Chromatography of glycosaminoglycans on ECTEOLA-cellulose columns (original) (raw)
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Journal of Chromatography A, 2012
Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize glycosaminoglycan composition and structure and has a direct relationship with glycosaminoglycan biological functions. In this study, glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of glycosaminoglycan composition and structure.
Fractionation and characterization of glycosaminoglycans of mammalian origin
Pharmacological research communications, 1979
Glycosaminoglycans (GAG), occurring.as complex mixtures in many animal tlssues, can De separatedlnto inaividual components taking advantage of differences in charge density or complexing properties. Each mixture represents a special problem because of different degrees of heterogeneity of components as regards the degree of sulfation and the molecular weight. The fractionation of GAG extracts from a typical source {pig duodenum), performed in order to make individual GAG available for pharmacological tests, is described. The composition of the extracts was monitored at various steps of fractionation by ~uantitative cellulose acetate electrophoresis and NMR spectroscopy. The purified isolated GAG (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfates and hyaluronic acid) were characterized by various physico-chemical and enzymicmethods. ! NTRODUCT ION Glycosaminoglycans (GAG, also called mucopolysaccharides) are widely distributed in animal tissues, usually as proteoglycans, i.e., linked to a protein matrix (Muir and Hardingham, 197S). As a common structural feature, GAG are characterized by a co-polymeric backbon~ made up of alternating sequences of a uronic acid and a hexosamine. Individual GAG differ from each other by nature of the u ronic acid (D-glucuronic or L-iduronic) and the hexosamine (D-giucosamine or D-galactosamine), the way o Visiting Scientist, Istituto G. Ronzoni, 1977-78.
Analysis of Glycosaminoglycans by Electrophoretic Approach
Current Pharmaceutical Analysis, 2008
Glycosaminoglycans (GAGs) are non branched polysaccharides which are raising a great interest among the scientists for their biological roles. In fact GAGs play a pivotal role in several biological events, since they participate in and regulate cell adhesion, migration and proliferation. The quantification and analysis of the fine structure of GAGs are increasingly important not only for understanding many biological processes, but also for elucidate many critical aspects in human pathology development. Chondroitin sulfate (CS), heparan sulfate (HS) and keratan sulfate (KS) are commonly described as sulfated GAGs and these molecules are linked to a core protein forming proteoglycans; the sulfation pattern shows a high level of complexity and it is associated with specific function in the tissues. The only GAG without protein core is hyaluronan (HA), which is produced in almost all tissues, often with a molecular weight of 10 6 Daltons. Several human tissues contain high amount of GAGs and the change of the quantity and the structure of these macromolecules are described in tissue development and it is commonly associated with diseases. Electrophoretic methods based on the gel separation of 2-aminoacridone labelled HA and CS sulfate-disaccharides, derived from GAG digestion with specific eliminases, have been recently proposed. These new techniques represent a suitable method for GAG fast and sensitive analysis. In this review we will describe the recently achieved methods on the GAG analysis based on the electrophoretic approach in comparison with the more standard chromatographic techniques (HPLC).
Separation of glycosaminoglycan saccharide and glycoside mixtures by gel filtration
Analytical Biochemistry, 1969
Our recent studies of the binding properties of lysoayme, employing difference spectroscopic (1) and magnetic resonance (2-6) methods, have made extensive use of chitin oligosaccharides as enzyme inhibitors. Also, parallel investigations (7-9) of the catalytic mechanisms of various lysozymes have utilized the higher members of the same saccharide series and their methyl, phenyl, and p-nitrophenyl glycosides as substrates. Additional studies (10) of highly purified bovine hyaluronidase have involved use of oligosaccharides obtained from hyaluronic acid. All these investigations have necessitated the purification of homologous series of saccharides, peracetylated sugars, and glycosides as well as the separation of glycosides from reducing sugars. Chromatography of chitin oligosaccharides has been successfully performed previously on charcoal-Celite columns (11). Such methods have, however, proved inadequate for some of the separations we desired. This communication describes the use of gel filtration methods, using various chromatographic materials and solvents, to fractionate effectively complex mixtures of reducing sugars and glycosides.
Analytical Biochemistry, 1980
Quinoxalines derived from various homoglucans by the alkaline OPDmethod were subjected to GLC and GC-MSanalyses. The main quinoxalines derived from amylose and curdlan were identified as 2-hydroxymethyl-3-(2/,3 /-dihydroxypropyl)quinoxaline (M-l) and 2-(2/,3 /,4/-trihydroxybutyl)quinoxaline (G-l), respectively, using a 2% OV-210 column after trimethylsilylation. Twoquinoxalines M-l and G-l were formed from barley glucan and lichenan, and the approximate molar ratios of the 1,4-to 1,3linkage in both glucans were estimated to be 1.6 and 2.2, respectively. From scleroglucan, G-l and 2-(2/,3 /-dihydroxy-4/-D-glucopyranosyl)quinoxaline were produced, reflecting a 1 ,3-and branched 1,6-linkage in the glucan. The ratios of the 1,4-to 1,3-linkage of barley glucan and lichenan, or the 1,6-to 1,3-linkage of scleroglucan estimated from the formed quinoxalines were in accordance with those obtained by conventional methylation analysis.