fragments were prepared in a mixture of porcine mucosal heparin 50 mg ml and purified heparinase heparin lyase ec 4 2 2 7 derived from flavobacterium heparinum 5u mg 1 u 1 u03bcmol bonds cleaved min both in buffer consisting of 0 25 m sodium acetate and 0 0025 m calcium acetate at ph 7 this mixture was shaken at 30 u00b0 c until the reaction reached completion the ph of the mixture was then adjusted to ph 4 and the enzyme was removed by passage over a column containing sp sephadex c 50 the eluent containing the polysaccharide mixture was adjusted to ph 7 and freeze dried the polysaccharide mixture obtained as described in example i was dissolved in water 500 mg in 2 ml and was loaded onto a column packed with fractogel tsk f40 80 u00d7 2 5 cm and eluted with 1 m ammonium bicarbonate buffer at 0 3 ml min the eluent was diluted with water and its absorbance was measured at 232 nm fig1 six distinct peaks were observed representing from last to first eluted disaccharide mixed tetrasaccharide fragments mixed hexasaccharide fragments mixed octasaccharide fragments mixed decasaccharide fragments and mixed higher oligosaccharide fragments these peaks were identified on the basis of elution volume as compared to heparin and chondroitin derived standards of known molecular weight and by their absorbence at 232 nm based on a reported molar absorptivity of 5 2 u00d7 10 3 m 1 each sized fragment mixture 1 mg in 0 01 ml was then applied to a strong anion exchange high performance liquid chromatography column and eluted using 180 ml linear gradient y 1 5 u00d7 10 4 m sec u00d7 0 2 m sodium.