889 research outputs found
Separation of Glycosaminoglycan Derived Oligosaccharides by Capillary Electrophoresis Using Reverse Polarity
No full textAnalytical Biochemistry, 221, 182-188,Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.A comparative study on compositional analysis of two sets of eight unsaturated disaccharide standards derived from heparin/heparan sulfate and chondroitin/dermatan sulfate was carried out using capillary electrophoresis performed in both normal and reverse polarity modes. While these heparin/heparan sulfate disaccharides (S. A. Ampofo, H. M. Wang, and R. J. Linhardt (1991) Anal. Biochem. 199, 249-255) and chondroitin/dermatan sulfate disaccharides (A. Al-Hakim and R. J. Linhardt (1991) Anal. Biochem. 195, 68-73) have previously been fractionated using normal polarity capillary electrophoresis, multiple buffer systems and conditions were required to separate certain disaccharide isomers and these separations often resulted in poor peak symmetry and significant tailing. This paper demonstrates that reverse polarity capillary electrophoresis completely resolves disaccharide mixtures into all components using a single buffer, 20 mM phosphoric acid-sodium phosphate at pH 3.48. This improved resolution is due primarily to an increase in the sharpness of peaks and improved peak symmetry. Separation of heparin-derived oligosaccharides, ranging from disaccharide to hexasaccharide, had also previously been reported using normal polarity capillary electrophoresis (U.R. Desai, H.M. Wang, S.A. Ampofo, and R.J. Linhardt (1993) Anal. Biochem. 213, 120-127). This paper now demonstrates the separation of 13 heparin-derived oligosaccharides of sizes ranging from disaccharide to tetradecasaccharide using both reverse and normal polarities. An enzymatic digestion of bovine lung heparin containing many of these larger oligosaccharides was also compared in both normal and reverse polarity modes. Mixtures containing oligosaccharides primarily differing in size (number of saccharide units) were better resolved using normal polarity.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1006/abio.1994.139
Regulation Activity of Heparin in Complement System
No full textin the Chemistry and Biology of Heparin and Heparan Sulfate, Elsevier Ltd., Oxford, H. G. Garg, R. J. Linhardt, and C. A. Hales (Eds.), Chapter 11, pp.313-343Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Heparin (HP) can bind to a variety of proteins, including growth factors, pro-inflammatory chemokines and cytokines, extracellular matrix proteins, and complement proteins. HP has a variety of biological activities, many of which are of interest because of their potential therapeutic utility. By regulating the activity of HP-binding proteins, HP and the related glycosaminoglycan (GAG), heparan sulfate (HS), can influence various biological processes giving HP therapeutic applications as an antithrombotic, antiatherosclerotic, anticomplement, antiinfective, anticancer, and anti-inflammatory agent. Monosaccharide and disaccharides with structural similarities to dextran did not cause a detectable decrease in C3b-factor H binding, while sugar polymers caused large decreases in the affinity between C3b and factor H as a result of the polysaccharide occupying the binding site in C3b or in factor H, preventing their interaction. HP and the structurally similar HS regulate multiple steps in the complement system including ones in both the classical and alternative pathways. Quantitative data in the form of association rates, dissociation rates, and affinity constants for complex formation are provided for many of these interactions.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/B978-008044859-6/50012-
Disaccharide Compositional Analysis of Heparin and Heparan Sulfate Using Capillary Zone Electrophoresis
No full textAnalytical Biochemistry, 199, 249-255,Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.National Institutes of Healthhttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/0003-2697(91)90098-
Industrial production of glycosaminoglycans
No full textReference Module in Life Sciences 2017, 1-10Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Glycosaminoglycans (GAGs) are naturally occurring polysaccharides, composed of alternating sugar units; these include an amino sugar (e.g., N-acetyl glucosamine, GlcNAc or N-acetyl galactosamine, GalNAc) and either a galactose (Gal) or an uronic acid (GlcA or IdoA) (Fig. 1). The carbon backbone of the GAG chain may undergo no further modifications (e.g., hyaluronan) or may be further modified through sulfation, de-acetylation, and/or epimerization (e.g., heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate). Simple non-sulfated GAGs (e.g., hyaluronan) and the precursors of highly sulfated GAGs (e.g., heparosan and chondroitin) are produced in certain bacteria as well as in animals (Cress et al., 2014) These simple GAGs are thought to contribute to pathogenicity of the host bacteria. Simple, non-sulfated GAGs as well as the highly modified sulfated GAGs (e.g., heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate) are produced in all animals, including humans. In humans, hyaluronan is part of the connective tissue extracellular matrix (ECM) and is involved in biological functions such as lubrication of joints. Highly sulfated GAGs have tissue-specific sulfated domains that are binding sites for various proteins (Capila and Linhardt, 2002) and, therefore, play critical roles in biological functions such as homeostasis, cell growth, cell migration, development, morphogenesis, tissue repair, and angiogenesis (Linhardt and Toida, 2004; Linhardt, 2003). The current industrial production of GAGs can be broadly classified into three categories: (1) industrial production from animal source, (2) industrial production using microbial cells, and (3) industrial production using eukaryotes. Various GAGs have been routinely extracted and purified from animal sources, for example, hyaluronan from rooster combs, and heparin from pig intestines or bovine lung. An increased knowledge of GAG biosynthesis, as well as the advances in metabolic engineering strategies, bioprocess optimization, downstream processing approaches and analytical tools have led to the production of GAGs from microbial cells and exploration of eukaryotic cells, for example, CHO cells, toward heparin production.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/B978-0-12-809633-8.12224-
Separation of Hydroxyl-Protected Heparin Derived Disaccharides using Reversed-Phase High Performance Liquid Chromatography
No full textJournal of Chromatography A, 705, 369-373Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.A simple and efficient method for the separation of hydrophobic derivatives of glycosaminoglycan-derived disaccharides is described. Hydroxyl-protected derivatives of a trisulfated disaccharide, prepared from heparin using heparin lyase, were separated by reversed-phase high-performance liquid chromatography. These disaccharide derivatives differed by the number, position, and stereochemistry of acetyl and pivaloyl groups. Separation was achieved on a C18 column using a reversed gradient of ammonium sulfate in water. This method has application in the purification of disaccharide derivatives being used as chiral synthons in the preparation of higher oligosaccharides.National Institute of General Medical Scienceshttps://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/0021-9673(95)00293-
Purification and Characterization of Heparin Lyases from Flavobacterium heparinum
No full textJournal of Biological Chemistry, 267, 24347-24355,Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate
Influence of Heparin Chemical Modifications on its Antiproliferative Properties
No full textin the Chemistry and Biology of Heparin and Heparan Sulfate, Elsevier Ltd., Oxford, H. G. Garg, R. J. Linhardt, and C. A. Hales (Eds.), Chapter 18, pp 513-532Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.This chapter focuses on the mechanisms contributing to heparin inhibition of smooth muscle cell growth. Chronic pulmonary hypertension is characterized by structural changes in the pulmonary vasculature, which along with variable degrees of vasoconstriction, are responsible for the high pulmonary vascular resistance and associated right heart failure. Circulating HP binds to endothelial cells and is taken up by the reticuloendothelial system where it enters a cellular pool to be released at a later stage. Fully sulfated HP and other glycosaminoglycan (GAGS) are prepared by treating tributylammonium salt of these with sulfur trioxide. Sulfo groups in HP appear to play an important role in the growth inhibitory effect on smooth muscle cell proliferation. Removal of N-sulfo groups from HP reportedly negates its growth inhibitory effect on smooth muscle cells (SMCs). No appreciable difference was found between heparin and fully sulfated heparin on the growth of pulmonary artery smooth muscle cells. Chondroitin and dermatan sulfates stimulated the pulmonary artery SMCs. Hyaluronan was not antiproliferative but full sulfation made HA strongly antiproliferative against pulmonary SMCs.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/B978-008044859-6/50019-
Regio and Stereoselective Synthesis of Derivatives of L-Idopyranuronic Acid and D-Glucopyranuronic Acid from D4-Uronates
No full textTetrahedron Letters, 38, 923-926Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.The stereoselective synthesis of β-d-glucopyranosiduronic, α-l-idopyranosiduronic, and α-l-altropyranosiduronic acids has been performed from different Δ4-uronate monosaccharides. Bromination of the C-4,5 double bond provided the trans-diaxial bromohydrin derivatives, which were converted to the corresponding epoxides in high yields. Direct reduction of the epoxides using borane−tetrahydrofuran complex led to the corresponding glucuronic acids in low to good yields. Glucuronic acids were also obtained in satisfactory yields through a two-steps procedure involving bromination of the epoxide with titanium(IV) bromide followed by reduction using tributyltin hydride. Lewis acid-catalyzed rearrangement of these epoxides led to the corresponding α-l C-4 ketopyranosides adopting the 1C4 chair conformation. Hydride reduction afforded the α-l-idopyranosiduronic or the α-l-altropyranosiduronic acids, the stereoselectivity of the reduction being controlled by the appropriate substitution pattern.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1021/jo981477
Analysis of glycosaminoglycan-derived, pre-column 2-aminoacridone-labeled disaccharides using liquid chromatography-fluorescence and -mass spectrometric detection.
No full textGlycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1-2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1-2
Electrophoresis for the analysis of heparin purity and quality.
No full textThe adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment
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