SIMONA CAVALU_Raman and Surface Enhanced Raman Spectroscopy of 2,2,5,5-Tetram...
POSTER 2013.ppt
1. Heparin Interactions Using Capillary Electrophoresis
Noemi Garcia, Meredith M. Dinges, Consuelo N. Beecher and Cynthia K. Larive
Department of Chemistry, University of California – Riverside, Riverside, CA 92521
Abstract
Heparin is a microheterogeneous and highly sulfated glycosaminoglycan that acts as
anticoagulant by binding to antithrombin-III, a protein inhibitor of the enzyme thrombin.
Heparin is stored with high concentrations of histamine in granules of mast cells. Heparin
binds histamine with μM affinity through electrostatic interaction involving a specific
tetrasaccharide sequence. Low molecular weight heparin (LMWH) drugs have improved
bioavailability and reduced side effects including lower risk of osteoporosis, deep vein
thrombosis, and pulmonary embolism. The LMWH enoxaparin is produced by chemical
digestion generating a complex mixture of variously sized oligosaccharides. Our group is
interested in the molecular structure of isolated enoxaparin oligosaccharides. Size-exclusion
chromatography (SEC) and strong anion exchange chromatography (SAX) are used to separate
the enoxaparin oligosaccharides based on size and charge prior to characterization by NMR
and mass spectrometry. However, the complexity of enoxaparin makes it difficult to resolve
oligosaccharides with minor structural differences. To enhance the separation of
oligosaccharides of similar size and charge we are exploiting the interaction of histamine to
add a third mechanism of separation based on differences in histamine affinity. Capillary
electrophoresis (CE) separations using histamine in the running buffer allow us to examine the
ability of histamine to resolve closely related oligosaccharides. CE provides rapid charge-based
separations with very low sample requirements allow us to explore conditions for resolving
oligosaccharides by histamine complexation prior to porting the method to preparative-scale
SAX separations. The results of CE experiments showing the effect of histamine on heparin
mobility will be presented.
DiscussionResults
Instrumentation and Reagents
Acknowledgments
References
Binding Constant Determination
Future Plans
Enoxaparin tetrasaccharide
(1→4)-(Δ-IdoA2S)-(1→4)-(GlcNS6S)-(1→4)-(IdoA2S)-(1→4)-(GlcNS6S)
Methods
Linear Equation Kd
Determination
Kd
Double
Reciprocal
Slope/Intercept 5.714x10-7
Linear Isotherm 1/Slope 7.092x10-7
Rabenstein 6.803x10-7
Size-exclusion chromatography (SEC) separates molecules
based on size. Due to the porous gel inside the column large
molecules elute first (3).
Strong anion exchange chromatography (SAX) separates
molecules by charge. The negatively charged enoxaparin
oligosaccharides stick to the positively charged beads in the
column. As the salt gradient increases, the negatively
charged oligosaccharides with the lowest net negative
charge will elute first (4).
Affinity Capillary Electrophoresis (ACE) is a technique for the analysis of receptor-ligand
interactions and the determination of binding constants. The basic principle involves measuring
the change in electrophoretic mobility of an analyte in buffer solutions containing dissolved
ligands (7).
The instrument that was used to separate the overlapping oligosaccharides
was a Beckman PA-800 Capillary Electrophoresis System with UV as the
mode of detection. The study used a 50 mM sodium phosphate buffer at
pH 3.5. A 5 mM benzyl alcohol solution was used as the neutral marker and
6 mM enoxaparin tetrasaccharide as the binding receptor. Histamine was
added to the running buffer at increasing concentrations (0 μM to 10 μM )
as the ligand..
Histamine is found bound to heparin in mast cell granules and is
responsible for activating the inflammatory response (1). At pH 5.2-6.0
histamine is a diprotonated dication that binds to heparin.
Benzyl alcohol is used as the neutral marker in this study to account for the
EOF in capillary. It is useful due to its low vapor pressure, low toxicity,
polarity, and it does not interfere with binding interaction.
The SEC Chromatogram shows the resolution of peaks for
different sized oligosaccharides by the relation of the
absorbance at 232 nm and fraction number
The SAX Chromatogram shows the separation of an enoxaparin
tetrasaccharide fraction obtained by SEC. Because of the
complexity of enoxaparin, it is not possible to resolve all of the
tetrasaccharides using SAX. For example, at about 48 min a peak
of interest is identified that contains the overlapping peaks of
tetrasaccharides with different structures.
This electropherogram shows the change in effective mobility of the enoxaparin tetrasaccharide as the
concentration of histamine is increased in the running buffer. Benzyl alcohol is used to account for the
capillary EOF. As the concentration of histamine in the running buffer is increased, the migration time of the
enoxaparin tetrasaccharide also increases.
The binding isotherm represents the correlation between the effective mobility of the enoxaparin
tetrasaccharide and concentrations of the histamine added. The linear portion of this graph can be used for
the linear isotherm and double reciprocal graphs (2).
The double reciprocal graph above is used to determine the
mobility of the complex. The linear equation provided can then
be used to find the binding constant (Kd
) (2).
The linear isotherm graph pictured above is produced using
the linear portion of the binding isotherm, taking into
consideration the mobility of the complex which can be
determined from the double reciprocal graph.The Kd
can then
be obtained by the linear equation produced.
This chart is representative of the calculated Kd
values obtained from the binding of
histamine and the enoxaparin tetrasaccharide. The chart shows the equations used to
calculate the Kd
values using the double reciprocal and linear isotherm graphs.
Our results obtained using ACE are comparable to those previously published by the
Rabenstein lab using NMR. In order to determine the binding constant, we first have to
calculate the effective mobility of the enoxaparin tetrasaccharide using the equation above.
The linear equations of the double reciprocal and linear isotherm, shown in the table above,
can then be used to obtain the binding constant.
(1) Rabenstein, Dallas L., Peter Bratt, and Jie Peng. "Quantitative Characterization of the
Binding of Histamine by Heparin." Biochemistry 37.40 (1998)
(2) Varenne, A., P. Gareil, S. Colleic-Jouault, and R. Daniel. "Capillary Electrophoresis
Determination of the Binding Affinity of Bioactive Sulfated Polysaccharides to Proteins: Study
of the Binding Properties of Fucoidan to Antithrombin." Analytical Biochemistry 315.2 (2003):
152-59
(3) My Scientific Blog - Research and Articles: GEL FILTRATION. Digital image. My Scientific Blog
- Research and Articles: GEL FILTRATION. Web. 19 Aug. 2013.
(4) "Biotechniques Den." : ION-EXCHANGE CHROMATOGRAPHY. Digital image. Web. 19 Aug.
2013.
(5) Skoog, Douglas A., F. James Holler, and Timothy A. Nieman. Principles of Instrumental
Analysis. Philadelphia, Pa. : Saunders College Publ., 1998.
(6) Chu, Yen-Ho, Luis Z. Avila, Jinming Gao, and George M. Whitesides. "Affinity Capillary
Electrophoresis." Accounts of Chemical Research 28.11 (1995): 461-68.
(7) Linhardt, Robert J. "2003 Claude S. Hudson Award Address in Carbohydrate Chemistry.
Heparin: Structure and Activity." Journal of Medicinal Chemistry 46.13 (2003): 2551-564.
(8) Eldridge, Stacie L., Layne A. Higgins, Bailey J. Dickey, and Cynthia K. Larive. "Insights into the
Capillary Electrophoresis Separation of Heparin Disaccharides from Nuclear Magnetic
Resonance, P, and Electrophoretic Mobility Measurements." Analytical Chemistry 81.17
(2009): 7406-415.
(9) Langeslay, Derek J., Elena Urso, Cristina Gardini, Annamaria Naggi, Giangiacomo Torri, and
Cynthia K. Larive. "Reversed-phase Ion-pair Ultra-high-performance-liquid Chromatography-
mass Spectrometry for Fingerprinting Low-molecular-weight Heparins." Journal of
Chromatography A 1292.31 (2013): 201-10.
• Department of Chemistry Kuwana-
Sawyer Award
• NSF CHE–1213845
• Larive Research Group
Future plans include using the ACE method to study the binding of histamine to other
oligosaccharides. We plan to explore the use of CE with histamine in the run buffer to separate
oligosaccharides that cannot be resolved using SAX. If successful we will transition this project
from CE to SAX to attempt to separate overlapping oligosaccharides on a preparative scale
based on differences in histamine affinity. After desalting each sample we will obtain the
structure of the purified oligosaccharides using NMR. In the longer term, isolated
oligosaccharides with defined structure will be used to elucidate heparin motifs that are
required in specific protein binding.
Capillary Electrophoresis (CE) is a unique separation method
that uses an applied voltage to separate molecules based on
differences in electrophoretic mobility. Buffer solution flows
through the fused silica capillary in which a small band of
sample is injected. The analytes in the sample migrate at
different speeds based on the ratio of their charge and size.
Due to the absence of a stationary phase there is less peak
broadening than in chromatographic methods and high
resolution separations can be carried out on exceptionally
small sample volumes.
Effective Mobility