A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans
Authors: Lauder Robert 
Published: August 2004

Introduction  Chondroitin and dermatan sulfate (CS/DS) show considerable species, tissue, age and pathology‐related structural heterogeneity. In addition within chains their sulfation patterns are not random. To elucidate their structure/function relationships, methods for complete characterization are required.A method for keratan sulfate (KS) fingerprinting (Whitham et al. 1999) has been extended for the linkage, repeat and chain cap regions of CS/DS, including the acquisition of CS/DS ratios.Methods  Chains were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or ACII lyase at 50 mg/ml in 0.1 m ammonium acetate, pH 8 and for 15 h at 37 °C. Alternatively, chains were de‐N‐acetylated by hydrazinolysis at 98 °C for 24 h at 10 mg/1 ml in anhydrous hydrazine with 100 mg/ml hydrazine sulfate. Then, they were depolymerized by 3.9 m sodium nitrite/0.28 m acetic acid at 0 °C for 4 h. Unreduced chains were released from their protein core in 0.5 m LiOH at 4 °C for 12 h.Materials were fluorescently labelled with 2‐AA as previously described (Whitham et al. 1999) and characterized by HPAEC using a Dionex AS4‐SC column at 50 °C and 2 ml/min with constant 15% 1 m NaOH. A 5‐min isocratic period of 85% H2O/0% 2 m NaCl was followed by a linear gradient of 0–30% 2 m NaCl over 60 min. The oligosaccharides were monitored using a λex of 315 nm and a λem of 400 nm.Results and discussion  This method resolves repeat region di‐, tri‐ and tetrasaccharides, capping oligosaccharides and linkage regions and can be used to profile known and unknown oligosaccharides. Unsulfated oligosaccharides elute between 2 and 10 min, monosulfated between 7 and 30 min, disulfated between 25 and 40 min and trisulfated between 49 and 54 min. Allied with data on size, oligosaccharide identification is facilitated.Hydrazinolysis/nitrous acid depolymerization of CS/DS chains results in disaccharides from CS with 4‐ or 6‐sulfation and from DS with 4‐sulfation which retain IdoA and GlcA structures and which can be distinguished chromatographically. The methodology was used to examine CS/DS from shark, whale, bovine and human tracheal, articular and meniscal cartilage and cornea.Tracheal cartilages show predominantly 4‐sulfation with porcine sources being more highly 4‐sulfated (ca. 75%) than bovine (ca. 65%). Articular cartilage comprises mainly 6‐sulfated GalNAc (ca. 95% in the adult), while adult meniscal cartilage shows only ca. 85%. Tracheal and articular cartilage aggrecan showed no IdoA; however, it represented ca. 20% of the uronic acids of bovine meniscal aggrecan, showing the presence of DS.Corneal CS/DS has a very low level of 6‐sulfation (<ca. 5%) but shows an equal abundance of unsulfated and 4‐sulfated residues and contains high levels, ca. 50%, of IdoA residues.Shark cartilage shows ca. 75% 6‐sulfation with significant levels of uronic acid 2‐sulfation found only between a 4‐sulfated residue and a 6‐sulfated residue, reflecting sulfotransferase specificity. Shark cartilage contains modest (ca. 1–5%) levels of DS that may be contaminants of preliminary isolation.This method extends a previous method to now allow the complete examination of KS, CS and DS chains by a single rapid chromatographic method.