Physicochemical studies of fractionated bovine heparin

Lasker, Sigmund E.; Stivala, Salvatore

doi:10.1016/0003-9861(66)90286-4

Cited by 120 publications

(21 citation statements)

References 40 publications

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“…Hoparin and dermatan sulphate fractions were centrifuged for 19h and for 21 h at 36000 and 22000rev./min respectively. The values for partial specific volumes used were 0.47ml/g for heparin (Lasker & Stivala, 1966) and 0.54ml/g for dermatan sulphate (Luscombe & Phelps, 1967 The total content of glycosaminoglycuronan in the tissue was determined as follows. Homogenized, defatted, dried liver capsule (2.0g) was suspended in 20ml of 0.05M-sodium acetate buffer, pH5.5, containing 0.3 M-NaCl, 0.01 M-EDTA and 0.01 Mcysteine-HCl.…”

Section: Methodsmentioning

confidence: 99%

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

Jansson¹,

Ogren²,

Lindahl³

1975

Biochemical Journal

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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.

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Section: Methodsmentioning

confidence: 99%

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

Jansson¹,

Ogren²,

Lindahl³

1975

Biochemical Journal

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“…The rapid long-column meniscus-depletion method (Chervenka, 1970) was used. All calculations of molecular weights are based on a partial specific volume for heparin of 0.45 ml/g, which is an average of the two literature values reported for the polysaccharide (Laurent, 1961;Lasker & Stivala, 1966).…”

Section: Methodsmentioning

confidence: 99%

“…The molecular weights obtained from these experiments may be 10-20% in error. This is due to the uncertainty associated with the partial specific volume of heparin (Laurent, 1961;Lasker & Stivala, 1966), to the slight residual polydispersity of the preparations, and to the influence on the determinations of the high negative charge of the polysaccharide (Tanford, 1961).…”

Section: Molecular Weightsmentioning

confidence: 99%

Binding to antithrombin of heparin fractions with different molecular weights

Danielsson

Björk

1981

Biochemical Journal

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The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34 700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

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“…Moreover, viscosity (g) provides a measure of the viscosity averaged molecular weight of linear polymers. Indeed, the molecular weight of heparin measured by sedimentation is linearly related to its viscosity in solution [33]. Thus, the change in viscosity measures the change in polysaccharide chain length and affords a measure of the average molecular weight of GAGs, such as heparin, that behave as Newtonian solutions at the dilute concentrations studied [34].…”

Section: Viscosity Profiles Of Enzymatic Depolymerizationmentioning

confidence: 99%

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Capila

Rethwisch

et al. 2002

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through h-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords f 10-fold higher K m values but no significant change in V max , consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage. D

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Physicochemical studies of fractionated bovine heparin

Cited by 120 publications

References 40 publications

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

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

Binding to antithrombin of heparin fractions with different molecular weights

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

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