Selective cleavage of heparin using aqueous 2-hydroxypyridine: production of an aldose-terminating fragment with high anticoagulant activity.
Unfractionated heparin (UFH) was partially depolymerized by heating at 115 degrees C with aqueous 2-hydroxypyridine. Compared to starting UFH, no significant loss of anticoagulant (anti-Xa) activity was observed. Products consisted of polysaccharide fragments and small quantities of ammonia, sulfate, and hexuronic acid. Fragments with aldose termini that reacted with [3H]NaBH4 (fragment A) were of relatively uniform size (6000 D) and increased as depolymerization time increased. Fragment A contained the anticoagulant activity, with 90-94% and 24-31% binding to Sepharose-thrombin and Sepharose-antithrombin, respectively. In contrast, a non-reducing fragment B that did not react with [3H]NaBH4 was more heterogeneous (6000-10,000 D) and did not have anticoagulant activity or Sepharose-antithrombin affinity. Given the polysaccharide 3H-incorporation, small release of monosaccharide products, and fragment A end-group analysis, thermolysis of UFH is likely limited to one site per molecule when protected by 2-hydroxypyridine. Thus, an anticoagulant fragment A is hydrolytically released from UFH leaving a variable-length fragment B complete with linkage region.
Berry
Leslie R
LR
Henderson Research Centre, McMaster University, Department of Pediatrics, Hamilton, Canada L8V 1C3.
Parmar
Nagina
N
Hatton
Mark W C
MW
Chan
Anthony K C
AK
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006493:Heparin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006358:Hot Temperature; D008958:Models, Molecular; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism*; D011728:Pyridones / Q000494:pharmacology*; D012997:Solvents; D013552:Swine; D014867:Water
D000925:Anticoagulants; D010446:Peptide Fragments; D011134:Polysaccharides; D011728:Pyridones; D012997:Solvents; D014867:Water; C017679:2-hydroxypyridine; D006493:Heparin