Anion-exchange High-performance Liquid Chromatography Research Articles

Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography

The carbohydrate-dependent microheterogeneity of recombinant coagulation factor VIIa (rFVIIa) has been characterized by capillary electrophoresis (CE) of the native protein and by high-performance liquid chromatography (HPLC) of tryptic peptides and of oligosaccharides released by hydrazinolysis. The development of the CE analysis is reported here. We have found that application of 1,4-diaminobutane (putrescine) as additive to the CE separation buffer is essential for the separation of the various glycoforms. Under optimum conditions rFVIIa migrates as a cluster of six peaks or more. By CE of neuraminidase-treated rFVIIa a faster-moving double peak is observed. This indicates that the separation obtained is primarily based upon the different content of N-acetyl-neuraminic acid of the oligosaccharide structures in rFVIIa. By reversed-phase HPLC of tryptic digested neuraminidase treated rFVIIa the glycopeptides containing the heavy chain N-glycosylated site elute as two peaks compared to the four peaks corresponding to glycopeptides with 0 to 3 N-acetyl-neuraminic acids seen for untreated rFVIIa. In high-pH anion-exchange HPLC of the oligosaccharides released from native rFVIIa by hydrazinolysis the major peaks elute as oligosaccharides with 1 or 2 N-acetyl-neuraminic acids. Oligosaccharides released from neuraminidase treated rFVIIa elute earlier compared to oligosaccharides from native rFVIIa, but separated into several peaks, indicating heterogeneity for the oligosaccharide structures without N-acetyl-neuraminic acid.

Single-Run Separation and Detection of Multiple Metabolic Intermediates by Anion-Exchange High-Performance Liquid Chromatography and Application to Cell Pool Extracts Prepared fromEscherichia coli

A method is described for analysis of the intracellular concentrations of metabolic intermediates of the Embden–Meyerhof–Parnas pathway, the Entner–Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle in cell pool extracts ofEscherichia coli.A single anion-exchange HPLC run of 40 min allowed resolution of 27 anionic metabolite standards. Detection limits of 0.1 nmol per injection were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor and a uv detector. A boiling water extraction procedure was used to prepare cell pool extracts. Cochromatography of cell pool extracts and metabolite standards was used to confirm the identities of metabolites in the cell pool. As many as 16 metabolites could be detected and quantified in the cell pool extracts by using the described HPLC method. An analysis of metabolite concentrations inE. colishowed the dynamics of glucose metabolism during a 2-min transition from starvation to steady-state metabolism following addition of glucose. The ease and power of this method suggests general utility forin vivometabolite analysis in a variety of experimental systems.

Developmental Differences in Distribution of Cyclic Nucleotide Phosphodiesterase Isoforms in Cardiomyocytes and the Ventricular Tissue from Newborn and Adult Rats

We characterized cyclic nucleotide phosphodiesterase (PDE) activities in neonatal rat cardiomyocytes and in whole ventricle from newborn and adult rats, to determine whether cyclic nucleotide hydrolytic activities might be influenced by the developmental stage of the animal or by cell isolation and culture procedures. Using anion-exchange high-performance liquid chromatography (HPLC), we obtained four soluble PDE activities from adult rat ventricle. Peak 1, a cyclic GMP-specific PDE was stimulated by Ca2+/calmodulin; peak 2 hydrolyzed cyclic AMP and cyclic GMP, the addition of cyclic GMP stimulating the hydrolysis of cyclic AMP. Peaks 3 and 4 selectively hydrolyzed cyclic AMP, peak 3 being very sensitive to inhibition by rolipram and peak 4 consisting of two different enzyme activities, one inhibited by cyclic GMP (peak 4b) and the other inhibited by rolipram (peak 4a). In cardiomyocytes and whole ventricle from newborn rats, the response of the two cyclic GMP-sensitive PDE isoforms (cyclic GMP-stimulated and cyclic GMP-inhibited PDE) to the effector cyclic GMP was markedly reduced as compared with that of the corresponding isoforms (peaks 2 and 4b) present in the heart ventricle of adult rats. The other peaks were analogous to peaks 1, 3, and 4a of the whole ventricle. The profile of PDE activities in nonmyocardial cells did not differ from that of myocytes. Therefore, the lack of cyclic GMP effects was not due to culture conditions. Differences in PDE activities observed between cardiomyocytes from newborn rats and ventricle from adult rats might be age dependent.

Chromium speciation by anion-exchange high-performance liquid chromatography with both inductively coupled plasma atomic emission spectroscopic and inductively coupled plasma mass spectrometric detection

Development of a new method for the determination of Cr(III) and Cr(VI) is described. Anion-exchange high-performance liquid chromatography (HPLC) was used to separate Cr(III) and Cr(VI) with on-line detection by inductively coupled plasma atomic emission spectroscopy (ICP-AES) at 2766 Å in preliminary studies, and inductively coupled plasma mass spectrometry (ICP-MS) with single-ion monitoring at m/ z 52 and m/ z 53 for final work. A mobile phase consisting of ammonium sulfate and ammonium hydroxide was used, and a simple chelation procedure with EDTA was followed to stabilize the Cr(III) species in standard solutions. ICP-MS results indicated the feasibility of using chromium isotope m/ z 53 instead of the more abundant m/ z 52 isotope due to a high mobile-phase background most significantly from the SO + polyatomic interferance. The absolute detection limits based on peak-height calculations were 40 pg for Cr(III) and 100 pg for Cr(VI) in aqueous media by HPLC-ICP-MS. The linear dynamic range extended from 5 ppb (ng/ml) to 1 ppm (μg/ml) for both species. By HPLC-ICP-AES, detection limits were 100 ng for Cr(III) and 200 ng for Cr(VI). Cr(III) was detected in NIST-SRM 1643c (National Institute of Standards and Technology-Standard Reference Material, Trace Elements in Water) by HPLC-ICP-MS at the 20 ppb level.

HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides

This unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion-exchange high-performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge-to-mass ratio can also contribute to elution position.

Aquatic soluble unreactive phosphorus: HPLC studies on concentrated water samples

Soluble unreactive phosphorus (SUP) in concentrated forest stream (Walker Branch, Tenn.) and lake water (Crystal Lake, Ill.) samples was analyzed with an anion-exchange high performance liquid chromatography (HPLC) system containing a phosphorus-specific detector. The detector, which utilized the ascorbic acid-molybdate reaction, consisted of a flow-injection system, a post-column reactor, and a u.v.-vis detector. Before HPLC analysis, samples were concentrated and molecular size fractionated with a series of ultrafiltration and reverse osmosis membranes. The SUP composition was found to be a function of season in both Crystal Lake and Walker Branch, as well as a function of stream length for Walker Branch. Seasonal and spatial SUP variations could be explained using contemporary knowledge of nutrient cycling in streams and lakes. A signal, seen in many of the HPLC traces and which eluted with the solvent front, was characterized but not fully identified using several extraction and degradation methods, as a function of sample concentration.

Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.

High-performance liquid chromatographic system for separating sugar phosphates and other intermediary metabolites.

A simple method for separating intermediates of carbohydrate metabolism, including the difficult-to-resolve sugar phosphates, using anion-exchange high-performance liquid chromatography is described. A gradient of decreasing borate concentration (10 to 0 mM) and increasing ionic strength (0 to 150 mM NH4Cl) separates phosphorylated sugars based on the strength of the ester complex that they form with borate anion. Lyophillized samples are reconstituted in a low ionic strength aqueous medium (5 mM triethanolamine-HCl, pH 8.1) and chromatographed on a commercially available anion-exchange column (Hamilton PRP-X100). The process requires only 3 h and permits nanomole detection of the phosphorylated intermediates of the glycolytic and pentose shunt pathways.

Contribution of high-performance liquid chromatographic analysis of carbohydrates to authenticity testing of honey

A high-performance anion-exchange liquid chromatography method with pulsed amperometric detection was developed to investigate the oligosaccharide composition of honey. Quantification was achieved by reference to a maltotetraose internal standard. The oligosaccharide profiles of 91 authentic UK honey samples were obtained and multivariate statistical techniques employed to investigate whether these profiles could be used as a basis for identification of flower source. Canonical Discriminant Analysis proved most successful. Results indicated that honey oligosaccharide profiles have a potentially valuable role to play in the assessment of floral origin of honey though it is unlikely that this procedure alone will allow unambiguous determination of all floral types.

Characterization of cancer cell dissociation factor in a highly invasive pancreatic cancer cell line.

Two pancreatic cancer cell lines, the highly invasive and metastatic cell line PC-1.0 and the weakly invasive and rarely metastatic cell line PC-1, were established from a pancreatic ductal carcinoma induced by N-nitrosobis (2-oxopropyl) amine in a Syrian golden hamster. The cancer cell dissociation activity in serum-free conditioned medium of PC-1.0 cells was partially purified using a heparin column, a hydroxylapatite column, anion exchange, and gel filtration high-performance liquid chromatography. Several biologic properties of the partially purified activity were evaluated. Two cell lines exhibited different growth morphologic changes in vitro: the weakly invasive cell line PC-1 formed islandlike colonies, and the highly invasive cell line PC-1.0 grew mainly as single cells. The conditioned medium of PC-1.0 cells induced dissociation of islandlike colonies and morphologic changes of PC-1 cells to elongated cells, with a high frequency of pseudopodia formation similar to the morphologic findings of PC-1.0 cells. The dissociation activity did not bind to the heparin column and had an apparent molecular mass of > 400 kDa, as deduced from gel filtration. Several immunoreactive proteinous bands were observed in immunoblotting analysis using a polyclonal blocking antibody. The partially purified activity enhanced cell motility, chemoinvasion, and cell adhesion to plastic plates and fibronectin. Highly invasive and metastatic PC-1.0 cells produce a soluble proteinous factor, called "dissociation factor" (DF), which induces cell dissociation of weakly invasive and rarely metastatic PC-1 cells. It seems likely that DF has a role in tumor invasion and metastasis.

Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation.

HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high performance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-alpha-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-alpha-O-benzyl was metabolized by the cells to Gal beta 1-3GalNAc-alpha-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan alpha-2,3-sialyltransferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sialylation.

Metabolism of cyclic ADP-ribose in opossum kidney renal epithelial cells.

We have previously shown that NAD+ inhibits renal Na(+)-Pi symport; however, the biochemical mechanism of NAD+ in this action is not clarified. We now propose that NAD+ acts indirectly by first being converted to cyclic ADP-ribose (cADPR), a potent stimulator of intracellular Ca2+ mobilization. In permeabilized opossum kidney (OK) cells, a cell line often employed as a model for study of proximal tubular epithelial transport, cADPR is synthesized from beta-NAD+ in a substrate concentration (0.01-1 mM) and time-dependent manner. That cADPR was generated from beta-NAD+ by OK cells was verified by coelution with authentic cADPR on anion exchange high-performance liquid chromatography and by homologous desensitization of the Ca2+ release bioassay to authentic cADPR. cADPR synthesized by permeabilized OK cells was not influenced by the addition of parathyroid hormone. The OK cell also contains the enzyme activity necessary to catalyze catabolism of cADPR. Identification of these two key enzyme activities of cADPR metabolism in OK cells is consistent with a possible role of cADPR in regulation of the Na(+)-Pi symporter by NAD+ in response to metabolic stimuli.

Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards.

The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside, were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-[1-3H]mannitol derivatives by deamination with nitrous acid followed by reduction with NaB3H4. The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/NaB3H4 treatment of heparan sulphate isolated from human brain. The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure. Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard. The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position. No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard. It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3. By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.

Alkaline stability of the oligosaccharides present in beet medium invert sugar

The stability of the components of beet medium invert sugar, d-glucose, d-fructose, sucrose, and oligosaccharides, in the eluent used for anion-exchange high-performance liquid chromatography (HPLC) analysis with pulsed amperometric detection has been determined. The monosaccharides undergo degradation when stored in 100 m m sodium hydroxide but the degradation products produced elute before the beet medium invert sugar fingerprint oligosaccharides. Sucrose and the fingerprint beet medium invert sugar oligosaccharides do not produce detectable degradation products when stored under similar conditions. The presence of sodium acetate, the eluting salt in the HPLC method, does not alter the degradation product spectrum. The fingerprint peaks in the oligosaccharide region, are components of beet medium invert sugar and not artefacts of the anion-exchange HPLC method; they are stable under the HPLC analysis conditions used.

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using direct injection fast anion-exchange chromatography and capillary gel electrophoresis.

The analysis of antisense phosphorothioate DNA (SODN) in human plasma via direct injection using anion-exchange high-performance liquid chromatography (AE-HPLC) is presented. The method relies on the ability to selectively extract phosphorothioate DNA from undigested serum, plasma and urine on anion-exchange resins. The automated HPLC method can analyze a sample every 5 min with a limit of detection of 50 ng/ml (ppb). The DNA was collected, desalted and analyzed by capillary gel electrophoresis. Due to the high resolving power of this technique, a qualitative assessment of enzymatic degradation of the antisense oligonucleotide can be made.

Preparative isolation of the lectin jacalin by anion-exchange high-performance liquid chromatography

The lectin jacalin from Artocarpus integrifolia was purified to homogeneity in a single step by preparative anion-exchange high-performance liquid chromatography (HPLC). Selection of the optimum Chromatographie parameters in gradient elution allowed a rapid procedure to be obtained for the qualitative and quantitative isolation of the most important α- and α'-jacalin components. A recovery of 27–33% was obtained from a total soluble extract using a polyacrylate-DEAE HPLC column. The identities of the two isolated polypeptides were established by N-terminal amino acid sequence analysis and from the IgA 1 binding lectin activity.

Biosynthesis of heparin. Different molecular forms of O-sulfotransferases.

O-Sulfotransferases involved in heparin biosynthesis were purified > or = 10,000-fold from detergent extracts of mouse mastocytoma tissue by sequential chromatographies on DEAE-Sephacel, heparin-agarose, blue Sepharose, and 3',5'-ADP-Sepharose. The resultant preparation catalyzed the transfer of 35S from 3'-phosphoadenosyl-5'-phospho-[35S]sulfate into N,O-desulfated, re-N-sulfated heparin. Anion-exchange high performance liquid chromatography of disaccharides obtained by deaminative cleavage of the 35S-labeled polysaccharide product revealed O-35S-sulfation at C-2 of L-iduronic acid and at C-6 of D-glucosamine units. SDS-polyacrylamide gel electrophoresis of semipurified enzyme followed by extraction of gel segments and renaturation of proteins consistently showed two distinct fractions of O-sulfotransferase activity, corresponding to proteins of approximately 20 and approximately 60 kDa. The approximately 60-kDa enzyme(s) catalyzed both the 2-O- and 6-O-sulfotransferase reactions, whereas the approximately 20-kDa fraction promoted iduronosyl 2-O-sulfation only. These results are discussed in relation to previous findings, indicating that some of the enzymes involved in heparin biosynthesis catalyze more than one reaction.

Separation and Analysis of 4′-Epimeric UDP-Sugars, Nucleotides, and Sugar Phosphates by Anion-Exchange High-Performance Liquid Chromatography with Conductimetric Detection

An anion-exchange HPLC method coupled with conductimetric detection was developed for the analysis of UDP-sugars, nucleotides, and sugar phosphates, each in a single chromatographic run. The analysis was applicable to concentrations over 50 pmol. The utility of this technique was demonstrated by the measurement of UDP-sugar 4′-epimerase activity of cell-free extracts from wild-type and mutant Neisseria meningitidis serogroup B strains, Additionally, the method has been applied to the analysis of the intermediates of glycolysis in human erythrocytes.

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Analytical and Preparative Separation of Anionic Oligosaccharides by Weak Anion-Exchange High-Performance Liquid Chromatography on an Inert Polymer Column

The use of ammonium formate buffers for analytical and preparative weak anion-exchange high-performance chromatography of anionic sugars is described. The method can be used for structures containing sulfate, phosphate, sialic acid, and uronic acid moieties. Excellent separation of the anionic sugars was achieved from pH 5.5 to 9. In contrast to silica matrix columns the polymer matrix column used can tolerate a wide range of pH values for the eluting buffer and it does not shed material into the eluent. The use of a single volatile buffer avoids additional purification steps to recover separated sugars suitable for further structural and functional analyses.