Heparin Interactions Using Capillary ElectrophoresisNoemi 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.

Discussion Results

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

DeterminationK

d

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 (K

d) (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 K

d 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 K

d 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