Fractionation in a Density Gradient of some Glycoproteins from Human Ovarian Cyst Fluids

Bhaskar

Donald

et al. 1974

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH4)2SO4 and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH4)2SO4.

Section: Explanation Of Plate I Immunodiffusion Platessupporting

confidence: 82%

Section: Explanation Of Plate I Immunodiffusion Platesmentioning

confidence: 99%

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation

Bhaskar

Donald

et al. 1974

“…(i) The glycoprotein-1 fractions contain a relatively high proportion of peptide, ranging between 22 and 30 %. This is considerably higher than the average of 15 % characterizing, for example, the purified soluble blood-group-specific glycoproteins ofhuman ovariancyst fluids (Watkins, 1972;Donald, 1973), but similar to the values for the sparingly soluble or insoluble cyst-fluid glycoproteins, for which values of 30% peptide are common (Dunstone & Morgan, 1965;Dunstone, 1969).…”

Section: Analyticalcharacterization Oftheglycoproteinfractionsmentioning

confidence: 71%

The separation and characterization of bronchial glycoproteins by density-gradient methods

Bhaskar

Horton

et al. 1977

104

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.

“…The buoyant density of proteins in aqueous caesium chloride is approx. 1.3g/ml Ifft & Vinograd, 1962), whereas that of carbohydrates is approxiimately 1.6g/ml (Erikson & Szybalski, 1964). Accordingly, isopycnic equilibrium methods (see, e.g., Vinograd & Hearst, 1962) should be particularly useful for the preparation and characterization of blood-groupspecific glycoproteins, where a buoyant density intermediate between the extremes quoted may be expected.…”

mentioning

confidence: 99%

“…Accordingly, isopycnic equilibrium methods (see, e.g., Vinograd & Hearst, 1962) should be particularly useful for the preparation and characterization of blood-groupspecific glycoproteins, where a buoyant density intermediate between the extremes quoted may be expected. In the general field of glycoprotein fractionation the value of the method has been demonstrated by the applications to proteinpolysaccharide complexes from aortic tissue and nasal cartilage (Franek & Dunstone, 1966, to hyaluronic acid (Silpananta, Dunstone & Ogston, 1967 and to the blood-group-specific substances derived from water-insoluble cyst glycoproteins (Dunstone, 1969).…”

mentioning

confidence: 99%

The use of equilibrium-density-gradient methods for the preparation and characterization of blood-group-specific glycoproteins

Denborough

1970

1. The method of sedimentation equilibrium in a gradient of caesium chloride has been applied to the preparation of blood-group-specific glycoproteins from human ovarian-cyst fluids: it is shown that virtually complete separation from contaminating protein is easily accomplished in a single step. 2. The glycoproteins isolated in this way have been characterized by analytical density-gradient experiments in both caesium chloride and caesium sulphate and values of the buoyant density, selective solvation and apparent molecular weight have been obtained. 3. In some cases, materials prepared from the same cysts by solvent extraction methods have also been characterized in these terms. 4. The selective solvation values are about 0.1 and 0.5g of water/g of glycoprotein in caesium chloride and caesium sulphate respectively. 5. The apparent molecular-weight values are much lower than the weight-average molecular weights, and it is shown that the origin of the discrepancy is heterogeneity in density of the glycoproteins. 6. Some sources of error in the interpretation of density-gradient schlieren patterns are examined.