Preparative Anion-exchange Chromatography Research Articles

Isolation and Characterization of a Novel 20-kDa Sulfated Polysaccharide from the Extracellular Slime Layer of Staphylococcus epidermidis

Slime-producing coagulase-negative staphylococci have emerged as important pathogens especially in immunocompromised hosts and patients with implanted devices. Although the extracellular slime layer is considered an important virulence factor, the chemical composition of the slime polysaccharide(s) remains unknown. The crude slime product derived from two reference Staphylococcus epidermidis strains (ATCC 35983 and 35984) and two clinical isolates was found to contain protein (11-20.5%), hexosamines (8-19%), neutral sugars (12.2-14%), phosphates (4-9.5%), uronic acids (1-13%), and small amounts of sulfates (0.5-3%). Preparative anion-exchange chromatography separated a main carbohydrate component which was isolated by combined chromatographies on DEAE-Sephacel and Sepharose CL-6B. HPLC and electrophoreses on polyacrylamide gel and cellulose acetate membrane revealed the presence of one species of low-sulfated polysaccharide with a relative molecular mass of 20-kDa. Chemical analyses of the polysaccharide showed that it is rich in glucosamine (46%) and neutral sugars (30-34%) with small amounts of sulfates (5.7-6.5%) and glucuronic acid (2.9-3.4%). Ten percent of the glucosamine is sulfated at the amino group. The neutral monosaccharides present are glucose, fucose, and xylose with glucose as the predominant one. It is estimated that the polysaccharide consists of 61-65 molecules of glucosamine (6-7 of which are N-sulfated), 30-35 neutral monosaccharides, 3-4 molecules of glucuronic acid, and 1-3 of fucose and xylose. Isolation and characterization of such a polysaccharide from the extracellular slime layer of S. epidermidis has not been previously reported. Its role to pathogenicity remains to be elucidated.

Drug-Sensitivity and DNA-Binding of a Subform of Topoisomerase Iiα in Resistant Human HL-60 Cells

Topoisomerase II alpha (170 kDa) expressed in human HL-60 cells is heterogeneous in charge. By two-dimensional electrophoresis and chromatofocussing two major subforms with pI of 6.5 and 6.7 can be resolved. By preparative anion-exchange chromatography we separated the known topoisomerase II isoenzymes (170/180 kDa) and in addition a late-eluting 170 kDa form, which has not been described before. The catalytic optimum of this late-eluting form is shifted to pH 9.4. It is more than 100-fold resistant to orthovanadate, amsacrine or etoposide, and has an increased salt stability. SDS-treatment induces covalent attachment of this enzyme fraction to calf thymus DNA in the absence of drug. The latter observations indicate an increase in DNA-binding. In the tightly DNA-bound state the late-eluting enzyme is not targeted by cleavable complex forming drugs. Accordingly, cells may become drug-resistant by expressing this form predominantly.

Determination, Purification, and Characterization of α-NADH

α-NADH can be determined in the presence of high concentrations of β-NADH using anion exchange HPLC combined with oxidation of β-NADH by lactate dehydrogenase. The method is suited for the detection of a large number of impurities. Residual absorption is a poor measure of α-NADH. By combination of preparative anion exchange chromatography and reversed-phase HPLC, α-NADH can be prepared with 95-98% purity starting from β-NADH. Commercially available preparations contain only 80-90% α-NADH. The anomerization kinetics of α- and β-NADH has been investigated at pH 7.0 and 7.3 taking into account the irreversible conversion reactions of α- and β-NADH. The H + ion activity related anomerization rate constants k 1 (α to β) and k 2 (β to α) have been found to be 170 and 29 M −1 s −1, respectively, at pH 7.0 and 192 and 36 M −1 s −1 respectively, at pH 7.3. The equilibrium constants k 1/ k 2 are 5.8 and 5.3 at pH 7.0 and pH 7.3, respectively. Absorption and fluorescence properties of α-NADH preparations have been determined newly. At 25°C, maximum absorption was found at 260.1 and 346.6 nm, whereas at 37°C the wavelengths were 260.1 and 345.3 nm. Maximum fluorescence excitation was found at 348 nm, and maximum emission was at 453 nm.

Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughput. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 ,um). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-μm preparative column, led to the conclusion that the overloaded 20-μm column gave a higher throughput than the 40-μm column.

Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deaminoneuraminic acid) units in glycoconjugates

We have evaluated methods for separation, preparation, and characterization of α-2→8-linked oligomers of sialic acids (Neu5Ac and Neu5Gc) and deaminated neuraminic acid (KDN; 2-keto-3-deoxy- d- glycero- d- galacto-nononic acid) recently found as a naturally occurring novel type of sialic acid analogue. (A) We examined preparative anion-exchange chromatography for fractionation and preparation of oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN). (B) We also examined the TLC method for separation and differentiation of the partial acid hydrolysates of colominic acid, as well as polysialoglycoproteins (PSGP) and poly(KDN)-glycoproteins (KDN-gp) isolated from rainbow trout eggs, and for discrimination of lower oligomers of Neu5Ac, Neu5Gc, and KDN. (C) We developed the high-performance adsorption-partition chromatographic method for (a) separation of monomers and oligomers of three nonulosonates according to the difference in substituents at C-5 and the presence or absence of 9- O-acetyl groups in oligo(KDN) and (b) separation of three homologous series of lower oligomers according to the degree of polymerization. (D) We examined and compared high-performance anion-exchange chromatographic separation of 3H-labeled oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN) alditols by using Mono-Q HR 5 5 resin. (E) We examined a method of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of Neu5Ac by treating them with cetylpyridinium chloride. We also used PSGP and KDN-gp to test both the sensitivity and the selectivity of this method.

Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from [formula omitted] 3T3, B16.F10 melanoma, and cutaneous fibrosarcoma cell lines

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3–34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.

Isolation and purification oil glucuronides with preparative anion exchange chromatography in methanol

Abstract890 ml of urine of rats treated with [14C] 205–734 was desalted over an Amberlite XAD‐2 adsorption column. The methanol extract dissolved in 10 ml methanol was injected onto an anion exchange column (Nucleosil 10 SB 250 × 16 mm i.d.) equilibrated in methanol. A gradient of decreasing methanol proportion versus a 0.2 M ammonium hydrogen carbonate buffer eluted a pure glucuronide of 205–734. Its structure was established by NMR, MS, UV, and IR analyses. This procedure is simple, rapid, and very selective for the compound of interest.

Influence of pore and particle size on the frontal uptake of proteins : Implications for preparative anion-exchange chromatography

Several silica-based anion-exchange packings were synthesized with nominal pore sizes of 250, 500 and 1000 Å in 10-, 20- and 50-μm particles. The static (“equilibrium”) adsorption capacities for bovene serum albumin (mol.wt. 69 000), α-lactalbumin (17 500) and ferritin (440 000) were first measured using bulk material. The media were then packed into columns for frontal uptake experiments to measure adsorption from a flowing mobile phase. In general, frontal uptake was inversely related to both flow-rate and particle size. However, the magnitude of these relationships was strongly dependent on the pore to protein diameter ratio. More specifically, the uptake of bovine serum albumin was significantly more sensitive to linear velocity and particle size than α-lactalbumin. A mathematical model of the chromatographic process was used to calculate radial adsorption profiles across the chromatographic particle during frontal uptake. It was shown that restricted intraparticle diffusion due to insufficient pore size causes incomplete utilization of internal surface area. Under such conditions, protein is only bound within a finite shell on the outermost side of the particle; therefore, the effective loadability of the packing is greatly reduced. These data suggest that a pore size of at least 500 Å will be required for the preparative chromatography of proteins with a molecular weight higher than ca. 1000 000. This observation is especially evident when using particle sizes greater than 10 μm.

Factors contributing to intrinsic loading capacity in silica-based packing materials for preparative anion-exchange protein chromatography

Properties of the matrix and stationary phase which affect the intrinsic loading capacity of silica-based packing materials for preparative anion-exchange chromatography of proteins were investigated. Polyethyleneimine-coated controlled porosity glass beads ranging from 100 to 2000 Å in pore diameter were used to evaluate the effects of pore diameter and surface area. Protein binding was found to depend on accessible, rather than total, support surface area. Consequently, wide-pore, high surface area media provide maximum intrinsic loading capacity. Increasing the number of positively charged sites on the stationary phase by increased coating or by quatemization of amines increases hemoglobin-binding capacity.

Structure assignment to biologically produced malathion monocarboxylic acid isomers with 13C-nuclear magnetic resonance

Chemical synthesis of malathion α- and β-monocarboxylic acid yields a mixture of the two structural isomers. These two isomers were separated by preparative anion-exchange chromatography on QAE-Sephadex. 13C-Nuclear magnetic resonance of the pure components shows that the main product is the β-isomer, with the α-isomer being present in much smaller quantities. This result was used for identification of hydrolytic malathion metabolites produced by rat tissues. On incubation of malathion with rat liver fractions in vitro it was found that α- and β-monoacid are formed in a ratio of 3:2, whereas this ratio is 9:2 for the metabolites excreted into the urine after intraperitoneal injection of malathion in rats.