Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

Abstract

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.

Untitled

“…The GAGs separated electrophoretically and transferred onto NC may also be released and recovered from the cationized membranes at the mg-level for further analysis, such as disaccharide pattern evaluation after treatment with specific lyases [35], molecular mass determination [35], characterization of specifically sulfated derivatives or sequences [41,42] inside the polysaccharide chains. According to Karlsson et al [24], the immobilized GAGs are efficiently released from membranes using a nonionic detergent at high ionic strength.…”

Analysis of Glycosaminoglycans by Electrophoretic Approach

“…In our laboratory, it has been established the PAGEFS technique for a quick separation of AMAC-derivatized -disaccharide obtained from HA and CS/DS with detection using a CCD-camera [28,39,40]. Capillary electrophoretic (CE) technique has been previously used with success for the separation of AMAC-derivatized -disaccharide obtained from different GAGs [41], as well as for the separation of AMAC-derivatized oligomeric domains obtained from chondroitin sulfate, using both UV detector and LIF detector. This method was optimized and improved by Zinellu et al [42], for the analysis of both HA and CS/DS disaccharides in human plasma.…”

“…In quest for higher sensitivities, current studies have shown that derivatization with AMAC of the HA-and CS/DS-derived -disaccharides and analysis with reversed polarity CZE using a UV detector at 260 nm (absorbance of AMAC), leads to 100-fold increase of sensitivity [41]. A related developed method including similar CZE conditions and AMAC derivatization was used to attempt higher sensitivity for the analysis of all known HE/HS-deriveddisaccharides [43].…”

“…A related developed method including similar CZE conditions and AMAC derivatization was used to attempt higher sensitivity for the analysis of all known HE/HS-deriveddisaccharides [43]. When a laser-induced fluorescence (LIF) detector is used, the detection limits of the above methodologies may decrease up to 1000-fold [41,42,80]. The HS/HE analysis CE protocols, previously reviewed by Karamanos et al [81], include different conditions of polarity and applied voltage, as well as buffer composition, concentration and pH.…”

Advances in Chondroitin Sulfate Analysis: Application in Physiological and Pathological States of Connective Tissue and During Pharmacological Treatment of Osteoarthritis

“…Quantitative derivatization is generally achieved with 100-fold excess of this reagent. The 2-AMAC molecule has been used for the derivatization of acidic GAGs-derived di-and oligosaccharides and their subsequent HPCE analysis [13,51,75]. The stability of derivatized unsaturated disaccharides has been studied by preparing various mixtures of the standard ∆-disaccharides and keeping them at various temperatures.…”