Heparin sequencing.
Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.
Stringer
Sally E
SE
Drug Development Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. sallyelizabethstringer@yahoo.co.uk
Kandola
Balbant S
BS
Pye
David A
DA
Gallagher
John T
JT
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006493:Heparin / Q000032:analysis*; D007068:Iduronidase / Q000378:metabolism; D008969:Molecular Sequence Data; D009608:Nitrous Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D010316:Particle Size; D017421:Sequence Analysis / Q000379:methods*; D013429:Sulfatases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009844:Oligosaccharides; D006493:Heparin; D013429:Sulfatases; C028553:disulfoglucosamine-6-sulfatase; D007068:Iduronidase; D009608:Nitrous Acid