Anion-exchange High-performance Liquid Chromatography Research Articles

Determination of carbon sources in fermentation media using high-performance anion-exchange liquid chromatography and pulsed amperometric detection

The technique of high pH anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) has been adapted for the rapid determination of common carbohydrates present in fermentation broths. Simple water dilution of filtered fermentation broth samples was the sole sample preparation step required. The samples were analyzed using a Dionex CarboPac PA1 column with 150 m M NaOH as the mobile phase at a flow-rate of 1 ml/min and a total run time of 20 min. A gradient method was also developed to resolve species which exhibited similar t R in the isocratic procedure. Among the analytes examined in this study were ethanol, glycerol, galactose, glucose, mannose, fructose, raffinose, ribose and lactose. Examples from several microbial fermentations using chemically defined or complex medium are presented.

Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography.

A new anion-exchange HPLC method for separation of gangliosides has been developed using TMAE (trimethylaminoethyl)-Fractogel as the stationary phase and a gradient system of ammonium acetate in methanol for elution. Chromatography of mouse brain gangliosides resulted in separation according not only to their degree of sialylation into mono-, di-, tri- and tetrasialogangliosides, but also in separation of positional isomers within the four elution domains, e.g. GD1a (IV3Neu5Ac,II3Neu5Ac-GgOse4Cer) and GD1b (II3(Neu5Ac)2-GgOse4Cer) of the disialoganglioside fraction. Furthermore, base-line separation of alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides e.g. IV3Neu5Ac-nLcOse4Cer and IV6Neu5Ac-nLcOse4Cer, isolated from human granulocytes, was achieved and pure fractions of each ganglioside were obtained. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the isolation of pure gangliosides on a preparative scale by one-step column chromatography.

Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II.

A group of low molecular weight protein substrates was found to co-purify with casein kinase II from broccoli (Brassica oleracea var italica). These substrates showed very high affinity toward casein kinase II and were efficiently phosphorylated even in the presence of an excess of exogenous substrates. The broccoli substrates were purified from cytosolic extracts as a double band of related proteins migrating at 18.7 and 20 kD. Further microheterogeneity was revealed by anion-exchange high-performance liquid chromatography and mass spectroscopy. The actual molecular masses of the three major components identified by mass spectroscopy were determined to be 12,691, 13,256, and 14,128 D. The substrates showed characteristic amino acid composition with a high content of polar amino acids, including about 20% each of acidic and basic amino acids. They were soluble in 2% trichloroacetic acid. The substrates cross-reacted with an antibody against wheat high-mobility group protein d (HMGd) but not HMGa. The isolated broccoli HMGs showed general DNA-binding activity without preference for AT-rich DNA. The presence of these HMG proteins in the cytosolic fraction is similar to the distribution characteristics of the animal HMG-1 subgroup. On the basis of amino acid composition and DNA-binding specificity, the isolated broccoli HMGs resemble other plant HMGs homologous to the HMG-1 subgroup.

Heterogeneity of smooth muscle myosin light chain kinase Characterization of isoelectric variants

Purified chicken gizzard myosin light chain kinase (MLCK) analyzed by anion-exchange high-performance liquid chromatography (HPLC) can be consistently resolved into three well separated peaks, termed α, β, γ. These peaks are shown to correspond to differently charged forms of MLCK with the charge difference between α and β twice as large as between β and γ. The isoelectric point and elution position of the peaks as well as their amplitudes are modified by phosphorylation or by autophosphorylation of MLCK suggesting that the observed charge differences are related to their different phosphate content. The three forms appear to have similar apparent affinity for both the substrates, ATP and the isolated regulatory light chain, but their specific activities are different.

Isolation and characterization of naturally and artificially induced hepatic metallothioneins from two species of flatfish: Limanda limanda and Microstomus kitt

The separation of hepatic metallothioneins of Limanda limanda and Microstomus kitt by gel-filtration chromatography, anion-exchange high-performance liquid chromatography (HPLC) and reversed-phase HPLC is described. Two isoforms of metal-binding proteins were isolated by DEAE HPLC chromatography from fish caught in the field and fish injected intraperitoneally with Cd. The amino acid composition of these proteins further purified by reversed-phase HPLC chromatography revealed the existence of metallothioneins. The route of contamination (natural or artificial) of the fish has no effect on the nature of these proteins and one of the two protein isoforms from each species has similar ionic properties.

Single-step purification of recombinant melanoma growth-stimulating activity by anion-exchange high-performance liquid chromatography.

The cytokine melanoma growth-stimulating activity (MGSA) is a growth factor for melanoma cells and a chemotaxin for neutrophils. Known purification procedures of MGSA from human sources or expression systems give a low yield and require multiple chromatography steps. Here, a fast and high-yield method for the purification of recombinant MGSA is described. Approximately 500 micrograms MGSA were recovered from the bacterial lysate of a 10 liter culture within a day. For this purpose, total mRNA of Hs294T melanoma cells was isolated and cDNA of MGSA was obtained by reverse transcription and polymerase chain reaction. The cDNA of MGSA was subcloned into the expression vector pGEX-2T, generating a fusion with the Schistosoma japonicum glutathione S-transferase gene. The fusion protein was expressed in E. coli DH5a and purified from the bacterial lysate using glutathione-sepharose beads. MGSA was cleaved from the complex of fusion protein and glutathione-sepharose beads with thrombin and purified to homogeneity by anion-exchange high-performance liquid chromatography with a Mono-S-column. The bioactivity of the recombinant MGSA was assessed by chemotactic migration and triggered [Ca2+]i-transients in human neutrophils. In addition, [125I]MGSA bound specifically to undifferentiated human leukemia cells HL-60 transfected with the cDNA of the interleukin-8 (IL-8) receptor beta with similar properties as [125I]IL-8. Thus, this described method might be a powerful tool to generate large amounts of cytokines in a short time.

Isolation and partial characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation: evidence of activation peptide of human prothrombin.

In order to clarify the characteristics of crystal matrix protein (CMP), which exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis, we have isolated CMP from macromolecular matrix substances of newly-formed calcium oxalate crystals. Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis, anion exchange chromatography and high-performance liquid chromatography. CMP showed the protein band of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CMP was identical to that of human prothrombin. Both anti-CMP polyclonal antibody and anti-human prothrombin antibody cross-reacted well with human prothrombin and CMP in Western blotting. Its amino acid composition and its molecular weight of 31 kDa strongly suggest that CMP is the activation peptide of human prothrombin.

14] High-performance liquid chromatography of oligosaccharides

Publisher Summary This chapter proposes separation methods primarily for oligosaccharides that are radiolabeled by metabolic incorporation of 3H- or 14C-labeled sugar, metabolic incorporation of 35SO4 or 32PO4, or chemical incorporation of 3H at the reducing terminus by reduction with NaB3H4. The most common strategy is to first separate the radiolabeled oligosaccharides into different groups on the basis of their anionic character, using anion-exchange high-performance liquid chromatography (HPLC). Oligosaccharides differing in the number and/or character of their anionic groups are then further fractionated on the basis of size, using ion suppression-amine adsorption (ISAA) HPLC. The effects of chemical modifications and enzymatic digestions on individual species are subsequently monitored by anion-exchange HPLC and ISAA–HPLC along with other methods such as lectin affinity HPLC. Four methods for separation of anionic oligosaccharides are illustrated in the chapter. Fractionation of oligosaccharides bearing zero to four anionic moieties, using MicroPak AX-10 or AX-5 columns and a phosphate buffer (pH 4.0), is effective for oligosaccharides bearing terminal sialic acid. Oligosaccharides-bearing sulfate or phosphate can be fractionated under the same conditions; however, the concentration of the initial eluant is reduced to 2.5 mM in order to retain monosulfated species.

Quantitative Determination of Oligosaccharides in Commercial Soybean Lecithin Products by High-Performance Liquid Chromatography.

A method to quantitatively determine the oligosaccharides in a commercial soybean lecithin product by high-performance liquid chromatography (HPLC) was studied. The lecithin product was dispersed in chloroform/methanol/water (10/8/9, v/v) containing an internal standard, and a portion of the water phase was injected onto the anion exchange HPLC column. Oligosaccharides were selectively monitored by a fluorescence detector after a post-column chemical reaction with arginine. This method was highly sensitive (<0.1μg) and also applicable for the determination of saccharide content in soybeans. By this method, we demonstrated that the oligosaccharides in the product were changed into fluorometrically undetectable components by a browning reaction, and that monosaccharides of soybean were not contained in the product.

A practical reagent for reversible amino-protection of insulin, 3,4,5,6-tetrahydrophthalic anhydride.

Of the examined dicarboxylic acid anhydrides, 3,4,5,6-tetrahydrophthalic anhydride was found to be the best reagent in practical use for reversible amino-protection of insulin in terms of the rapidity of acid-deprotection. Twelve Gly(A1),Phe(B1),Lys(B29)-triacyl-insulins were prepared by reaction of porcine insulin with the dicarboxylic acid anhydrides and time courses for the deprotection of the acylated insulins with dilute acetic acid were investigated by means of capillary zone electrophoresis, and the tetrahydrophthalyl(THP)-insulin obtained with the reagent was the most rapidly deacylated (6 h, 25 degrees C, 0.1 M acetic acid). Isolation of triacyl-insulins and Gly(A1)-THP-, Gly(A1),Phe(B1)-diTHP- and Gly(A1),Lys(B29)-diTHP-insulins using DEAE anion-exchange high-performance liquid chromatography is also described.

Identification and Characterization of Two Isozymic Forms of Arylamine N-Acetyltransferase in Syrian Hamster Skin

Arylamine N-acetyltransferase (EC 2.3.1.5) activity was examined using skin from Syrian hamster. Two isozymes of arylamine N-acetyltransferase, designated NAT-1 and NAT-2, were detected on anion-exchange high-performance liquid chromatography analysis. Both enzyme activities had indistinguishable molecular masses (30 kDa), but differed significantly in their specificity toward the aromatic amines including serotonin, dopamine, methoxytryptamine, tryptamine, para-phenetidine, para-aminobenzoic acid, and sulphamethazine. Specifically, NAT-2 but not NAT-1 catalyzed acetylation of dopamine to N-acetyldopamine and acetylation of serotonin to form N-acetylserotonin, a direct precursor of melatonin. The two isozymes were also distinguishable based upon their sensitivity toward methotrexate inhibition (50% inhibiting dose for NAT-1 = 380 microM; NAT-2 > 2 mM). The presence of these two activities in the skin raises new questions about the physiologic role of this enzyme in general and in the skin-specific functions in particular.

Nickel—chromium alloy electrode as a carbohydrate detector for liquid chromatography

A nickel—chromium alloy electrode was applied as an electrochemical detector for the analysis of carbohydrates by high performance anion-exchange liquid chromatography. This alloy electrode exhibited excellent sensitivity, reproductivity and stability. The cabohydrates are oxidatively detected by a surface catalyzed process, proposed to involve nickel(III) oxyhydroxides, which are formed in the range of ca. 0.45 to 0.5 V vs a Ag/AgCl reference electrode. The cyclic voltammograms of the NiCr electrode are compared to pure Ni in order to understand the basic mechanism of the oxidation. The effect of the temperature, the sodium hydroxide concentration, the mobile phase flow-rate, and the working potential on the electrode response were analyzed to optimize the detection conditions. The reproducibility of the electrode response to the injection of 100 pmol of glucose was studied continuously for a period of 21 days. A limit-of-detection (LOD) of approximately 500 fmol of glucose (signal-to-noise ratio of 3) makes this alloy electrode well suited for the high sensitive detection of carbohydrates.

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE). O- and N-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection. N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.

Purification of monoclonal antibodies with light-chain heterogeneity produced by mouse hybridomas raised with NS-1 myelomas: application of hydrophobic interaction high-performance liquid chromatography

Monoclonal antibodies (mAbs) of IgG1 class produced by hybridomas raised with NS-1 myelomas, which were purified homogeneously by anion-exchange high-performance liquid chromatography (HPLC), contained two types of immunoglobulin light (κ) chain. Since NS-1 myeloma synthesizes the light (κ) chain, the mAb was presumed to be the mixture of hybrid mAbs formed by the random association of heavy (γl) and light chains from antigen-immunized spleen cells and ligth chain from NS-1 cells. Hydrophobic interaction HPLC using TSKgel Phenyl-5PW was applicable to separate 3 species of hybrid mAb from mAb fractions obtained by anion-exchange HPLC. mAbs in the fractions were adsorbed onto the gel equiliberated with phosphate-buffered saline containing 1 M ammonium sulfate and eluted by reducing the concentration to 0 M. The hybrid mAbs were purified separately. The hydrophobic interaction HPLC could discriminate a small difference in hydrophobicity between κ chains from spleen and NS-1 cells. The immunoreactivities of hybrid mAb bearing light chains only from spleen cells and that bearing those from both spleen and NS-1 cells were almost the same, and hybrid mAb bearing light chains derived only from NS-1 cells showed a relatively lower immunoreactivity than the others. The method described here could be useful for purification of hybrid mAbs.

Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, in

Structures of N-Linked Sugar Chains Expressed Mainly in Mouse Brain

Sugar chains were liberated from mouse tissues (liver, heart, spleen, kidney, thymus, cerebrum, cerebellum, and brain stem) by hydrazinolysis. After acetylation of the free amino groups, the sugar chains released were pyridylaminated. Pyridylamino (PA-) derivatives of the sugar chains from each tissue were fractionated by anion-exchange high-performance liquid chromatography (HPLC) according to their negative charges. Fractions containing PA-neutral sugar chains thus obtained were separated successively by size-fractionation HPLC and reversed-phase HPLC. Two sugar chains that were more abundant in neural tissues than in other tissues were purified. The structures of the two sugar chains were determined by sugar composition analysis, sequential exoglycosidase digestion, and methylation analysis. The proposed structures are shown below. A structure with n = 1 was found in the cerebrum, cerebellum, and brain stem. A structure with n = 0 was abundant in the cerebrum and brain stem, but less so in the cerebellum. [formula: see text]

Determination of zinc-65, copper-64 and sulphur-35 labelled rat hepatoma tissue culture metallothioneins by high-performance liquid chromatography with on-line radioactivity detection

Molecular size exclusion (MSE), reversed-phase (RP), and anion-exchange (AE) high-performance liquid chromatography (HPLC) techniques were employed in combination with on-line radioactivity detection, in a study on the kinetic behaviour of 65Zn-, 64Cu- and [ 35S]cystein-labelled metallothionein (MT) in rat hepatoma tissue culture (HTC) cells. MSE-HPLC of [ 35S]cysteine-labelled HTC cell cytosol resulted in co-eluting MT-I and MT-II isoforms ( t R 19.80 min; V e/ V o: 1.85). AE-HPLC of 65Zn-treated HTC cell cytosol yielded separated 65Zn MT-I ( t R 11.5 min; I = 64 m M) and 65Zn MT-II ( t R 14.5 min; I = 104 m M). RP-HPLC of 64Cu-treated HTC cytosol resulted in separated 64Cu MT-I ( t R 26.4 min) and 64Cu MT-II ( t R 23.4 min). Determination of the amino acid composition, apparent molecular mass and cysteine content of HTC MT-I and MT-II isoforms showed the characteristics of class I metallothioneins. The rate of dissociation of Zn 2+ from Zn-MT could be determined from the losses of 65Zn from MT during a single AE-HPLC run, showing a Zn-MT dissociation half-life of 0.66 h. RP-HPLC showed a delay in incorporation of newly accumulated 64Cu into MT, possibly owing to the appearance of reduced glutathione as an intracellular copper-transfer compound. Application of compartmental analysis in [ 35S]-cysteine accumulation experiments permitted the determination of the actual rate of MT degradation; when 200 μ M of Zn were applied, the MT degradation half-life was 2.0 ± 0.8 h. These results indicate the potential of combined HPLC techniques and application of radionuclides in studies on the synthesis and degradation of MT and metal-MT complexes.

Acyl carrier proteins from developing seeds of Cuphea lanceolata Ait

The structure of acyl carrier protein (ACP) may determine the fate of the acyl moieties linked to it in the course of de-novo fatty acid synthesis in higher plants. To investigate a possible correlation between the structure of ACP and the synthesis of medium-chain fatty acids, we isolated and characterized ACP from the seeds of Cuphea lanceolata Ait. (subgenus Eucuphea/Section Heterodon), an annual crop that contains up to 90% decanoic (capric) acid in seed triacylglycerols. After a cell-free extract prepared from developing seeds was treated to 65% saturation with ammonium sulfate, two ACP-isoforms (ACP 1 and ACP 2) were identified in the supernatant that could be purified to homogeneity by anion-exchange chromatography and subsequent reversed-phase high-performance liquid chromatography. The molecular mass determined by matrix-assisted ultraviolet-laser desorption ionization mass spectrometry of ACP 1 was 9315 Da, whereas further heterogeneity was observed for ACP 2 with molecular masses of 8598 and 8703 Da. Aminoterminal sequencing was performed showing a high homology in the primary structures of ACP 1 and ACP 2. Both isoforms were present in the embryo, whereas in the chloroplast-containing seed coat ACP 2 was found in minute amounts, if at all. The expression of ACP 2 correlated with the production of capric acid during the phase of storage-lipid accumulation. These data indicate that ACP 2 is part of the machinery for the synthesis of medium-chain fatty acids, whereas ACP 1 appears to be a constitutive protein.

Determination of alkylbenzenesulphonates in environmental water by anion-exchange chromatography

A simple and selective method for the determination of μg/1 levels of alkylbenzenesulphonates (ABSs) in environmental waters is presented. Selectivity for ABSs was obtained by using anion-exchange pre-column concentration followed by anion-exchange high-performance liquid chromatography separation and ultraviolet detection. The method is quantitative and easily applicable to the analysis of real samples. The concentrations of ABSs in tap water and river water were determined to be ca. 0.1 and 100 μg/1, respectively.

Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells.

In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.