrome [2,11].
Circulating autoantibodies against β2gpI are strongly associated with thrombosis of
patients suffering from antiphospholipid syndrome [3,12]. The binding of β2gpI to
7
negatively charged phospholipids in one end of the protein is required for the
binding of antibodies to β2gpI at the other end, probably after a conformational
change in β2gpI [3,17]. The docking mechanism to a membrane is thought to
involve two steps: electrostatic interaction between positive charges on domain V
of β2gpI and negatively charged head groups of phospholipids, followed by an
insertion of the surface exposed hydrophobic loop of β2gpI into the hydrophobic
part of the membrane [2,5,11,12,20]. When bound to a membrane, domains I and
II are positioned far away into the blood, which enables smooth interactions of
these domains with antibodies [2]. Domains III and IV are heavily glycosylated,
functioning either as protected bridging domains [11] or as antibody recognition
sites [3]. Clearly, interaction studies of β2gpI with different components from
blood would be an important tool in the understanding of β2gpI mechanisms in
health and disease.
3. Capillary electrophoresis
Electrophoresis is the movement of ions in an electric field. Separation is based on
the ability of charged molecules to migrate at different velocities in an electric field.
A scheme of a CE instrument is shown in Fig. 2. The basic parts of a CE
instrument are the separation capillary, buffer reservoirs, a high voltage supply and
a detector. The capillary is typically made of fused-silica with an internal diameter
of 20-100 µm and a length of 40-100 cm. This is filled with an electrolyte buffer
connected to a high voltage supply. When an electric field is applied, ions migrate
towards the electrode of opposite charge unless the electroosmotic flow (EOF)
pulls the ions in the opposite direction (cf. below). The ions are commonly detected
by UV absorption or laser-induced fluorescence (LIF) online. The detector is
normally placed near the cathode end of the capillary. The use of more
information-rich detectors such as mass spectrometry (MS) is of great value and
various schemes have been developed [21].
8
Figure 2. Scheme of a capillary electrophoresis instrument.
The surface of the capillary wall contains ionizable silanol groups, which becomes
deprotonated at pH above approximately 2:
- +SiOH SiO +H� (1)
This creates negative charges at the capillary wall which attract positively charged
buffer ions, Fig. 3. An electrical double layer is created consisting of a rigid layer of
positive ions (Stern layer) and a mobile diffuse layer with an excess of positive
charges. When an electric field is applied, the diffuse part of the double layer
migrates towards the cathode. Because the diffuse layer contains an excess of
positive buffer ions, a net flow is formed which drags the buffer solution towards
the cathode. This flow, called the electroosmotic flow (EOF), has a pluglike profile
and this leads to very high separation efficiencies, i.e. narrow peaks. Due to the
EOF both positive and negative analytes can be analyzed in a single run.
The velocity of the EOF, υEOF, depends on the electric field strength, E, and the
mobility of the EOF, µEOF [22]:
EOF EOFυ =µ E (2)
The µEOF in turn is dependent on the zeta potential, ζ, the dielectric constant, ε,
and inversely dependent on the viscosity of the buffer solution, η:
EOF
ζεµ =
η (3)
The zeta potential is the potential difference between the outer boundary of the
Stern layer and the free solution at an infinite distance [23]. As the pH is increased,
more silanol groups become deprotonated and hence the surface becomes more
negatively charged, attracting even more positive buffer ions and a higher zeta
9
potential is obtained. Thus, a higher EOF is obtained. Also, the viscosity,
temperature and the ionic strength of the buffer influence the velocity of the EOF.
Figure 3. The electrical double layer and the resulting electroosmotic flow.
The velocity of a migrating molecule, υep, depends on the electrophoretic mobility,
µep, of the molecule and of the applied electric field strength [22,24]:
ep epυ =µ E (4)
The µep in turn is dependent on the charge of the molecule, q, and inversely
dependent on η and the hydrodynamic radius, r, of the molecule:
ep
qµ =
6πηr (5)
The electrophoretic mobility is approximately dependent on the charge to mass
ratio. In a CE separation, the µEOF and the µep both act at the same time, which
gives the molecule an apparent mobility, µapp:
app EOF epµ = µ + µ (6)
Under standard conditions with the cathode at the detector end, positively charged
molecules will have two positive contributions to the µapp and will therefore have
the shortest migration times, while negatively charged molecules have longer
migration times. However, negatively charged species only pass the detector if µEOF exceeds µep. This is usually the case.
10
Charged molecules can be separated using the separation technique described
above. Neutral molecules have no electrophoretic mobility because they do not
have a charge and will therefore move only with the EOF. However, by addition
of a surfactant forming micelles to the electrophoresis buffer, a pseudostationary
phase is formed. This phase provides a mechanism for retention of neutral
analytes. Neutral molecules can partition within the micelle and achieve an
apparent mobility which will be determined by the electrophoretic mobility of the
micelle, µEOF and the degree of partitioning. The approach was introduced by
Terabe et al. [25] in 1984. Additives such as organic solvents, urea, salts,
cyclodextrins and other chiral selectors can be added both alone and in different
combinations. With this approach very high selectivities can be achieved.
4. Analytical recovery of β2gpI
Purified β2gpI was not recovered in CE experiments using plain fused-silica
capillaries and neutral pH buffers (data not shown). A simple way to overcome
capillary adsorption problems is to perform a running buffer pH scan. This may
show that CE analysis is feasible at a slightly different pH value, still acceptable for
subsequent binding studies. However, pH values close to physiological pH were
found to be incompatible with the recovery of β2gpI.
4.1. Coated capillaries (Paper I)
As mentioned earlier, a major virtue of CE is the ability to achieve high separation
efficiencies of both high- and low-molecular weight compounds. Ideally, the only
contribution to band broadening would be diffusion and analytes of high
molecular weight, such as proteins, have a low diffusion coefficient and therefore
tend to be separated as narrow zones. However, in practice, other factors often
contribute to band broadening in CE. Analyte adsorption at the negatively charged
wall of the fused-silica capillary can be a major source of band broadening and lead
to tailing, as well as to irreproducible peak migration times and disappearance of
peaks. This charge-dependent adsorption can be minimized by the use of coated
capillaries [26,27]. A pioneering work within this field was made by Hjertén, who
stated that in ideal electrophoresis in free solution neither electroosmosis nor
adsorption of solutes onto the capillary wall should occur [28,29]. He described a
procedure to permanently coat capillaries and could thereby diminish both of these
phenomena. Today numerous types of coatings are commercially available. Coating
11
of capillaries may be permanent, e.g. by synthesizing a polymer film in situ on the
capillary wall, or dynamic, i.e. by using an additive in the electrophoresis buffer that
covers the wall reversibly and/or shields the proteins in the solution from the wall
by additive-protein interactions [30,31]. However, additives may influence analytes,
e.g. denature proteins and can therefore be difficult to use for interaction studies
where an active protein is desired during the separation. Dynamic coating is simple
but complicates MS detection. For the analysis of β2gpI compatibility with MS
detection is desired. The preparation of permanently coated capillaries is somewhat
laborious and prone to variability, but for a given capillary permanent coating
stable performance for quite a long time [28,32-34] can usually be expected. Other
methods to avoid wall interactions include the use of extreme pH or high salt
concentrations, but are not suitable options for interaction studies at near
physiological conditions.
An attempt to suppress wall interactions for β2gpI was carried out by the use of
permanently coated capillaries in paper I. Bonded dimethylacrylamide (DMA) as a
coating has been shown to provide good performance for the separation of a
selection of basic proteins [32]. Therefore, DMA coated capillaries were prepared
and used for electrophoresis of β2gpI at physiological pH. However, for β2gpI
poor recovery and reproducibility was obtained (data not shown). A more
hydrophilic polymer, acrylamide (AA) was implemented instead and the method
described by Hjertén (cf. above) was used [28]. With AA-coated capillaries at
physiological pH we found partial but inconsistent recovery of β2gpI (data not
shown). The reason for the bad recovery is not known, but β2gpI has a
hydrophobic region exposed to the solvent (see Fig. 1) and has well-known lipid
binding capabilities and thus affinity for hydrophobic surfaces. The higher
adsorptivity of β2gpI on the DMA-coating compared to the AA-coating may be
due to the somewhat more hydrophobic character of DMA. Another explanation
could be that the adsorption problems are due to incomplete coverage of the
surface and to the presence of precipitated proteins that originate from previous
runs [34].
4.2. Mobilization using ligand addition (Paper I)
Another approach to overcome wall interactions may be to add a known ligand to
the running buffer and thus confer a suitable charge to the analyte, e.g. a negative
12
charge upon complexation with basic analytes. The CE analysis of human
lactoferrin has been shown to require the addition of heparin [35]. The protein
β2gpI is, like lactoferrin, a heparin-binding protein and in paper I heparin was
added to the electrophoresis buffer and electrophoresis in uncoated and coated
capillaries were performed, Fig. 4. Here, the analyte peak begins to emerge when
heparin is added to the electrophoresis buffer at more than 0.1-0.2 mg/mL. Thus,
β2gpI was recovered using complex formation with heparin in both uncoated and
coated capillaries. However, the presence of interactions with a soluble ligand as
well as with the wall makes ligand binding analyses complicated.
0 5 10 15
0.00
Time (min)
0 mg/ml Heparin
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.50 mg/ml
1.0 mg/ml
2.0 mg/ml
4.0 mg/ml
5.0 mg/ml
A 200 nm
(A)M β2gpI
0 mg/ml Heparin
0.05 mg/ml
0.2 mg/ml
0.1 mg/ml
1.0 mg/ml
2.0 mg/ml
0.4 mg/ml
0.5 mg/ml
5.0 mg/ml
4.0 mg/ml
3.0 mg/ml
Time (min)10 20 30 40 50
mAU
0
50
100
150
200
250
300
0.01(B)
M β2gpI
0 Figure 4. Mobilization of β2gpI by affinity-CE with bovine lung heparin (BLH) added to the electrophoresis buffer. (A) Uncoated fused silica capillary, 50 cm to detector (57 cm total length); voltage 15 kV; temperature 20 °C. Electrophoresis buffer: 0.13 M Tris base, 0.5 M glycine pH 8.6 with added BLH at concentrations given in figure. (B) Acrylamide coated capillary, 32 cm to detector (40 cm total length); constant current conditions: -120 µA; temperature 22 °C. Electrophoresis buffer: 0.1 M phosphate pH 7.4 with added BLH at concentrations given in figure.
13
5. The pH hysteresis effect
5.1. The slow equilibrium
As described in section 3, the electroosmotic mobility depends on the surface
charge density at the silica wall. As the pH is raised, the equilibrium between
protonated and deprotonated silanol (Eq. 1) is shifted to the right and the number
of negative charges at the surface of the silica is increased. In 1990, Lambert and
Middleton measured the µEOF as a function of pH with both alkaline and acidic
pretreatments of the fused silica capillary [36] and found that the values of µEOF
were consistently lower after an acidic pretreatment than the values of µEOF
obtained after an alkaline pretreatment. They also discovered that the equilibration
of the surface charge on the fused silica surface appears to be a relatively slow
phenomenon, especially at intermediate pH. The physicochemical basis of this
phenomenon is not entirely clear, but a porous gel model of silica-solution
interface has been suggested by Huang as explanation [37]. At low pHs a gel layer
is formed close to the silica surface due to hydrolysis of SiO2, but Churaev et al.
[38] noted that a gel layer is formed also at neutral pH. When a porous gel layer is
formed at the silica-solution interface, the magnitude of the zeta potential is
reduced by counterions that are trapped in this gel layer. This would change the
electrokinetic behavior of the capillary and reduce the EOF. An alkaline
pretreatment would dissolve this gel layer and maintain a constant value of the zeta
potential, and such a pretreatment is a general recommendation when performing
CE. However, after an acidic pretreatment of the capillary fewer deprotonated
silanol groups are available and it would be possible to suppress charge dependent
wall interactions. Due to the slow deprotonation process this surface would stay
the same also at subsequent CE analysis at neutral pH. The pH hysteresis effect
has earlier been used to manipulate the µEOF [39].
In paper I, the acidic pretreatment approach was successful in eliminating recovery
problems in the CE analysis of β2gpI (Fig. 1 C-D in Paper I). The capillary was
thus preconditioned for 3 min with 0.1 M HCl and 1 min with the electrophoresis
buffer prior to each run and the reproducibility of the migration times and peak
areas was acceptable (RSD=5.7 and 13.6 % respectively). After establishing the
utility of the pH hysteresis effect, this approach was used for the binding studies of
β2gpI to heparin and anionic liposomes (cf. section 6). With this approach it was
14
possible to avoid analyte-wall interactions at the same time as physiological binding
experiments were feasible and meaningful.
5.2. Dependency of the EOF on the pretreatment method (Paper II)
The pretreatment approach was further characterized to investigate how to achieve
as much suppression as possible and to investigate the rate of the deprotonation
process in our buffer system. The µEOF was used as a measure of the surface charge
density and measured after different pretreatments of the capillary. The
pretreatment time, the type of acid, and the concentration of the acidic solution
were varied in an attempt to find the conditions giving lowest value of µEOF. The
type of acid had an impact on the degree of protonation of the silanol groups of
the fused-silica capillary. The stronger acid used, the stronger the magnitude of
suppression of the µEOF. HCl was the strongest acid tested and the molarity of HCl
showed a direct influence on the degree of suppression. Different protocols for
pretreatment were investigated and 35 measurements of µEOF after each
pretreatment were performed at pH 7.4 and the results are shown in Fig. 5. µEOF
(10-8 m2 V-1s-1)
A
B
C
D
E
4.0
4.5
5.0
5.5
6.0
6.5
0 5 10 15 20 25 30 35
Measurement number
Figure 5. Mobility of the electroosmotic flow (µEOF) after different pretreatments in fused-silica capillaries. (A) Capillary pretreated for 1 h with 1 M HCl and with intermediary 1 min 1 M HCl-washes between each measurement; (B) intermediary 1 min 1 M HCl-washes between each measurement; (C) pretreatment with 1 h wash with 1 M HCl but no intermediary washes between measurements; (D) capillary left for 15 h after being filled with 1 M HCl, no intermediary washes; (E) prolonged prewash with NaOH and intermediary washes with 1 M NaOH between each measurement.
15
The acidic pretreatments were compared to the generally recommended alkaline
pretreatment (stars in Fig. 5). Lower values of µEOF were obtained with acidic
pretreatment than with alkaline pretreatment, signifying that the approach does
result in fewer deprotonated silanol groups at a subsequent neutral pH. The rinse
time had only a minor impact on the suppression of µEOF. Thus, a 1 min
pretreatment with 1 M HCl was found to be sufficient to effectively suppress the
µEOF. The reproducibility was high for this procedure (RSD= 0.8 and 1.1 % (n=35)
for closed and opened circles in Fig. 5 respectively.) It is important to preserve the
protonated surface for as long time as the analysis is going on, i.e. the conditions
should be kept relatively stable. Consecutive measurements of µEOF at pH 7.4
without intermediary washes after acidic pretreatment of the capillary showed that
the surface remains relatively uncharged during the time of a routine CE separation
of 10-30 min. The procedure chosen for pretreatment was: 1 min wash with 1 M
HCl and 2 min wash with electrophoresis buffer.
About the same time as paper II was published, another research group published
their investigation on the same phenomenon [40]. They have drawn slightly
different conclusions about the pretreatment procedure, but the principle is the
same and in this study it was used for suppression of µEOF, while the applicability
to protein separation was not examined.
5.3. Utilization of the pH hysteresis effect for separation of some basic
proteins (Paper II)
Because the approach with acidic pretreatment was promising for the CE analysis
of β2gpI, the procedure could be useful for CE analysis of other proteins as well.
The established pretreatment procedure needed for efficient pretreatment of the
capillary was tested again for β2gpI and a higher reproducibility for the migration
time was obtained now, Fig. 6A (RSD=2.0 %, n=10) compared to the results in
paper I (cf. above). A comparison with an alkaline preconditioned capillary was
performed, Fig. 6B, and with this capillary poor recovery of β2gpI was in
accordance with our earlier studies.
16
(A) (B)
Time (min)10
A200 nm
0
20
40
60
80
100
120
Time (min)10 20 30
A200 nm
0
10
20
30
40
50
β2gpI
DMSO DMSO
Figure 6. Reproducibility of β2gpI analysis. Ten consecutive analyses of β2gpI on (A) HCl- and (B) NaOH-pretreated fused-silica capillary. Pretreatment: (A) 1 min 1 M HCl, 2 min electrophoresis buffer; (B) 1 min 1 M NaOH, 2 min electrophoresis buffer. Electrophoresis buffer: 50 mM phosphate buffer pH 7.4.
A mixture of basic protein standards was then subjected to analysis at pH 4.8 in
differently pretreated capillaries, Fig. 7. The acidic pretreatment offered
considerably better peak shapes of these proteins compared to the alkaline
pretreatment where the recovery problem is obvious. The RSD of the migration
times for the basic proteins in Fig. 7 ranged from 0.8-1.1 % (n=8) [Table 1 in
paper II]. Hjertén and coworkers discussed that the accumulation of proteins at the
capillary wall can be due to precipitation of proteins by free moving non-cross-
linked polyacrylamide chains at the wall [34]. These precipitates were efficiently
removed by HCl rinsing between runs. The possibility that the acidic pretreatment
effect in our case may be due to better rinsing of the silica surface is addressed in
Fig. 7B. Here, the capillary is rinsed with HCl prior to an alkaline preconditioning.
The results show that for the protein test mixture this is not the case, at least not
under the conditions used.
The approach with acidic pretreatment of the capillary is a simple and effective way
of suppressing wall interactions at the same time as facilitating subsequent
interaction studies at physiological pH. It should also be compatible with MS
detection, which is potentially a very rewarding combination with CE.
17
Time (min)10 20
A200 nm
0
50
100
150
200
250
(A)
(B)
(C)
lys cyt c
RnA
Trg
α-CTrg
Figure 7. Electropherograms of basic protein standards on differently pretreated fused-silica capillaries. Pretreatment: (A) 1 min 1 M HCl, 2 min electrophoresis buffer, (B) 1 min HCl, 1 min 1 M NaOH, 2 min electrophoresis buffer, (C)1 min 1 M NaOH, 2 min electrophoresis buffer. Electrophoresis buffer: 50 mM acetate/Tris pH 4.8.
6. Migration shift affinity capillary electrophoresis
Molecular interactions are present everywhere in biological systems [41]. Signal
transduction, enzyme-substrate binding, regulation of enzyme activity and
immunoreactions are some examples. Identification and characterization of such
interactions are important in order to elucidate biological mechanisms. Further,
proteins interfere with drugs in different ways. A specific and strong binding of
drugs (slow dissociation) to receptor proteins offers a pharmacological effect with
a prolonged duration, while strong binding to plasma proteins prevents the drug
from diffusing from the blood to the site where the pharmacological effect takes
place [42]. Equilibrium binding constants provide a measure of the affinity of a
ligand to a substrate molecule [43,44]. The equilibrium for a 1:1 molecular
reversible binding interaction [45] can be described by Eq. 7:
18
off
on
k
k
RL R + L���⇀↽��� (7)
where RL is the complex between the receptor R and ligand L molecules and koff
and kon are the dissociation and association rate constants. The equilibrium
dissociation constant, KD, (referred to as binding constant hereafter) depends on
the kinetics of dissociation/association:
on
off
Dk
kK = (8)
Strong interactions are characterized by slowly dissociating complexes, i.e. low
values of koff, while weak interactions are characterized by relatively fast
dissociation, i.e. high values of koff [46,47]. Measuring the equilibrium
concentrations of the interacting species, KD can be determined according to:
[ ] [ ][ ]RL
LRK D = (9)
Common methods for determination of binding constants include equilibrium
dialysis, gel filtration, filter assays, spectroscopy [48], calorimetry, surface plasmon
resonance techniques, chromatography [49] and enzyme-linked immunosorbent
assays (ELISA) [50]. Capillary electrophoresis is a promising technique for studying
molecular interactions. As mentioned above, the benefits of CE for this purpose
are many: fast separations, high resolution, ease of automation, on-line detection,
low sample consumption and the possibility to use non-denaturing conditions in
solution (i.e. immobilization of any species is not required) at physiological pH and
ionic strength. Labeling of ligands is no requirement in this technique, as compared
to e.g. ELISA, facilitating analysis of non-modified ligands.
In migration shift ACE one of the species, normally the ligand, is added to the
electrophoresis buffer and the other, the receptor, is injected as a narrow sample
zone. During electrophoresis, if interaction of the species occurs, the complex will
experience another electrophoretic mobility than the receptor molecule, due to a
change in charge and/or size. This will lead to a shift in migration time as
compared to an electrophoresis run without ligand added. The observed migration
time of the peak will be a weighted average of the ongoing associations and
dissociations. The time spent as a complex will depend on the concentration of the
ligand. The more ligand added, the more time the receptor will be in the
complexed form and a greater change in migration time will be observed.
19
There are some requirements that must be fulfilled for the application of this
approach. First, there must be a change in electrophoretic mobility upon
complexation in order to get a response [51]. The mobility change must be due
specifically to the association of receptor and ligand [49]. Second, the analysis time
must be long enough to achieve equilibrium conditions [46,49,51-53]. This is
practical only for low-to-intermediate affinity interactions with fast on-off-kinetics
[46]. More stable complexes are normally analyzed after pre-equilibration [46],
where the equilibrium is not sustained during electrophoresis, but the analysis time
is short enough to avoid dissociation of the complex. Also, for detection purpose
sufficient concentrations of both free ligand and complex should be present [51].
Equations for the determination of binding constants have been described and
used by several researchers [35,48,49,51,54-56]. The general rectangular hyperbolic
binding isotherm for the simple 1:1 binding [57] is:
dxy=
f + ex (10)
where y is the dependent variable, x is the independent variable (free ligand
concentration), and d, e and f are constants or parameters [51]. In affinity
electrophoresis, y is the change in electrophoretic mobility ∆µ. Equation 10 can be
rearranged to [48,58]:
max D
∆µ∆µ = ∆µ - K
[L] (11)
where ∆µmax is the change in electrophoretic mobility at saturating ligand
concentration. The electrophoretic mobility, µ, is inversely proportional to the
migration time of the analyte according to [24,49]:
Et
lµ
m
d= (12)
where ld is the capillary length to the detector, tm the migration time of the analyte
and E the electric field strength. The difference in the inverse peak appearance
time can therefore be used as a measure of ∆µ when capillary dimensions, buffer
conditions (except for ligand addition), current, temperature and electric field
strength are kept constant [58]. A non-interacting marker should be added to the
sample, to ensure that shifts in migration time are due specifically to binding of
20
ligand and to correct for possible changes in EOF [49,54,59]. The difference in
corrected peak appearance times, ∆(1/t) can be calculated according to:
0mMmM t
1
t
1
t
1
t
1
t
1∆
−−
−=
+L
(13)
Here tM is the migration time of the marker and the subscripts denote experiments
with, +L, and without 0, ligand added. A direct plot of ∆(1/t) against [L] should
show a saturable dependence (according to Eq. 11) [49]. The binding constant can
be calculated using a linearization method, but an estimation using non-linear curve
fitting is less biased [45].
6.1. Binding experiments with heparin (Paper I)
Binding studies with the negatively charged heparin was performed by adding
different amounts of the ligand to the electrophoresis buffer. Binding of β2gpI to
heparin caused a migration shift to a longer migration time compared with the
neutral marker Fig. 8A. The analyte peak was split in three not fully separated
peaks (labeled I, II and III in the figure) upon addition of heparin. This was also
seen in experiments in coated capillaries (Fig. 4B). Since the first peak (labeled I in
Fig. 8A) showed pronounced broadening at higher heparin concentrations,
quantitative binding data was difficult to obtain for this fraction. Also, at heparin
concentrations above 3 mg/mL the two last peaks (II and III) started to co-
migrate. The quantitative analysis was therefore restricted to data from analyses
below 3 mg/mL heparin. To extract binding data the average values of three
replicate runs for peaks II and III were plotted against the heparin concentration,
Fig. 8B. A one-binding site hyperbola function was fitted to each data set (R2> 0.9
in both cases) and binding constants of 0.73 mg/mL (49 µM) and 0.23 mg/mL (15
µM) for peaks II and III respectively were obtained. These values are only
estimates due to uncertainties in defining the precise peak positions and because
the binding isotherms are covered only partly. However, the binding constant for
peak II agrees with the binding constant estimated by Guerin et al. (43 µM) [60].
The results illustrate the unique capability of CE to fractionate heterogeneous
analyte species at the same time as qualitatively and quantitatively probing for their
ligand binding characteristics.
21
5 mg/ml
2.5 mg/ml
1.0 mg/ml
0.5 mg/ml
0.05 mg/ml
0 mg/ml Heparin
(A)
Time (min)
II
III
[Heparin] (mg/ml)0 1 2 3
0.01
0.02
0.03
0.04
0.05
0.00
∆ 1
/t (
min
-1)
(B)
Figure 8. Interaction of β2gpI with heparin. (A) Electropherograms of β2gpI. Conditions: constant current conditions: 120 µA; temperature 22 °C. Electrophoresis buffer: 0.1 M phosphate pH 7.4 with added BLH in the concentrations given in figure. Fractions of β2gpI (labeled I, II and III) were resolved. (B) Graph
showing ∆1/t (average + SD of triplicate experiments) as a function of heparin concentration for peaks II and III (see A) up to 2.5 mg/mL added ligand. Non-linear curve fitting using a one binding site hyperbola as a model gave the binding isotherms shown (R
2>0.9 for both).
22
The multiple peaks of β2gpI-species that were resolved upon addition of heparin
could be caused by several factors. There could be contaminating proteins,
different conformations of β2gpI and/or structural variants of β2gpI that are not
resolved without increases in selectivity. It could also be that heparin itself is
heterogeneous with different binding affinity to β2gpI or that the different
glycoforms [61] of β2gpI have different ligand binding characteristics.
Supplementary studies, e.g. with MS detection and deglycosylated β2gpI, will be
necessary to understand the causes of the splitting of the peaks.
6.2. Binding experiments with phospholipids (Paper I)
Biological membranes define the external boundary of cells and regulate the
molecular traffic across that boundary, as well as promoting signaling transduction
and cell-to-cell communication [62]. Phospholipids are a major constituent in these
membranes. Phospholipids consist of a hydrophilic head group and a hydrophobic
tail. When suspended in aqueous solutions phospholipids spontaneously form
micelles, lipid bilayers or spherical vesicles (liposomes). Liposomes are the most
stable form of phospholipids in aqueous enviroments [62] and are widely used as
models for biological membranes [44]. Natural constituents of membranes, such as
cholesterols, lipids, proteins and carbohydrates, can be incorporated in liposomes.
The protein β2gpI has affinity for specific anionic phospholipids, e.g.
phosphatidylserine (PS), phosphatidic acid and cardiolipin, but less affinity for
other phospholipids [2,7,11,18,63]. Chonn et al. suggested that β2gpI acts as a
mediator in the clearance of apoptotic cells and foreign particles [18].
The possibility of using CE for detecting and characterizing these interactions was
examined in paper I. The zwitterionic phospholipid phosphatidylcholine (POPC),
which has zero net charge at neutral pH, and the anionic phospholipid PS were
used in the study. Large unilamellar vesicles composed of different ratios of POPC
and PS but with the same total concentration of phospholipid were prepared
according to the procedure described by Wiedmer and co-workers [64-67]. The
partial filling technique, where the liposomes were injected as a discrete sample
plug, had to be used. This was because the colloidal liposomes scatter light and
thus caused detection problems. Liposomes were injected prior to β2gpI to ensure
mixing of sample and liposomal zones. The resulting electropherograms are
showed in Fig. 9.
23
100
0
Time (min)0 5 10 15 20
100
0
0
100
0
100
0
100
0
100
0
100
mAU
0
100
M
M
M
M
M
M
M
M
β2
gpI
β2
gpI
β2
gpI
β2
gpI L
L
L
L
L
L
L
L
100/0 mol-%
*100/0 mol-%
90/10 mol-%
*90/10 mol-%
*80/20 mol-%
*POPC/PS 70/30 mol-%
70/30 mol-%
80/20 mol-%
Figure 9. Interaction of β2gpI with phospholipids using partial filling ACE. Injection: marker, 5 s at 50 mbar; liposome, 25 s at 50 mbar; sample, 5 s at 50 mbar; buffer, 5 s at 10 mbar. Fused-silica capillary pretreated with 0.5 M HCl for 10 min at 4 bar between runs; constant current conditions: 120 µA; temperature 22 °C. Electrophoresis buffer: 0.1 M phosphate pH 7.4; (*) denotes control runs, i.e. buffer was injected instead of the β2gpI sample.
Different migration patterns were obtained for β2gpI in the presence of liposomes.
The data indicates retardation of β2gpI in the presence of PS-containing liposomes,
indicating affinity for this, but the migration patterns are too complicated to
reliably extract binding data. In the absence of the β2gpI sample plug, the different
liposome plug compositions showed vastly different electroosmotic flows which
were normalized in the presence of β2gpI. Forthcoming studies will require a more
thorough characterization with not only fixed concentration of phospholipids and
24
different compositions of phospholipids but different total concentrations of
phospholipids as well.
7. Future perspective
The present work has focused on development and evaluation of a CE-based
binding assay for β2gpI and negatively charged ligands. The binding studies
performed so far have demonstrated the potential of this method. A more
thorough characterization of the interactions of β2gpI with different ligands is
desired. The glycosylation of β2gpI may have influence on interaction with
different ligands, such as hiding the epitope for binding to antibodies. The
structural role of β2gpI for various interactions has not yet been fully characterized.
By implementing MS detection in binding studies of glycosylated and
deglycosylated β2gpI with various ligands, such as monosaccharides, different
antibodies and phospholipids, our future studies will contribute to the
understanding of the function of β2gpI in health and disease.
25
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26
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Karlstad University StudiesISSN 1403-8099
ISBN 91-7063-074-7
Faculty of Technology and Science Chemistry
Karlstad University Studies2006:43
Maria E. Bohlin
Capillary electrophoresis
of b2-glycoprotein I
Capillary electrophoresis of b2-glycoprotein I
Human b2-glycoprotein I (b2gpI) is a phospholipid- and heparin-binding plasma glyco-protein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against b2gpI. With the final goal of assessing autoantibody influence on binding interactions of b2gpI we have developed capillary electrophoresis (CE)-based assays for interactions of ligands with b2gpI. The analysis of peptides and proteins by CE is desirable due to low sample con-sumption and possibilities for non-denaturing yet highly effective separations. However, adsorption at the inner surfaces of fused silica capillaries is detrimental to such analyses. This phenomenon is especially pronounced in the analysis of basic proteins and pro-teins containing exposed positively charged domains. The problem with these analytes is that they stick to the wall, which is negatively charged at neutral pH. To avoid wall interactions numerous procedures have been devised. Here, some of these methods were evaluated. Capillaries permanently coated with acrylamide and dimethylacryl-amide did not permit recovery of this basic protein (pI about 8) at neutral pH, unless the negatively charged ligand heparin was added to mobilize the protein. However, we found the pH hysteresis behavior of fused silica surfaces useful in avoiding b2gpI adsorption problems. The protonated surface after an acid pretreatment counteracted protein adsorption efficiently. This simple approach made estimates of heparin-b2gpI interactions possible and the principle was shown also to work for detection of b2gpI binding to anionic phospholipids. We also investigated the effects of different pretreat-ment techniques on the electroosmotic flow and the rate of the deprotonation process and show the more general utility of this approach for CE of various basic proteins in plain silica capillaries at neutral pH. The realization of a successful generic approach to facilitate protein analysis by CE is an important foundation for carrying out functional studies on b2gpI and other basic proteins.
Method development and binding studies
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