ISOLATION AND CHARACTERIZATION OF HEPARAN-SULFATE FROM CRUDE PORCINE INTESTINAL MUCOSAL PEPTIDOGLYCAN HEPARIN

A method for the preparation of heparan sulfate from peptidoglycan hep arin is described. The objective of this research was to provide a bas is for the development and validation of an industrial process to supp ort the preclinical development of heparan sulfate and/or heparan sulf ate derivatives. In the preparation of heparan sulfate, heparin was re covered by alcohol fractionation and dermatan sulfate was isolated by selective precipitation. The remaining crude heparan sulfate was fract ionated by anion-exchange chromatography into five subfractions. The b iological activities of these subfractions were examined by anticoagul ant and amidolytic assays. Molecular weight and molecular size were de termined using capillary viscometry and polyacrylamide gel electrophor esis. Charge density and degree of sulfation were determined by cellul ose acetate electrophoresis and elemental analysis. Oligosaccharide an d disaccharide analysis relied on enzymatic depolymerization using hep arin lyases followed by polyacrylamide gel and capillary electrophores is. H-1 NMR analysis provided detailed structural information on each subfraction.