Long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge

Chervenka, Charles H.

doi:10.1016/0003-2697(70)90082-5

Cited by 227 publications

(47 citation statements)

References 6 publications

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“…Molecular weights of glycosaminoglycans were determined by meniscus-depletion equilibrium ultracentrifugation as described by Chervenka (1970), at a polysaccharide concentration of 2mg/ml of 1 MNaCl-0.08M-Tris-HCI, pH 7.4. A 12mm40 capillarytype, synthetic-boundary, double-sector cell was used at 200C.…”

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 molecular weight was determined in a Spinco Model E analytical ultracentrifuge using the Yphantis meniscus depletion method as modified by CHERVENKA (9). The analysis was performed at 25 ~ in a sodium phosphate buffer, pH 6.9, ~t =0.2, at 20,410 rev.…”

Section: Molecular Weight Determinationmentioning

confidence: 99%

Isolation of a carboxypeptidase from malted barley by affinity chromatography

Breddam

Sørensen

1983

Carlsberg Res. Commun.

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A carboxypeptidase was isolated from malted barley by affinity chromatography in a yield of approximately 30%. The specific activity was higher than previously obtained for similar enzymes. The enzyme was a dimer where each of the monomers consisted of two peptide chains linked by disulfide bridges. Each monomer contained a single suifhydryl group, which was inaccessible to p-hydroxymercuribenzoate and ELLMAN' S reagent when the enzyme was in its native state. The two peptide chains were separated and the N-terminal sequence of each of them determined. The enzyme contained 6 amino sugar residues and 8% neutral sugar. Isoelectric focusing indicated that the enzyme existed in two forms with pI of 5.65 and 5.73, respectively, but N-terminal sequence determination indicated that they had identical peptide chains. Diisopropyl phosphorofluoridate completely inhibited the enzyme while Hg++ only inhibited the enzyme after deprotonation of an ionizable group on the enzyme with a pK a of approximately 6.7.

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“…The analysis was carried out using the longcolumn meniscus-depletion method [13] and was evaluated with the interference optical system. The molecular weight was calculated according to Yphantis [14].…”

Section: Sedimentation Analysismentioning

confidence: 99%

Structural Studies on the Four Repetitive Fc-Binding Regions in Protein A from Staphylococcus aureus

Sjödahl¹

1977

Eur J Biochem

121

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The covalent structures of the four highly homologous Fc-binding regions in protein A, regions D, A, B, and C, have been studied by enzymic fragmentations of previously isolated fragments originating from these regions and subsequent isolation of the generated peptides by ion-exchange chromatography, molecular-sieve chromatography, high-voltage paper electrophoresis and paper chromatography. The complete sequence of region B was elucidated by combining the results of Edman degradations on isolated fragment B peptides with the previously reported N-terminal sequence of the same fragment. Furthermore, Edman degradations of fragment D, A and C peptides differing from the region B sequence provided the structures of subregions not identical to corresponding subregions within region B. Thus, it is possible to propose a highly probable covalent structure for the N-terminal 27 000-molecular-weight portion of protein A responsible for the IgG-Fc-binding activities. However, it was not possible to assign the activities to specific structures within the regions.The sequence data indicate that not only mutual homology between the four regions exists, but also internal homologies within the regions. Furthermore, the data strongly supports the hypothesis of a stepwise gene fusion procedure being involved in the evolution of the protein.

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Long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge

Cited by 227 publications

References 6 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

Isolation of a carboxypeptidase from malted barley by affinity chromatography

Structural Studies on the Four Repetitive Fc-Binding Regions in Protein A from Staphylococcus aureus

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