Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Liposomal Delivery Systems for Encapsulation of Ferrous Sulfate: Preparation and Characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 μm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.     

Stability of Barium Sulfate Turbidity Standards

McFarland barium sulfate standards prepared in 1952 were compared spectrophotometrically with freshly and identically prepared standards; the results were nearly identical.     

Liposomal delivery systems for encapsulation of ferrous sulfate: Preparation and characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 microm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.     

Crystallization of free aspartate aminotransferase from chicken heart cytosol]

Homogeneous aspartate aminotransferase (purity--99%, yield--70%) has been prepared from chicken heart cytosol. The purification procedure included fractionation with ammonium sulfate and ethanol and crystallization. Crystals (0.3 x 0.5 x 2 mm) of the free enzyme were prepared from ammonium sulfate solution and studied by X-ray analysis at 2.5 A resolution.     

Purification and properties of Streptococcus lactis beta-galactosidase

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.     

Crystallization of water-soluble chlorophyll-proteins from Lepidium virginicum

Water-soluble chlorophyll-proteins were prepared from leaves of Lepidium virginicum, by means of ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. After intensive purification the chlorophyll-proteins were crystallized by dialysis against an ammonium sulfate solution.     

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.     

不同硫取代度昆布多糖硫酸酯的合成及理化性质初步研究

目的:对昆布多糖进行不同硫取代度的硫酸酯化修饰,并对其产物的硫酸基含量、糖含量与分子量进行检测,为研究不同硫取代度昆布多糖硫酸酯的生物活性奠定物质基础。方法:采用氯磺酸-吡啶法对昆布多糖进行硫酸化修饰,通过改变硫酸化修饰条件,来制取不同硫酸基取代度的昆布多糖硫酸酯;利用盐酸水解-硫酸钡比浊法测定昆布多糖硫酸酯的硫酸基含量,并通过公式求得其硫取代度;用苯酚-硫酸法测定昆布多糖硫酸酯的多糖含量,并使用HPGPC法测定其分子量。结果:两种不同硫取代度昆布多糖硫酸酯的硫酸基含量分别为37.8%、45.92%,取代度分别为1.07、1.51,糖含量分别为44.52%、37.19%,分子量分别为13000、16000。结论:利用氯磺酸-吡啶法对昆布多糖进行硫酸酯化修饰,该方法可以获取不同取代度产物,酯化率高。     

Preparation and quantification of 3'-phosphoadenosine 5'-phospho[35S]sulfate with high specific activity

The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAP35S), prepared from inorganic [35S]sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of [35S]sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl[35S]sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed.     

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.     

Skeletal keratan sulfate chain molecular weight calibration by high-performance gel-permeation chromatography

A method has been developed for the molecular sizing of skeletal keratan sulfate chains using an HPLC gel-permeation chromatography system. Keratan sulfate chains and keratanase-derived oligosaccharides were prepared from the nucleus pulposus of bovine intervertebral disc (6-year-old animals). A Bio-Gel TSK 30 XL column eluted in 0.2 M NaCl and at 30 degrees C was calibrated with keratan sulfate oligosaccharides of known size as well as 3H-end-labeled keratan sulfate chains to yield the relationship.     

The glucuronyl C5-epimerase activity is the limiting factor in the dermatan sulfate biosynthesis.

An early step in the biosynthesis of dermatan sulfate is polymerization to chondroitin, which then is modified by the D-glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of D-glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20--30%, and fibroblast 80% of the uronosyl residues in the l-iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d-glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d-glucuronyl C5-epimerase is the main factor for formation of dermatan sulfate in tissues.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Metal Chelates as Fungicides

A remarkable improvement in the emulsifying properties of proteins was observed in mixtures of serum albumin-dextran sulfate, lysozyme-dextran sulfate, serum albumin-chondroitin sulfate, α-lactalbumin-chondroitin sulfate and lysozyme-chondroitin sulfate. The emulsifying properties of these protein-polysaccharide complexes were affected by the molecular size of the polysaccharide and by such conditions as the presence of salt, acidic or alkaline pH, and heating. On the other hand, the emulsifying properties of ovalbumin-dextran hybrids prepared by covalent attachment were more enhanced in alkaline pH or by heating. The molecular size of the ovalbumin-dextran hybrids greatly affected the emulsifying properties.     

Studies on the pyrophosphorylases of wheat

Adenosine diphosphoglucose and uridine diphosphoglucose pyrophosphorylase activities (class EC 2.7.7.9) were assayed radiochemically in ammonium sulfate fractions prepared from mature and germinating wheat grain. Both enzymes showed similar pH optima and metal ion requirements but differed markedly in their response to activation by d-glycerate 3-phosphate and inhibition by sulfate and phosphate.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.     

A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.     

Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro

Polysaccharide extracted from Ulva pertusa (Chlorophyta) is a group of sulfated heteropolysaccharide; for simplicity, the sulfated polysaccharide is referred to as ulvan in this paper. In this study, different sulfate content ulvans were prepared with sulfur trioxide/N,N-dimethylformamide (SO3-DMF) in formamide, and their antioxidant activities were investigated including scavenging activity of superoxide and hydroxyl radicals, reducing power and metal chelating ability. As expected, we obtained several satisfying results, as follows: firstly, high sulfate content ulvans had more effective scavenging activity on hydroxyl radical than natural ulvan. Secondly, comparing with natural ulvan, high sulfate content ulvans exhibited stronger reducing power. Thirdly, HU4 (sulfate content, 30.8%) and HU5 (sulfate content, 32.8%) showed more pronounce chelating ability on ferrous ion at high concentration than other samples.