Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage

Protocols for analyzing the fine structure of hyaluronan and chondroitin su lfate using fluorophore-assisted carbohydrate electrophoresis of 2-aminoacr idone-derivatized hyaluronidase/chondroitinase digestion products were adap ted for direct analysis of previously characterized cartilage-derived sampl es. The chondroitin sulfate disaccharide compositions for fetal and 68 year human aggrecan from FACE analyses were Delta Di4S (50%), Delta Di6S (43%), and Delta Dios (7%); and Delta Di4S (3%), Delta Di6S (96%), and Delta DiOS (1%), respectively. The nonreducing terminal structures included predomina ntly 4S-galNAc with minor amounts of 6S-galNAc and Di6S for the fetal aggre can sample and, in addition, included 4,6S-galNAc in the 68 year aggrecan s ample. FACE analysis of a proteinase K digest of rat chondrosarcoma tissue gave an internal disaccharide composition for its chondroitin sulfate chain s of Delta DiOS ('7%) and Delta D14S (93%) with no Delta Di6S and Delta Di4 ,6S detected, while Delta DIHA from hyaluronan was 5% of the total. Analysi s of nonreducing terminal structures indicated the presence of 4S-galNAc (5 1%), galNAc (27 %), and Di4S (22%) with no 4,6S-galNAc or Di6S detected. Un expectedly, FACE analysis detected putative linkage oligosaccharide structu res from the chondroitin sulfate chains including both unsulfated (85%) and 4-sulfated (15%) linkage oligosaccharides, Finally, the number averaged ch ain length estimated from the ratio of the molar fluorescence of the Delta disaccharides to that of the nonreducing termini or the linkage oligosaccha ride structures was calculated as similar to 16 kDa, A tissue glucose conce ntration of 0.72 g/l was also measured. These results for both samples as d etermined by FACE analysis were similar to results previously reported, usi ng more labor and time intensive procedures, validating the FACE protocols.