Ion-exchange Chromatographic Method Research Articles

Quantification of metallothionein isoforms in fish liver and its implications for biomonitoring

A rapid, reproducible, and sensitive ion-exchange chromatography (IEC) method combined with graphite-furnace atomic absorption spectrometry (GF-AAS) was developed to separate and quantify basal amounts of both metallothionein (MT) isoforms in dab (Limanda limanda) liver samples. Dab liver homogenates were saturated with Cd, and obtained cytosols were purified by a two-step acetone precipitation prior to chromatographic analysis. Metallothionein isoforms were separated by IEC and were subsequently quantified indirectly by GF-AAS via their Cd contents. The amount of Cd needed for saturation was optimized. The efficiency of Cd saturation and acetone precipitation was proven by gel permeation chromatography (GPC) and metal distribution analysis. Based on the method of standard addition, a recovery for MT was 98% after acetone precipitation and 68% after IEC. The repeated determination of MT isoforms in a dab liver homogenate resulted in coefficients of variation of approximately 12% for both isoforms. Based on the detection limit for GF-AAS, the calculated detection limit for MT isoforms is 2 ng/mg protein. Therefore, this method is suitable for monitoring purposes. The implications of isoform-specific measurement for biomarker monitoring are discussed.

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.

Development of ion exchange chromatography methods for monoclonal antibodies

Development of ion exchange chromatography methods for monoclonal antibodies

Conservation of functional site structure across different membrane transport protein families

Beuuveriu hsim, pwn in potato dextrose broth and measured their activity by their ability to agglutinate human erythrocytes. The lectins were purified by ion-exchange chromatography and affinity methods to give preparations that gave a single band in SDS-PAGE. Basic characterization has been carried out. The X. polvtnorphu lectin shows specificity for lactose and the B. hassiunu lectin for N-acetyl-Dglucosamine; both lectins have subunit masses of approx. 17 kDa. The N-terminal of each protein was determined. The X. pnlvmrphu lectin shows similarities with the galectin isolated from the fungus Coprims cinereus and is, possibly, a new member of the galectin family. B. hassiaM is insecticidal and it is interesting to note that its lectin has sequence homology with the N-acetyl-D-glucosamine-specific lectin from the fungus

Gene transfer in vitro and in vivo by cationic lipids is not significantly affected by levels of supercoiling of a reporter plasmid.

It is a common preconception that supercoiled plasmid DNA is more desirable for the transfection of cells that the relaxed form of the plasmid. This notion has led to the recommendation that a specification for the minimum amount of plasmid in the supercoiled form should exist in a gene therapy product. We have tested this notion by examining the effects of the degree of supercoiling on cationic lipid-mediated gene transfer in vitro and in vivo. An ion-exchange high performance liquid chromatography (HPLC) method was developed to accurately quantitate the relative amounts of supercoiled DNA in purified plasmid. A sample of the purified plasmid was fully relaxed using topoisomerase. Next, the ability of various levels of supercoiled plasmid to transfect mammalian cells was measured. This study suggests that there is no relation between the degree of supercoiling and lipofection efficiency. Subsequent transfection using several different lipofection agents, different cell types, and an in vivo model support these results. In considering a specification for the amount of supercoiled plasmid in a gene therapy product, it must be noted that the relaxed forms of the plasmid are no less efficient at gene delivery than the supercoiled forms.

Isolation and Characterisation of Four Distinct Cytotoxic Factors of Salmonella Weltevreden

Four distinct cytotoxins with different biological, physico-chemical and antigenic characteristics were isolated from a single Salmonella Weltevreden strain recovered from buffalo meat. The toxins were purified by means of salt precipitation, dialysis, gel filtration and ion-exchange chromatographic methods. Cytotoxin I was dermonecrotic, verocytotoxic and lethal for the mouse (LD50 120 micrograms). It induced accumulation of serosanguinous fluid in the rabbit ligated ileal loop (RLIL) and mucinous fluid in the stomach. It was active within a narrow pH range (5.0-8.0) and lost its activity on autoclaving for 1 min. Cytotoxin II was verocytotoxic, enterotoxic and lethal to for the mouse (LD50, 1 mg). It induced delayed vasopermeability in rabbit skin, was active at between pH 5.6 and 8.4 only and heat-sensitive (100 degrees C, 30 min). Cytotoxin III was neither dermatotoxic nor enterotoxic. It induced vacuolation, multinucleation and formation of syncytia in Vero cells. It was pH-sensitive beyond the range of 4.8 to 8.2. It was completely inactivated on boiling for 30 min. Cytotoxin IV was intensely necrotizing to rabbit skin within 6 h of inoculation and lysed Vero cells. It also possessed haemolytic and lecithinase/phospholipase-C activities. The cytotoxin lost its activity on heating at 90 degrees C (30 min) but remained active between pH 2.5 and 7.5. The PIs of the cytotoxins were estimated to be 9.0, 7.0, 5.6 and 3.0, respectively. All the four cytotoxins were immunogenic in rabbits but antigenically unrelated as the anticytotoxin neutralized only the homologous cytotoxin and did not cross-react with heterologous cytotoxins.

Measurement of phenylalanine and tyrosine in plasma by high-performance liquid chromatography using the inherent fluorescence of aromatic amino acids.

An isocratic high-performance liquid chromatography (HPLC) method is described using the natural fluorescence of phenylalanine and tyrosine compared with that of an internal standard N-methyl phenylalanine. Plasma precipitated with 6% perchloric acid was separated isocratically using a base-deactivated C18 column with 5% acetonitrile in water as the mobile phase. Fluorescent measurements at an excitation wavelength of 215 nm and emission 283 nm showed only three peaks for tyrosine, phenylalanine and the internal standard eluting within 9 min. Inter-batch coefficients of variation for phenylalanine were 2.9% and 1.8% at levels of 70 and 567 mumol/L, respectively, and 2.9% at a level of 63 mumol/L for tyrosine. The results for phenylalanine for this method showed a small mean positive bias (11 mumol/L) when compared with the target all-method means for UK National External Quality Assessment Scheme samples (n = 31). The results for tyrosine showed a small positive mean bias (10 mumol/L) when compared with an ion-exchange chromatographic method (n = 40). This method provides a quick and simple alternative to those using HPLC with pre- or post-column derivatization for monitoring patients with phenylketonuria. It is also less subject to interferences than HPLC methods using ultraviolet detection, particularly for the early eluting tyrosine peak.

Identification of L-amino acid/L-lysine alpha-amino oxidase in mouse brain.

Lysine, an essential amino acid is catabolized in brain through only the pipecolic acid pathway. During the formation of pipecolic acid, alpha-deamination of lysine, and the formation of the alpha-keto acid as well as its cyclized product are pre-requisites. The enzyme mediated alpha-deamination of L-lysine and the formation of the alpha-keto acid and the cyclized product are not demonstrated so far. Both lysine and pipecolic acid are known to increase in brain under the conditions of fasting, studies were therefore undertaken to identify the enzyme responsible for the alpha-deamination of L-lysine in the brain tissue of mice which were fasted. The detection of the alpha-keto acid of L-lysine -alpha-keto-epsilon-amino caproic acid and its cyclized product-delta-piperidine-2-carboxylate was facilitated by the use of L-[U-14C]-lysine as the substrate. The quantitation of the radioactivity in reaction products was done after separation by ion exchange chromatographic methods. The formation of the alpha-keto acid was enzyme mediated, the alpha-keto acid formed was established by reaction with N-methyl benzothiazolinone hydrazone hydrochloride. The cyclized product was accounted in a fraction which matched the resolution of authentic pipecolic acid on the Dowex column, and the cyclized product was confirmed by spectrophotometry. The hitherto undemonstrated alpha-amino deaminating enzyme of L-lysine in brain tissue, the alpha-keto acid of L-lysine and its cyclized product in a mammalian system could thus be demonstrated in the present study. These findings confirm the involvement of L-lysine oxidase/L-amino acid oxidase in the formation of pipecolic acid from L-lysine.

Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane

Two ion-exchange chromatographic methods are reported for the rapid isolation of antibacterial peptides from lactoferrin (LF). Using the first method, a pepsin hydrolysate of LF was fractionated by bead-based cation-exchange chromatography. After removal of weakly bound material by washing with ammonia, highly purified lactoferricin-B (LFcin-B) was obtained in a single step by elution with 2 M NaCl. Some other cationic peptides, copurified as minor components, were also characterised by N-terminal sequencing, mass spectrometry and antibacterial activity determination. With the second method, cheese whey was filtered through a cation-exchange membrane, and the selectively bound LF was directly hydrolysed in situ with pepsin. Inactive LF fragments were washed off the membrane with ammonia, and a fraction enriched in LFcin-B was obtained by further elution with 2 M NaCl. The membrane method is more rapid and offers several economic advantages.

Purification of coniferyl alcohol oxidase from lignifying xylem of sitka spruce using immobilised metal affinity chromatography

Summary Extracts of lignifying xylem of Sitka spruce, obtained by a combined extraction/affinity method that selects for cell-wall-associated glycoproteins, were enriched in oxidase and peroxidase activity but peroxidase activity was approximately 80 times more abundant than oxidase activity. Previous attempts to purify the oxidase using ion-exchange, hydrophobic interaction and gel filtration chromatographic methods have been unsuccessful and it has proved particularly difficult to separate completely oxidase activity from peroxidase activity. All of the oxidase activity in the xylem cell wall extracts bound to an affinity matrix loaded with immobilised copper (Cu2+) ions and was fractionated into two main peaks by eluting the bound proteins with a gradient of histidine. The second oxidase peak, which represents a subset of oxidase that has higher affinity for the bound metal ions, contained a third of the total oxidase activity at a 2-fold increase in specific activity. In addition, this fraction had greatly reduced although still detectable peroxidase activity. Oxidase activity was split into a bound and an unbound fraction when extracts were applied to a matrix of immobilised cobalt (Co2+) ions. In this case, a larger proportion of the applied oxidase activity than peroxidase activity bound to the Co2+-loaded matrix and the specific activity was higher than that in the unbound fraction. This suggested that this bound fraction of the oxidase population had a higher affinity for metal ions and could therefore be isolated from peroxidase. When applied to a Zn2+-loaded matrix, the oxidase activity also split into bound and unbound portions but effectively all of the peroxidase activity was eluted in the unbound fraction yielding a bound fraction enriched in oxidase activity with negligible peroxidase contamination. The nature of this «high affinity fraction» of oxidase and the heterogeneity in the oxidase population is discussed. Gel permeation on Superose-6 partially resolved oxidase from the residual peroxidase activity in the Zn-bound fraction and gave an apparent Mr of 62 kDa for the oxidase activity. However, an examination of the protein profile of fractions enriched in oxidase activity by SDS-PAGE strongly suggests that a protein band of apparent Mr 80 kDa is responsible for the oxidase activity. This apparent disparity is discussed. In summary, immobilised metal affinity chromatography on Zn-loaded matrix provides a rapid method to separate oxidase from peroxidase activities enabling studies of the substrate specificity and the physical properties of the oxidase to be carried out.

Purification in Quantity of the Secreted Form of WI-1: A Major Adhesin onBlastomyces dermatitidisYeasts

WI-1, a 120-kDa adhesin onBlastomyces dermatitidis,binds the yeast to macrophages and is a major target antigen of immune recognition in acquired resistance to the fungus. In past studies, WI-1 has been purified by extracting the protein from the yeast cell wall, which yields microgram quantities for biological assays. We report a strategy for generating and purifying the secreted form of WI-1 in quantity. Yeasts ofB. dermatitidisATCC strain 60636 cultured in HMM medium were found to secrete 10 μg/ml of WI-1 into a supernate relatively free of other medium and yeast components. Using a two-step method of ion exchange and hydrophobic interaction chromatography, we achieved a 7.1-fold purification of WI-1. Purified WI-1 was sequenced at the N-terminus which revealed that the secreted protein exists in two different forms. In functional assays, purified WI-1 also retained its adhesivity for human macrophages, and its antigenicity in binding anti-WI-1 antibodies and stimulating T-cells to proliferate, but it lost some capacity to elicit delayed-type hypersensitivity in mice. These findings advance our understanding of the WI-1 adhesin/antigen and our ability to express and purify WI-1 in quantity and will permit a study of the relationship between three-dimensional structure and activity of the molecule.

Determination of organic and inorganic anions in insect haemolymph and midgut contents by ion chromatography

A simple and rapid ion-exchange chromatographic method with conductimetric detection for the determination of carboxylic acids (succinic, malic and citric) and inorganic anions (chloride, phosphate and sulphate) in haemolymph and midgut content of Lepidoptera larvae is provided. The mobile phase is 0.975 m M phthalic acid at pH 4.1. The procedure of sample extractions was simple. Comparison of the results with whose reported in the bibliography showed that the values were similar. The ion-exchange chromatographic–conductimetric detection method permits the analysis of various organic and inorganic anions in small biological samples.

Fructans in Foods and Food Products, Ion-Exchange Chromatographic Method: Collaborative Study

Abstract Nine collaborating laboratories assayed 6 blind duplicate pairs of food samples containing the fructans inulin or oligofructose. The 6 sample pairs ranged from low (4%) to high levels (40%). Following the proposed method, the samples were treated with amyloglucosidase and inulinase enzymes and the released sugars were determined byion exchange chromatography. Repeatability standard deviation ranged from 2.9 to 5.8%; reproducibility standard deviation ranged from 4.7 to 11.1%. The ion-exchange chromatographic method for determinationof fructans in food and food products has been adopted first action by AOAC INTERNATIONAL (997.08)

Decreased cerebrospinal fluid levels of neutral and basic amino acids in patients with Parkinson's disease

We measured the CSF levels of 21, and the plasma levels of 26, amino acids in 31 patients with Parkinson's disease (PD) and in 45 matched controls. We used an ion-exchange chromatography method. When compared to controls, PD patients had lower CSF levels of taurine, alanine, valine, leucine, isoleucine, ethanolamine, citrulline, ornithine, lysine, histidine, arginine, and alpha-aminobutyric acid. PD patients not treated with levodopa or with dopamine agonists had higher CSF tyrosine and phenylalanine levels than those not treated with these drugs and also than controls. PD patients had higher plasma levels of phosphoserine, threonine, methionine, tyrosine, sarcosine and α-aminoadipic acid, and lower plasma levels of valine, leucine, and tryptophan, than controls. The CSF/plasma ratio of many of these amino acids was significantly lower in PD patients than those of controls, suggesting that PD patients might have a dysfunction in the transport of neutral and basic amino acids across the blood–brain barrier.

Derivative Spectroscopy in Conjunction with Thin Layer Chromatography Applied for Determination of Mixtures of Amino Acids in Baby Food.

The preparative thin layer chromatography using silica gel and solvent systems n. butanol : acetic acid: water (4:1:1 V/V) and 96% ethanol : water (70 : 30 v/v) were used to separate the amino acids mixture into groups that can be eluted and one or two of amino acids can be determined by derivative spectroscopy. This was done for determining histidine in presence of cystine, arginine in presence of lysine and ornithine, tryptophan in presence of tyrosine and phenylalanin, and methionine in presence of aspartic acid. The proposed method is applied for determination of these amino acids in baby food, Neslac, Dialac and Nestogen. The statistical analysis of the results were found to be good and in agreement with the ion exchange chromatographic method.

Simple liquid-liquid extraction of U and Th in tracer amounts from phosphate matrix for dating purposes

Existing procedures for 230Th dating of phosphate-rich materials (bone, teeth) are labor-intensive and troubled with variable, operator-dependent results. A liquid-liquid extraction procedure was developed, using a long-chained quaternary ammonium salt as the organic extractor. Quantitative separation of U and Th with chemical yields of 80–90% are attainable, at a tolerance of up to 200 g Ca 3(PO 4) 2 per litre dissolved sample. Liquid-liquid extraction is very fast, and appears not to produce any bias on the resulting dates, as compared with the more time-consuming ion-exchange chromatographic methods in common use. The technique also works well on conventional material, like carbonate fossils and speleothems.

HPLC methods for purification of antimicrobial peptides.

AbstractThe advent of high performance liquid chromatography (HPLC) has greatly accelerated the discovery, purification, and characterization of antimicrobial peptides. Virtually every modern study of an antimicrobial peptide includes or was preceded by a description of its purification. The increased pace of peptide discovery and characterization has resulted from the development of sophisticated column and solvent delivery technology over the last four decades. Interestingly, the modern methods described here derive directly from standard open-column (“low performance”) chromatographic modalities. Therefore, it is not surprising that virtually every method used in traditional column chromatography has been adapted to high performance methods. These include gel filtration, ion-exchange, and reversed-phase chromatography methods described in this chapter.KeywordsHigh Performance Liquid ChromatographyHigh Performance Liquid ChromatographyAntimicrobial PeptideHigh Performance Liquid Chromatography MethodHigh Performance Liquid Chromatography SystemThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The use of crude tissue section lysates for PCR assessment of gene copy number with human tumour biopsy samples

This paper reports the development of experimental procedures for the use of crude tissue section lysates, in conjunction with a non-radioactive polymerase chain reaction-high performance ion-exchange chromatographic ( PCR HPIEX ) method, for the quantitative assesment of gene copy number in human biopsy samples. In particular, these methods have been established for the assessment of gene amplification with the FGF-2, FGF-3, FGF-4 and c- erb-B2 genes with the a single copy IFN-γ gene used as an internal control. In principle, the same procedures could, in general, be simultaneously applied for monitoring the amplification or deletion of other oncogenes as well as normal genes in mammalian cells. Procedures to optimise the precision of the analysis have been examined, including the influence of the oligonucleotide primer concentrations, cycle number, extension temperature, and method of pre-treatment of the tissue sample with proteinase K. The results confirm that crude tissue lysates, rather than purified DNA samples, can be reliably employed, thus extending the scope of non-radioactive PCR HPIEX methods for the assessment of aberrant gene copy number to biological samples such as tumour biopsy tissues.

Comparison of APDC/FREON Extraction and Chelex-100 Ion Exchange Chromatography for Preconcentration of Heavy Metals from Seawater

Abstract Two methods for preconcentration of heavy metals from seawater: APDC/Freon extraction and CHELEX-100 ion exchange chromatography, have been compared. Retention of heavy metals on a CHELEX-100 resin in the acid form was not complete. Consistent and reliable results were obtained for Ni. Cd, Pb, and Zn by both studied preconcentration methods. CHELEX-100 ion exchange chromatography is not suitable for Fe and Cu preconcentration because of the low recoveries obtained, 79 and 72% respectively. Applying the CHELEX-100 ion exchange chromatography method, the preconcentration factor and the consuming time were higher than when the APDC/Freon extraction method was used.

Neurotransmitter amino acids in cerebrospinal fluid of patients with Parkinson's disease

We measured the CSF and plasma levels of glutamate, glutamine, aspartate (only in plasma), asparagine, glutamine, glycine and GABA in 31 patients with Parkinson's disease and in 45 matched controls. We used an ion-exchange chromatography method. When compared to controls, PD patients had similar CSF levels of glutamate, glutamine, asparagine, and glycine higher CSF GABA levels higher plasma levels of glutamine, asparagine, and glycine, and lower plasma levels of aspartate. The CSF levels of the amino acids measured were not correlated with the clinical features of PD. Our results that CSF GABA levels are not decreased in PD as previously suggested.