Ion-exchange High-performance Chromatography Research Articles

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS–PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of ∼25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZαA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771 Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a p I value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 μm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5–30 min, and even more rapid (1–2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.

Maltosyl-erythritol, a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase.

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-alpha-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts.

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.

Development and application of a sensitive high performance ion-exchange chromatography method for the simultaneous measurement of dopamine, 5-hydroxytryptamine and norepinephrine in microdialysates from the rat brain

A high performance liquid chromatography (HPLC) method based on cation exchange separation has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in microdialysates. The separation conditions have been optimised for using electrochemical detection. All three bioamines were resolved in less than 22 min using isocratic conditions. The optimum oxidation potential for the three bioamines was found to be +0.4 V vs. in situ Ag/AgCl reference electrode. Linear regression analysis of HPLC-peak area as a function of concentrations in the range 1–50 ng ml −1 gave coefficients of correlation between 0.998 and 0.999. The limit of detection for DA, 5-HT and NE was found to be between 50 and 100 pg ml −1 with a signal to noise ratio of 3:1. The method has been applied to the simultaneous measurement of the three monoamines in microdialysates from the medial prefrontal cortex under basal conditions and following the administration of the antipsychotic drug clozapine (10 mg kg −1 s.c.).

Two glycoforms are present in the GPI-membrane anchor of the surface antigen 1 (P30) of Toxoplasma gondii

SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite, the life cycle stage associated with the acute phase of infection. The protein is inserted into the parasite's plasma membrane by a glycosyl-phosphatidylinositol anchor, a modification that is present on all T. gondii surface proteins characterized so far. Here we describe a detailed structural analysis of this anchor. GPI anchor peptides were isolated from [ 3H]glucosamine labeled purified P30 by protease digestion and phase partitioning. Neutral glycans were prepared from this material by dephosphorylation and deamination. Two glycoforms were characterized by gel filtration and high performance ion exchange chromatography in combination with exoglycosidase treatment. Both forms were shown to carry an N-acetylgalactosamine bound to the first mannose of the conserved three-mannosyl core. Glycan B carries an additional terminal hexose linked to GalNAc. To identify the nature of this hexose, bulk anchor peptide was prepared and glycans were purified by aminopropyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-α1,2-Man-α1,6-Man-[GalNAc-β1,4-]-α1,4-GlcN-PI and Man-α1,2-Man-α1,6-Man [Glc-α1,4-GalNAc-β1,4-]-α1,4-GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein.

Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography

An efficient LC method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins based on ion-exchange chromatographic separation of the toxins followed by electrochemical post-column oxidation and fluorescence detection as well as mass spectrometric (MS) detection. The method can be applied to the determination of PSP toxins in phytoplankton and to control seafood for PSP content.

Rapid detection of perchlorate in groundwater using capillary electrophoresis

Perchlorate is a groundwater contaminant originating from facilities that manufacture and test solid rocket fuel. A new technology, capillary electrophoresis, has the potential to measure perchlorate rapidly and inexpensively in water samples. With its speed and simplicity, this method would complement existing methods. The perchlorate anion is routinely detected in water samples using high performance ion exchange chromatography, a very sensitive yet time consuming and expensive method. In this work, the parameters for detection of perchlorate are optimized to permit detection of 0.400 mgL−1 perchlorate in a standard solution. The usefulness of this technology is demonstrated for measuring perchlorate in several ground-water samples from the Western United States. The results demonstrate that CE can be used to rapidly screen environmental samples for perchlorate at intermediate to high levels (greater than 0.400 mgL−1). This technique allows faster, easier screening of potential contamination sites and could complement the use of ion exchange chromatography for groundwater testing.

Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10–11.

Delta-aminolevulinic acid in superficial basal cell carcinomas and normal skin-a microdialysis and perfusion study.

Delta-aminolevulinic acid (ALA) is used for photodynamic therapy of basal cell carcinoma (BCC) as it is converted to protoporphyrin IX in tumour tissue. During illumination with 635 nm light a photochemical reaction takes place, singlet oxygen is generated and the tumour destroyed. In this study we used the microdialysis technique to quantify the concentration of ALA at a certain depth in tumour and healthy skin. The penetration ability of ALA was investigated as a function of time in BCCs (n = 14) and in normal skin (n = 4) after topical application. The microdialysis catheters were inserted intracutaneously and the depth position recorded by means of ultrasound. Microdialysate sample concentrations of amino acids and ALA were determined by high performance ion-exchange chromatography. A laser Doppler perfusion imager measured perfusion in the BCCs. The data show that the average depth of the microdialysis catheters was 0.5 mm. The interstitial ALA concentration in the BCCs increased from 0 to 3.1 mmol/L 15 min after application of ALA, whereas no measurable amounts of ALA were found in healthy skin. The blood perfusion was 2.5-fold increased in the BCCs. The interstitial levels of amino acids were not significantly changed during the ALA treatment. In summary, we found that ALA rapidly penetrates tumour skin. We conclude that microdialysis seems to be well suited for pharmacodynamic studies of ALA in skin.

Evaluating the quality of oligonucleotides that are immobilized on glass supports for biosensor development

Three different analytical techniques were compared to assess methodologies that could evaluate the success of oligonucleotide assembly on glass substrates functionalized with hexaethylene glycol (HEG) linker molecules. Post-synthesis cleavage of the linker-oligonucleotide conjugates from the solid support was done to prepare samples for analysis. Samples were investigated by electrospray ionization mass spectrometry (ESI-MS), high performance ion-exchange liquid chromatography (HPIEC) and polyacrylamide gel electrophoresis after radiolabeling ( 32 P -PAGE). The data from ESI-MS served to identify the various species detected by HPIEC and 32 P -PAGE. All three techniques were shown to be very sensitive to the presence and location (terminal or internucleotide) of HEG conjugated to the oligonucleotide sequence. This allowed differentiation and quantification of linker-oligonucleotides from non-conjugate oligonucleotides that originated from undesired synthesis directly on the glass surface. Furthermore, shorter linker-oligonucleotide conjugates that were formed by incomplete nucleobase-coupling during DNA synthesis on the linker could be identified by HPIEC and 32 P -PAGE, allowing purity assessment of the assembled strands. Despite the inherent higher sensitivity of PAGE of radiolabeled samples, HPIEC was shown to be the method of choice due to high sample throughput and facile quantitative analysis of the products.

Purification of TaqI endonuclease from Thermus aquaticus

A purification procedure for the thermostable restriction enzyme TaqI was developed using high-performance ion-exchange liquid chromatography. The effects of various operating conditions on the separation behaviour of TaqI endonuclease from the cell extracts were investigated for optimisation and scaling up. The separation of the enzyme by HPLC was found to be strongly dependent on the sample volume, slope of linear gradient and order of the ion-exchange columns. The final yield of the enzyme is also dependent to a great extent upon the number of fractionation steps employed to purify the enzyme. In the present study, 4000 U TaqI endonuclease per mg protein was recovered from 2 g Thermus aquaticus cells with a two-step purification protocol in one day. The purification factor was 24. Compared to other classical methods of purification reported in literature with 4000 or 32 000 U enzyme from 200 g of Thermus aquaticus cells, HPLC yielded 190 000 U enzyme from 200 g cells using cation and anion HPLC columns sequentially and thus resulted in a higher efficiency.

Ion-exchange chromatography with an oxalic acid-α-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime

An oxalic acid-α-hydroxyisobutyric acid eluent has been used for the separation and determination of rare-earth elements by high-performance ion-exchange chromatography. Fifteen rare-earth elements were separated within less than 25 min on a 150×4.6 mm i.d. column packed with 5-μm sulfonic acid-bonded silica particles by elution with a combined gradient of 0.60–9.0 mM oxalic acid and 19.0–5.0 mM α-hydroxyisobutyric acid at pH 4.6. Detection and quantitation of the separated rare-earth elements was accomplished by visible-absorbance measurements at 600 nm after postcolumn reaction with arsenazo I. The gradient of the two complexing agents was optimized to enable the separation of yttrium(III) without interference from other elements, especially dysprosium(III) and terbium(III). Mass detection limits of the elements were in the range of 2–4 ng. Finally, the chromatographic system was applied to the quantitative analysis of rare-earth elements in monazite and xenotime.

Purification and Characterization of NADPH–Cytochrome P450 Reductase from Filamentous FungusRhizopus nigricans

We report here the isolation and partial characterization of a flavoprotein, NADPH–cytochrome P450 (cytochromec) reductase. The enzyme is a part of steroid 11α-hydroxylating system and is associated with the microsomal fraction of the fungusRhizopus nigricans.Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 μmol cytochromecreduced/min/mg protein was achieved. A single protein band was obtained on SDS–PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH–cytochromec(P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11β-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11α-hydroxylating activity from the separated components NADPH–cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.

High-performance ion-exchange chromatography and adsorption of plasma proteins

Resolution and recovery are primary concerns in protein chromatography. Separations are often by size, ion exchange, or hydrophobic–hydrophilic properties of the support, eluent and protein. Adsorption of a protein to a synthetic surface plays an essential role in this complex process. In this study, we examined the adsorption properties of three representative plasma proteins (albumin, fibrinogen, and immunoglobulin G) on nonporous column materials containing either quaternary amine or sulfopropyl functional groups. The adsorption properties were studied at 37°C and pH 7.4. Salt gradients were used to examine the adsorption/desorption properties of each of the proteins on each type of surface. The salt concentrations at desorption were measured and compared to the protein isoelectric points. In addition, we examined protein recoveries as a function of desorption time. Our results suggest that protein recoveries depend not only on the protein, eluent and surface, but also the residence time and overall charge concentration during the initial adsorption process. Finally, we correlated the number of charge sites on a molecule with the width of a chromatographic band at half height. The data produced as a result of this study may be used to determine the actual unfolding time of a protein, given a certain set of conditions. The data may also help in understanding the chemistry and dynamics of the protein adsorption processes in ion-exchange chromatography as well as provide key structural information about the proteins.

The relative merits of haemoglobin A1c and fasting plasma glucose as first-line diagnostic tests for diabetes mellitus in non-pregnant subjects.

HbA1c was measured by high-performance ion-exchange chromatography in 401 non-pregnant patients undergoing oral glucose tolerance tests (OGTT). All those with HbA1c>6.2% (reference range 3.8-5.5%) had diabetic OGTT (sensitivity 41%, specificity 100%). Although a fasting plasma glucose (FPG) cut-off > or =7.0 mmol l(-1), as recommended by the American Diabetes Association (ADA), had greater sensitivity (78%), false positives (12%) limited its usefulness, so more diagnostic confidence could be placed in a positive HbA1c. In agreement with the ADA, we found FPG gave only slightly lower diabetes prevalence than the OGTT, but this masked a significant number of individual discrepancies (false positives and negatives) cancelling out each other. The new ADA category of impaired fasting glucose did not correlate well with impaired glucose tolerance. HbA1c is insufficiently sensitive as a direct substitute for the OGTT. A third of subjects diabetic on OGTT had normal HbA1c values, so it cannot exclude diabetes as currently defined, but HbA1c screening could make sufficient positive diagnoses to reduce our non-pregnant OGTTs by one-fifth. If a 'risk threshold' for diabetic complications could be applied to HbA1c, it could replace the OGTT as a more pragmatic diagnostic/prognostic test.

Hemoglobin Old Dominion/Burton-upon-Trent, β143 (H21) His→Tyr, Codon 143 CAC→TAC—a Variant With Altered Oxygen Affinity That Compromises Measurement of Glycated Hemoglobin in Diabetes Mellitus: Structure, Function, and DNA Sequence

To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (HbA1c). Blood specimens from four unrelated persons with this hemoglobin variant were examined by conventional laboratory methods, including electrophoresis, high-performance ion-exchange chromatography, and isoelectric focusing; by amino acid sequence analysis, polymerase chain reaction-based DNA sequence analysis, and electrospray ionization mass spectrometry, to establish the molecular structure; and by studies of oxygen affinity under varied conditions, to define the functional characteristics of the hemoglobin variant. The unique hemoglobin variant observed in these four cases is due to the mutation CAC-->TAC, at beta-globin gene codon 143, corresponding to beta 143 (H21) His-->Tyr. This amino acid substitution affects an important 2,3-diphosphoglycerate binding site and slightly increases the oxygen affinity of the hemoglobin variant. A hitherto unrecognized hemoglobin variant, encountered in four unrelated persons of Irish or Scots-Irish ancestry, hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, has now been characterized at the molecular, structural, and functional levels. Although it is associated with a slight increase in oxygen affinity, it is without hematologic effect, and its only clinical significance is that it coelutes with HbA1c on ion-exchange chromatography and thereby causes a spurious increase in HbA1c and compromises the use of this analyte to monitor the treatment of diabetes mellitus.

Using chemical and physical parameters to define the quality of karstic freshwaters (Timavo River, North-eastern Italy): a chemometric approach

The Timavo River, rising in Monte Nevoso (Slovenia), sinks into a limestone fissure and proceeds subterraneously toward the Adriatic Sea, feeding many springs and ponds in the Karst region near Trieste (Italy). In order to characterize and discriminate the freshwaters of this complex hydrological system, 84 samples were taken in 14 selected sites during the autumnal flood. Six chemical-physical parameters were determined: temperature, pH and conductivity in situ; chloride, nitrate and sulphate contents in the laboratory, by high-performance ion-exchange chromatography (HPIEC). Univariate analysis of dispersion and centrality estimates of the populations of the experimental data allow us to discriminate a group of typically-karstic springs situated near S.Giovanni di Duino, as well as a group—to the north—of “mixed waters” that receive run-off from the northern Isonzo and Vipacco river-beds, and also seem affected by seasonal conditions. The multivariate cluster analysis (CA) confirms the discriminating ability of the considered parameters, and allow us to observe occasional intrusions of waters belonging to a group within a different group. The principal component analysis (PCA) supports the results of CA and permits us to assert the existence of a common watershed for the examined karstic freshwaters, which are characterized by 2 PC's: the ionic solutes are associated with the first component, whereas temperature and pH are associated with the second one. Factor scores show seasonal and meteorological effects on the chemical composition of the freshwaters.

The Influence of Wildfire on Aqueous-Extractable Soil Solutes in Forested and Wet Meadow Ecosystems Along the Eastern Front of the Sierra-Nevada Range, California

Information is lacking regarding the influence of pre-fire microsite conditions on post-wildfire soil chemical properties. Following a 1994 wildfire along the eastern front of the Sierra Nevada range, California, high performance ion exchange chromatography was used to quantify anions and cations in aqueous soil extracts by plant microsite (Pinus jeffreyi subcanopy, Calocedrus decurrens subcanopy, Carer microptera meadow, Salix scouleriana subcanopy), by depth (ash or litter, mineral soil horizon 0-5 cm), and by treatment (unburned, burned). As compared to the unburned treatment, post-wildfire ash and surface mineral horizons had significantly (P less than or equal to 0.05) higher concentrations of sodium, magnesium, formate, and chloride. Plant microsite interacted significantly with treatment for ammonium; all plant microsites had significantly higher ammonium in burned treatments and burned S. scouleriana and C. microptera microsites had significantly more ammonium than the C. decurrens or P. jeffreyi microsites. For pH and acetate there was a significant treatment × depth interaction in which concentrations were highest in burned ash layers. For sulfate, ortho-phosphate, and calcium, there was a significant plant microsite × treatment × depth interaction. In general, post-wildfire ash and soil mineral layers had significantly more sulfate and calcium than the unburned controls; ash layers contained significantly less ortho-phosphate than unburned litter layers. There was a significant treatment × depth interaction for magnesium, chloride, formate, acetate, potassium, and nitrate. Initial post-wildfire levels of short-chained aliphatic carboxylic acids were very high, especially in ash layers, and did not decline to preburn values by the following summer.