The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete rho(0) values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Le(a) specificity. All fractions had approximately equal Le(a) activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8-0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.     

Partial deglycosylation of blood-group-specific glycoproteins.

The time course for the partial deglycosylation of blood-group-specific glycoproteins from human ovarian-cyst fluids with 0.25 M-H2SO4/acetic acid and 6 M-HCl in methanol was studied. Either reagent readily removed about 80% of the carbohydrate from the glycoproteins to leave non-diffusible glycopeptides that contain N-acetylgalactosamine as the predominant sugar. Some changes in amino acid distribution were observed during the deglycosylation, which were attributed to an accelerated break-up of the nonglycosylated regions of the parent glycoprotein. The N-acetylgalactosaminyl-peptides isolated were judged to be polydisperse by gel filtration, and ion-exchange chromatography divided the glycopeptide population into several fractions with differing amino acid compositions. A Lumbricus terrestris hexosaminidase preparation was successful in removing almost all the remaining sugar from the glycopeptides, but caused further rupture of the peptide. When a per O-acetylated glycoprotein was treated with the H2SO4/acetic acid reagent the glycopeptide contained, in addition to N-acetylgalactosamine, about 50% of the sialic acid present in the parent glycoprotein, indicating that most of this sugar is located near the peptide end of the carbohydrate chains.     

Milk fat and primary fractions obtained by dry fractionation 1. Chemical composition and crystallisation properties

The chemical composition and crystallisation properties of milk fat and its primary fractions, obtained by dry fractionation at 21 degrees C, were investigated. The solid fraction (stearin) and the liquid fraction (olein) displayed a different triacylglycerol (TG) composition. Stearin fraction was enriched in long-chain fatty acids, whereas olein fraction was enriched in short-chain and unsaturated fatty acids. Crystallisation properties of milk fat, and both the stearin and olein fractions were studied on cooling at |dT/dt|=1 degrees C min(-1) by differential scanning calorimetry and time-resolved synchrotron X-ray diffraction (XRD) at small and wide angles. Two main types of crystals corresponding to double chain length structures were characterised in the stearin fraction: alpha 2L(1) (47.5 Angstrom) and beta' 2L(2) (41.7 Angstrom). A triple chain length structure was formed in the olein fraction: alpha 3L (72.1 Angstrom). Crystallization of milk fat showed the formation of two 2L (47.3 and 41.6 Angstrom) and one 3L (72.1 Angstrom) lamellar structures with an hexagonal packing (alpha form). A schematic representation of the 3L packing of olein fraction was proposed to explain how a wide diversity of TG can accommodate to form a lamellar structure with a thickness of 72 Angstrom. Furthermore, the sharpness of the small-angle XRD lines associated to the alpha form was explained by the formation of liquid crystals of smectic type.     

Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes.

The ability of the uncharged open-coil dextran molecules to penetrate tissue space, without coil-shape change, was utilized to probe (by partitioning experiments) the structural arrangement of the collagen-fibre network and the proteoglycan system. Hyaluronidase digests most of the proteoglycans away and enables the respective contributions to the exclusion volume to be evaluated by using a series of different-molecular-weight dextrans. It appears that the major part of the exclusion volume is due to the collagen-fibril as a rod and the dextran coil as an impenetrable sphere. The additional exclusion due to the proteoglycans could be accounted for by a set of points (regions of high proteoglycan-segment density) over which the dextran coild cannot pass. These points are an average of 50 nm apart and are indicative of local extensive entanglement of high-molecular-weight proteoglycans with each other. Reasons are given why these entanglements could not act as cross-links in long-term elastic loading of the tissue.     

Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds

In vitro antioxidant activities of three selected Indian red seaweeds - viz., Euchema kappaphycus, Gracilaria edulis and Acanthophora spicifera were evaluated. Total phenolic content and reducing power of crude methanol extract were determined. The antioxidant activities of total methanol extract and five different solvent fractions (viz., petroleum ether (PE), ethyl acetate (EA), dichloromethane (DCM), butanol (BuOH) and aqueous) were also evaluated. EA fraction of A. spicifera exhibited higher total antioxidant activity (32.01 mg ascorbic acid equivalent/g extract) among all the fractions. Higher phenolic content (16.26 mg gallic acid equivalent/g extract) was noticed in PE fraction of G. edulis. Reducing power of crude methanol extract increased with increasing concentration of the extract. Reducing power and hydroxyl radical scavenging activity of E. kappaphycus were higher compared to standard antioxidant (alpha-tocopherol). The total phenol content of all the seaweeds was significantly different (P<0.05). In vitro antioxidant activities of methanol extracts of all the three seaweeds exhibited dose dependency; and increased with increasing concentration of the extract.     

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

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.     

The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion.

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 X 10(6] were degraded into 'subunits' (Mr approx. 2 X 10(6] by reduction of disulphide bonds. Trypsin digestion of the 'subunits' produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, 'subunits' and 'domains' suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five 'domains'/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the 'subunits' are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.     

Purification and properties of blood-group-specific lectins from Vicia cracca.

The lectins from the seeds of Vicia cracca react specifically with human blood group A erythrocytes. They were purified by affinity chromatography on an adsorbent containing matrix-bound N-acyl-D-galactosamine. By a continuous pH gradient the lectins could be separated into two fractions each of which was shown to consist of several agglutinating species. The behaviour of both fractions in affinity chromatography was paralleled by the pH dependence of the interaction with the hapten sugar N-acetyl-D-galactosamine. Both lectin fractions have the same molecular (125000) and subunit (33000) weights, display the same pH dependence of their titre against A1 erythrocytes, and bind to N-acetyl-D-galactosamine at pH 8 with the same constant of about 6 X 10(3)M-1.     

Role of solvent properties of aqueous media in macromolecular crowding effects

Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volume effect is not the only factor affecting the behavior of biomolecules in a crowded environment. The observed inconsistencies are commonly explained by the so-called soft interactions, such as electrostatic, hydrophobic, and van der Waals interactions, between the crowding agent and the protein, in addition to the hard nonspecific steric interactions. We suggest that the changes in the solvent properties of aqueous media induced by the crowding agents may be the root of these “soft” interactions. To check this hypothesis, the solvatochromic comparison method was used to determine the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions of different polymers (dextran, poly(ethylene glycol), Ficoll, Ucon, and polyvinylpyrrolidone) with the polymer concentration up to 40% typically used as crowding agents. Polymer-induced changes in these features were found to be polymer type and concentration specific, and, in case of polyethylene glycol (PEG), molecular mass specific. Similarly sized polymers PEG and Ucon producing different changes in the solvent properties of water in their solutions induced morphologically different α-synuclein aggregates. It is shown that the crowding effects of some polymers on protein refolding and stability reported in the literature can be quantitatively described in terms of the established solvent features of the media in these polymers solutions. These results indicate that the crowding agents do induce changes in solvent properties of aqueous media in crowded environment. Therefore, these changes should be taken into account for crowding effect analysis.     

Assessing accumulated solvent near a macromolecular solute by preferential interaction coefficients

Biological macromolecules are often studied in mixed solvents. To understand cosolvent-macromolecule interactions, the preferential interaction coefficient, Gamma(3), may help determine surface solvent compositions. Gamma(3) measures the amounts of water, B(1), and cosolvent, B(3), within the "local domain," the (possibly far-reaching) region surrounding the macromolecule where the solvent is non-bulk-like. The local domain's boundary is, however, vague and it is unclear which molecules are counted in B(i). It is useful to explore a simple model system to make B(i) more concrete and to understand which aspects of the surface solvent distribution, rho(x), are sampled by Gamma(3). We performed computer simulations on a two-dimensional (2D) system consisting of a hard-wall solute (the macromolecule) in a mixed solvent (hard disks of different radii). We simultaneously calculated Gamma(3) and rho(x). We found that 1) in practice, the local domain's boundary is demarked by the outer limit of the first cosolvent (not water) layer; B(i) mainly counts the solvent near the macromolecule; 2) assuming B(1) to count only the waters within the first water layer is a poor approximation; 3) when determining B(1) and B(3), water and cosolvent molecules must be counted from the same region of space. We speculate that these 2D results may serve as a first-order approximation for the dominant contributions to Gamma(3) even in three dimensions, so long as the cosolvent is not strongly excluded from the macromolecular surface and there is no significant long-ranged solvent structure.     

Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].     

Characterization by gas-liquid chromatography-mass spectrometry and proton-magnetic-resonance spectroscopy of pertrimethylsilyl methyl glycosides obtained in the methanolysis of glycoproteins and glycopeptides.

The quantitative analysis by gas chromatography of monosaccharides present in glycoproteins and glycopeptides using methanolysis, followed by re-N-acetylation and trimethylsilylation, gives rise to several peaks for each monosaccharide. The identity of these peaks for xylose, fucose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid was established for alpha- and beta-methyl pyranosides and furanosides by combined g.l.c.-mass spectrometry and proton-magnetic-resonance spectroscopy. These data provide for the unambiguous interpretation of the gas chromatograms obtained in the application of this g.l.c. method, and supply basic information for the further application of mass spectrometry in this field.