Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans.
A rapid, highly sensitive and reproducible HPCE method is described for the determination of all non- and variously sulphated disaccharides present in hyaluronan and vertebrate chondroitin sulphates and dermatan sulphates. Following chondroitinase digestion of glycosaminoglycans or proteoglycans, the non-, di- and tri-sulphated delta-disaccharides are completely separated and readily determined within 14 min on a fused-silica capillary in 15 mM sodium dihydrogen orthophosphate, pH 3.00, using reversed polarity at 20 kV and detection at 232 nm. The determination of the various delta-disaccharides derived from either glucuronic or iduronic acid and the presence of glucuronic and iduronic clustered structures in dermatan sulphate can also easily be made, using digests with chondroitinase AC or B. A linear detector response was obtained for the entire interval tested (up to 10 mg/l of delta-disaccharides). Concentrations as small as 32, 65, 100 and 250 pmol/l (22, 38, 50 and 98 ng/l) of tri-, di- and nonsulphated delta-disaccharides, respectively, can be reliably detected.
Karamanos
N K
NK
Department of Chemistry, University of Patras, Greece.
Axelsson
S
S
Vanky
P
P
Tzanakakis
G N
GN
Hjerpe
A
A
D000818:Animals; D002478:Cells, Cultured; D002810:Chondroitinases and Chondroitin Lyases; D046508:Culture Techniques; D049832:Decapodiformes; D004187:Disaccharides / Q000032:analysis; D004586:Electrophoresis; D006801:Humans; D006820:Hyaluronic Acid / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D011134:Polysaccharides / Q000032:analysis*; D011509:Proteoglycans / Q000032:analysis*; D012754:Sharks; D013552:Swine
D004187:Disaccharides; D011134:Polysaccharides; D011509:Proteoglycans; D006820:Hyaluronic Acid; C007815:galactosaminoglycan; D002810:Chondroitinases and Chondroitin Lyases