Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule

Abstract

Glycosaminoglycans were extracted from bovine liver capsule with 4M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30 % respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentationequilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/ polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.

Structure-function relationships of heparin species

“…Preliminary experiments suggest that the hexitol end groups * It is assumed that uronic acid residues at the reducing end of heparin molecules are predominantly glucuronic acid. This is in accord with the findings of Jansson et al (15) Biochemistry: Rosenberg et al 3068 Biochemistry: Rosenberg et al…”

Size-exclusion chromatography of heparin oligosaccharides at high and low pressure

“…The uronic acid at the reducing end is a somewhat unexpected finding, because it can't originate from specific cleavage with HL-I leaving always glucosamine at the reducing end. This finding thus suggests that trisaccharides with the uronic acid at the reducing end originate from the protein linker region of heparin (reducing end) as proposed in 1975 [35,36]. According to this proposal, heparin is cleaved from its protein core by a tissue-specific endo-glucuronidase between uronic acid and the protein linker Gal-Gal-Xyl-Ser (Fig.…”

Metabolism of macromolecular heparin in mouse neoplastic mast cells

“…Attempts to isolate a heparin proteoglycan were unsuccessful, yielding essentially single polysaccharide chains, with mol.wts. ranging from 7000 to about 25000 (Lindahl, 1970;Ogren & Lindahl, 1971;Jansson et al, 1975). However, in rodent tissues heparin has been shown to -occur as a multichain macromolecular species (mol.wt.…”

“…The enzyme (hereafter termed 'heparin-liberating enzyme') was tentatively identified as an endoglucuronidase. Evidence has been obtained suggesting that the formation and degradation of macromolecular heparin may also occur in mammals other than rodents (see Jansson et al, 1975).…”

“…Further, heparin isolated from bovine liver capsule showed the structural properties expected (i.e. a molecular weight similar to commercial heparin and lack of a carbohydrate-protein linkage region) for the singlechain product released from macromolecular heparin by heparin-liberating enzyme (Jansson et al, 1975). Macromolecular hepar'i has been isolated not only from rodents but also from monkey and man (Horner, 1970).…”