Ion Exchange Chromatography Research Papers (original) (raw)

Crude extracts were partially purified by organic solvents fractionation: best results (96% of proteins, 91% of total caseinolytic activity) were obtained by adding four volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (>80%) at pH 5-9, and exhibited high thermal stability (>90% of residual activity after heating for 60 min at 60 • C). The enzyme was completely inhibited by E-64 trans-epoxysuccinyl-leucyl-amido(4-guanidino)-butane and iodoacetic acid and activated by the addition of cysteine or ␤-mercaptoethanol; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed five bands (pI 7.3 to <9.3), most of them proteolytically actives, but only three of which (pI 7.6, 8.2 and 8.8) proved to be important. Ion exchange chromatography in DEAE-Sephadex, was selected to separate this active bands.

- by
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- Chemistry, Environmental Biotechnology, Ion Exchange Chromatography, Enzyme

Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were puri®ed from the sea anemone Stichodactyla helianthus combining gel ®ltration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone. 7

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4000Å) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pHfractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.

- by Jean-Pierre Chervet
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- Engineering, Analytical Chemistry, Technology, Chromatography

Major hen egg white proteins have been widely studied for their functional properties but these studies still are unable to explain, alone, all of the biological properties of hen egg white. Hence, it is still interesting to produce pure and non-altered proteins to improve our knowledge on the biological properties of hen egg white. Presently, identification and characterization of both bioactive peptides and minor proteins from hen egg white is essential work for progressing in the understanding of hen egg white biological properties. With this objective in mind, a new process for a complete "mucin free" hen egg white fractionation based on ion exchange chromatography is proposed. "Mucin free" egg white is fractionated into six different fractions. Four of them are high-recovery yield purified fractions of lysozyme, ovotransferrin, ovalbumin and flavoprotein. The two other fractions are enriched in recently detected minor proteins in hen egg white.

- by Thomas Croguennec
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- Engineering, Technology, Mass Spectrometry, Chemical Analysis

In this work, a methodological approach is reported, aimed at assessing the electrochemical response of some model gluco-oligosaccharides (dextrans). Such strategy is based on the complementary use of both anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) and capillary zone electrophoresis coupled with UV detection (CZE–UV). Unlike HPAEC–PAD, CZE–UV required derivatization with a chromophoric dye (i.e., 8-aminonaphtalene-1,3,6-trisulphonic acid, ANTS) to enhance UV response and separation selectivity. From the comparison between chromophore response and PAD signal, the reliability of HPAEC–PAD for quantitative evaluation of dextran mixtures containing mainly oligomers with polymerization degree (DP) up to 18 could be proved, due to the fairly constant molar response. For higher DPs (up to 41), a maximum in the trend of the molar responses was observed followed by a steep decrease for DPs higher than about 30–35; indeed, an underestimation of weight-average...

- by Sergio Paoletti
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- Engineering, Technology, Electrochemistry, Capillary electrophoresis

A keratinase enzyme was isolated and purified from a feather-degrading culture of Aspergillus oryzae. Fractional precipitation of the crude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at... more

Background/Objectives: Free amino acids affect food palatability. As information on amino acids in frequently purchased prepackaged food is virtually absent, we analyzed free amino acid patterns of 17 frequently purchased ready-to-serve convenience food products, and compared them with the information obtained from the respective food labels. Subjects/Methods: Quantitative amino acid analysis was performed using ion-exchange chromatography. g-Aminobutyric acid (GABA) concentrations were verified using a stable isotope dilution gas chromatography/mass spectrometry (GC-MS) method. The patterns of free amino acids were compared with information obtained from food labels. Results: An obvious mismatch between free amino acid patterns and food label information was detected. Even on considering that tomatoes and cereal proteins are naturally rich in glutamate, the concentrations of free glutamate outranged the natural concentration of this amino acid in several products, and strongly suggested artificial enrichment. Free glutamate was found to be elevated even in dishes that explicitly state 'no glutamate added'. Arginine was markedly elevated in lentils. Free cysteine was generally low, possibly reflecting thermal destruction of this amino acid during food processing. The meat and brain-specific dipeptide carnosine (CARN) was present in most meat-containing products. Some products did not contain detectable amounts of CARN in spite of meat content being claimed on the food labels. We detected GABA at concentrations that contribute significantly to the taste sensation. Conclusion: This investigation highlights a marked mismatch between food label information and food composition.

- by U. Gonder and +1
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- Nutrition and Dietetics, Food Processing, Germany, Glutamate

The aim of this work was to isolate and identify antibacterial peptides present in a pepsin digest of ovine a s2 -casein. A protein digest with antibacterial properties was first separated by ion exchange chromatography. The fractions most active against Escherichia coli ATCC 25922 were fractionated by semi-preparative RP-HPLC, and the identification of the active peptides was carried out by on-line and off-line RP-HPLC-ESI-MS/MS. Following this strategy, 10 different peptides were identified, all corresponding to the C-terminal region of the ovine a s2 -casein. Four of them were chemically synthesized and showed antibacterial activity against several Gram-positive and Gram-negative bacteria. Among the synthesized peptides, ovine a s2 -casein f(165-181) exhibited the highest antibacterial potency against all bacteria tested. The antimicrobial activity was compared with that of other previously described peptides like lactoferricin and fragment f(183-207) of bovine a s2 -casein. r

- by Lourdes Amigo
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- Mass Spectrometry, Escherichia coli, Gram Positive, Antimicrobial activity

The quantitative characterization of pore structure of Sartobind Q, a strongly basic membrane anion exchanger that is formed by cross-linked cellulose support and a hydrogel layer on its pore surface, was made combining the results obtained by several experimental techniques: liquid impregnation, batch size-exclusion, inverse size-exclusion chromatography, and permeability. Mercury intrusion and nitrogen sorption porosimetry were carried out for a dry cellulose support membrane in order to get additional information for building a model of the bimodal pore structure. The model incorporated the distribution of the total pore volume between transport and gel-layer pores and the partitioning of solutes of different molecular weights was expressed through the cylindrical pore model for the transport pores and random plane model for the gel layer. The effect of composition of liquid phase on the pore structure was investigated in redistilled water, phosphate and Tris-HCl buffers containing up to 1 M NaCl. Evident differences in the bimodal pore structure were observed here when both the specific volume and size of the hydrogel layer pores significantly decreased with the ionic strength of liquid phase.

- by Milan Polakovic
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- Engineering, Technology, Chromatography, Adsorption

A new method based on ion exchange chromatography with integrated pulsed amperometric detection has been developed for simultaneous determination of underivatized biogenic amines and applied to the analysis of cadaverine, putrescine, spermidine, and histamine in fish products. The amines were extracted from fish products with a mixture of 5 % aqueous trichloroacetic acid, 6N hydrochloric acid, and nheptane. The extract was passed through a C18 cartridge and the biogenic amines separated by cation exchange on an IonPac CS10 column using gradient elution. Detection was performed by integrated pulsed amperometry, and resulted in estimated detection limits of 5 ng for putrescine, 12 ng for cadaverine and histamine, and 25 ng for spermidine. The method was linear in the range 0.025 to 5 Isg of injected amine. This method was applied to the analysis of products from fish such as Scombridae and Clupeidae. The average recoveries were greater than 92 %.

- by Silvano Cavalli
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- Analytical Chemistry, Ion Exchange Chromatography, Ion Exchange, Cation Exchange

The trihydrate of alendronate sodium (MK-0217) is an important bisphosphonate drug for the treatment of a variety of bone diseases. Determination and characterization of this compound in dosage formulations is chalIenging since it has no ~hromophore, and as a highly polar and thermally labile compound, it is not amenable to electron impact mass spectrometry. Ion chromatography coupled with an ion spray mass detector (IC-BP-MS) in the negative ionization mode was developed and applied to the characterization of this compound. Under these conditions alendronate (m/z 248, [M -H] _, M = parent alendronic acid) was readily observed. The anion can form cluster anions with acid molecules including that of the alendronic acid in the gas phase, which is a distinguishing feature of the IC-ISP-MS spectrum. K-ISP-MS-MS study of the anion shows that cleavage of the C-P bond(s) is the dominant fragmentation pathwa~(s~ of the anion, characteristic of its structure.

- by Eric Tsai
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- Engineering, Technology, Mass Spectrometry, Quantitative analysis
- by Valentina Kovaleva
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- Physiology, Mass Spectrometry, Phylogeny, Sequence alignment

This paper describes a suitable method for the optimum extraction and isolation of phycocyanin from the cyanobacterium Synechococcus sp. IO9201 isolated from Caribbean waters. Phycocyanin from this microorganism was purified to homogeneity and some of its properties were investigated. The purification steps consisted of extraction, hydrophobic interaction chromatography and ion exchange chromatography. Freezing at −21°C-thawing at 4°C, using an alkaline buffer was the best method for extracting phycocyanin from Synechococcus sp. IO9201. The best extraction was obtained using butyl-sepharose resin for hydrophobic interaction chromatography and 0.05 M Tris-HCl (pH = 7) containing 10% ethanol for phycocyanin elution. Finally, phycocyanin was further purified by ion exchange chromatography using Q-sepharose and eluted with a complex isocratic system. The estimated molecular weight of the phycocyanin purified from Synechococcus sp. IO9201 was 102 000 daltons by gel filtration and the isoelectric point was 4.6. When analyzed by SDS-PAGE, Synechococcus sp. IO9201 phycocyanin migrated as two bands having an apparent molecular weight of 21 360 and 18 980 Da. The first band corresponds to β phycocyanin subunits, whereas the second corresponds to α phycocyanin subunits. So, this phycocyanin was characterized as (αCPCβCPC)3.

- by Julio Cid
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- Protein Purification, Plant Biology, Plant Science, Method
- by Sergei Pechenov
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- Cell fusion, Escherichia coli, Staphylococcus aureus, Affinity chromatography

This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.5 or anion-exchange chromatography (AEX) at pH 7.2 without lowering the salt concentration in the CCS. The performance of both CEX and AEX was predicted to be equal for this particular MAb capture.

- by Michel Eppink and +3
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- Engineering, Technology, Cell Culture, High throughput screening

Phragmytes vallatoria belongs to the family poaceae. The complexity of leaf ethanolic extract can be simplified through column chromatography. Different solvent mixtures were used in elution systems i.e., hexane, ethyl acetate and methanol. The concentrated ethanolic leaf extract of 100 g was fractionated by column chromatography on silica gel (60-120 mesh). Major fractions were (hexane + ethyl acetate 9:1 and ethyl acetate + methanol 8.5: 1.5 fraction) and identified the major phytochemicals through NIST Electron Ionization Mass database. The identified compounds were 9, 12, 15-Octadecatrienoic acid, ethyl ester, (Z, Z, Z)-, Androstane-11, 17-dione, 3-hydroxy-, (3.alpha. 5. alpha)-, 9,12-Octadecadienoic acid ethyl ester and 9-12-15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)-. Only one peak was observed in ethyl acetate and methanol (8.5: 1.5) fraction and it was subjected to GC-MS and HPLC. The structures were (Benzenamine, 3-(methylthio)-, Morpholine, Phenanthrene and 3-Eicosene, (E).

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and wellunderstood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resinprotein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.

- by jonathan Coffman
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- Mechanical Engineering, Chemical Engineering, Robotics, Algorithms

Liposome-mediated intracellular delivery of clodronate is reported to selectively deplete mononuclear phagocytic cells such as macrophages that are important effector cells involved in the pathogenesis of neuropathies associated with demyelination and destruction of neuronal cells. Application of liposome-encapsulated clodronate (dichloromethylenediphosphonic acid disodium salt) is a method of choice to deplete macrophages to prevent such a neurodegeneration. In the present work, a comparison of an ion-exchange chromatography (IEC) and a capillary zone electrophoresis (CZE) method with indirect UV detection was performed and, based on the results of this comparison, a CZE assay was developed for quantitation of clodronate in mannosylated liposomes. This CZE method employed Nitroso-R salt (1nitroso-2-naphthol-3,6-disulphonic acid disodium salt) as background electrolyte with UV detection of the analyte at 254 nm. The assay for the determination of clodronate in mannosylated liposomes after their solubilization in 10 mM Triton X-100 showed acceptable within-day precision (repeatability), day-to-day precision (reproducibility) and linearity in the target quantitation range of 0.5 Á/10.0 mg ml (1 . The method reported here can be used as part of the quality control during the preparation of liposome-encapsulated clodronate as a drug formulation for macrophagemediated diseases. #

- by Laszlo Prokai
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- Analytical Chemistry, Capillary electrophoresis, Quality Control, Pharmaceutical

An isocratic, ion-exchange chromatographic method is described for the determination of nitrite and nitrate in dairy products. Following dissolution and molecular weight centrifugation (30 kDa), the supernatant is injected directly on to the column and analytes determined at 540 nm subsequent to coupled post-column reduction of nitrate and derivatisation via Griess chemistry. Optimisation and validation studies are presented, including comparison against conventional methods in common usage. Attributes of the described technique include enhanced sensitivity and freedom from endogenous UV-absorbing matrix interferences that compromise an unequivocal chromatographic analysis of infant formulas and protein hydrolysates.

- by Harvey Indyk
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- Chromatography, Dairy, Ion Exchange Chromatography, Clinical Sciences

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.

- by Susana Kalil and +1
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- Biochemistry, Protein Purification, Multidisciplinary, Process Design

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

- by Petruta Calina Cornea
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- Engineering, Chemical Engineering, Genetics, Microbiology

A new chromatography system,ÄKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at >90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine-and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.

- by Tuomo Frigård
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- Engineering, Technology, Protein Purification, Drug Discovery

The initial clinical trials for treatment of acute myeloid leukemia have demonstrated the effectiveness of the alpha emitter 213 Bi in killing cancer cells. Bismuth-213 is obtained from a radionuclide generator system from decay of 10days 225 Ac parent. Recent pre-clinical studies have also shown the potential application of both 213 Bi, and the 225 Ac parent radionuclide in a variety of cancer systems and targeted radiotherapy. This paper describes our five years of experience in production of 225 Ac in partial support of the on-going clinical trials. A four-step chemical process, consisting of both anion and cation exchange chromatography, is utilized for routine separation of carrier-free 225 Ac from a mixture of 228 Th, 229 Th and 232 Th. The separation of Ra and Ac from Th is achieved using the marcoporous anion exchange resin MP1 in 8 M HNO 3 media. Two sequential MP1/NO 3 columns provide a separation factor of 106forRaandAcfromTh.TheseparationofAcfromRaisaccomplishedonalowcross−linkingcationexchangeresinAG50−X4using1.2MHNO3aseluant.TwosequentialAG50/NO3columnsprovideaseparationfactorof10 6 for Ra and Ac from Th. The separation of Ac from Ra is accomplished on a low cross-linking cation exchange resin AG50-X4 using 1.2 M HNO 3 as eluant. Two sequential AG50/NO 3 columns provide a separation factor of 106forRaandAcfromTh.TheseparationofAcfromRaisaccomplishedonalowcross−linkingcationexchangeresinAG50−X4using1.2MHNO3aseluant.TwosequentialAG50/NO3columnsprovideaseparationfactorof10 2 for Ac from Ra. A 60-day processing schedule has been adopted in order to reduce the processing cost and to provide the highest levels of 225 Ac possible. Over an 8-week campaign, a total of 100mCiof225Ac(100 mCi of 225 Ac (100mCiof225Ac(80% of the theoretical yield) is shipped in 5-6 batches, with the first batch typically consisting of $50 mCi. After the initial separation and purification of Ac, the Ra pool is re-processed on a bi-weekly schedule or as needed to provide smaller batches of 225 Ac. The averaged radioisotopic purity of the 225 Ac was 99.6 7 0.7% with a 225 Ra content of p0.6%, and an average 229 Th content of (4 À4 +5 ) Â 10 À5 %.

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against dierent bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were puri®ed by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500-to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identi®ed, no modi®ed amino acids were found. No signi®cant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium dicile.

- by Didier Bogusz
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- Microbiology, Medical Microbiology, Food, Bacteriophages

Un ni iv ve er rs si it tá á d di i B Br re es sc ci ia a, , I It ta aly Abstract: Lactate is one of the principle products of anaerobic metabolism in living organism. Determination of lactate (lactic acid) is required in the diagnosis and medical management of various diseases such as tissue hypoxia, the individual causes include shock (hypovolemic, cardiogenic or endotoxic), systemic disorder (neoplastic diseases, liver or renal failure, diabetes mellitus), respiratory failure (asphyxia), severe congestive heart failure and also in food industry. Various methods are available for the measurement of lactic acid such as colorimetric, gas chromatography, mass spectrometry, ion exchange chromatography, near IR spectroscopy, HPLC, capillary electrophoresis, isotachophoresis, FIA, enzymic colorimetric and finally biosensor and biochip method. The principle, advantage and disadvantages of these methods have been compared and summarized in this review.

Pentosidine, a crosslink amino acid in which one arginine and one lysine residue are linked together by a pentose, has been detected in foods for the first time using ion-exchange chromatography with direct fluorescence detection and subsequent ninhydrin derivatization. The method allows the simultaneous quantification of pentosidine along with all other amino acids of acid hydrolyzates at levels lower than 50 g kg protein. Levels of pentosidine in all food samples investigated were very low (milk products between not detectable and 2-5 mg/kg protein; roasted coffee and some bakery products up to 35 mg/kg protein), indicating that pentosidine does not play a major part in the polymerization of food proteins.

A simple gradient apparatus, consisting of a perlstaltic pump in addltlon to a standard hlgh-pressure pump, Is descrlbed. The device is used to make a slngle-run ion chromatographic separation of sulfite, sulfate, and thiosulfate In less than 15 min. This separatlon requlred a step gradlent with 4.8 mM NaHC0,/4.7 mM Na,CO, as start eluent and 6.9 mM NaH-COJ8.6 mM Na,CO, as final eluent when two (4 X 50) mm Dionex anion precolumns In serles were used as separator. The eluent compositions were slmplex optlmlzed.

- by Darryl Siemer
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- Chemical Engineering, Analytical Chemistry, High Pressure, Ion Chromatography
- by وسام احمد
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- Engineering, Analytical Chemistry, Technology, Crustacea

The culture conditions for extracellular secretion of lignin peroxidase by Pleurotus sajor caju MTCC–141 in the liquid culture growth medium amended with lignin containing natural substrates have been studied. Secretion of lignin peroxidase has been found to be maximum in the presence of bagasse. Lignin peroxidase from the liquid culture filtrate has been purified to homogeneity. Two isozymes having relative molecular masses 38 and 40 kDa have been isolated. The enzymatic characteristics like Km, pH, and temperature optima of the major isozyme (40 KDa) has been determined using veratryl alcohol, n-propanol, and H2O2 as the substrate. The Km values for veratryl alcohol, n-propanol, and H2O2 have been found to be 57 μ M, 500 μ M, and 80 μ M, respectively. The pH and temperature optima of lignin peroxidase have been found to be 3 and 30°C, respectively. The inhibition of the enzyme activity by sodium azide has been studied and it has been found to be uncompetitive, with KI value of 4 mM.

- by Kapil Yadav
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- Engineering, Materials Engineering, Technology, Biological Sciences

One strain of nitrogen-fixing cyanobacterium (Nostoc sp. PCC 9202) was grown indoors in 1 litre glass air-lift reactors as well as 17 litre polyethylene bags. Temperature dependence of growth-kinetic parameters, as well as of biomass and phycobiliprotein productivities, were determined. Harvesting and phycobiliprotein extraction methodologies are presented. The purification of crude extracts was performed by means of ultrafiltration or (NH4)2S04 precipitation followed by gel filtration and ion-exchange chromatography. Final phycocyanin and phycoerythrin preparations were characterized by purity ratios above 4 and 5, respectively. Phycobiliprotein production cost determination for each purity grade and cumulative phycobiliprotein weight losses along purification steps are shown. Production cost for high purity phycoerythrin (30 US$ per g) seems to be far below the market price.

- by José Empis
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- Multidisciplinary, Growth Kinetics, Weight Loss, Nitrogen

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA 2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA 2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED 50 of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.

- by Osvaldo A Sant'Anna and +1
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- Mass Spectrometry, Biological Sciences, Mice, Physical sciences

Higginbotham, G. R.; Huang, A.; Firestone, D.; Verrett, J.; Ress, J.: Campbell. A. D. Nature (London) 1968, 220, 702. (22) Schwetz, 9. A.; Norris, J. M.; Sparschu, G. L.; Rowe, V. K.; Gehring, P.

- by Giorgio Fassina
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- Cell Culture, Scaling up, Affinity chromatography, Ion Exchange Chromatography

A sample preparation method based on sintering, followed by analysis by inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) for the simultaneous determination of chloride and bromide in diverse and mixed solid wastes, has been evaluated. Samples and reference materials of known composition were mixed with a sintering agent containing Na(2)CO(3) and ZnO and placed in an oven at 560 degrees C for 1h. After cooling, the residues were leached with water prior to a cation-exchange assisted clean-up. Alternatively, a simple microwave-assisted digestion using only nitric acid was applied for comparison. Thereafter the samples were prepared for quantitative analysis by ICP-SFMS. The sintering method was evaluated by analysis of certified reference materials (CRMs) and by comparison with US EPA Method 5050 and ion chromatography with good agreement. Median RSDs for the sintering method were determined to 10% for both chlorine and bromine, and median recovery to 96% and 97%, respectively. Limits of detection (LODs) were 200mg/kg for chlorine and 20mg/kg for bromine. It was concluded that the sintering method is suitable for chlorine and bromine determination in several matrices like sewage sludge, plastics, and edible waste, as well as for waste mixtures. The sintering method was also applied for determination of other elements present in anionic forms, such as sulfur, arsenic, selenium and iodine.

- by Ilia Rodushkin
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- Environmental Engineering, Mass Spectrometry, Arsenic, Waste Management

The present paper describes an efficient single step chromatographic method for purification of C-Phycocyanin from three cyanobacterial species, i.e., Spirulina sp. (freshwater), Phormidium sp. (marine water) and Lyngbya sp. (marine water). C-Phycocyanin from these cyanobacterial species was purified to homogeneity and some of their properties were investigated. The purification involves a multistep treatment of the crude extract by fractional precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-Sepharose CL-6B column. Pure C-Phycocyanin was finally obtained from Spirulina, Phormidium, and Lyngbya spp. with purity ratio (A 620 /A 280 ) 4.42, 4.43, and 4.59, respectively, further the purity and homogeneity were confirmed by native and SDS-PAGE. The estimated molecular weights of purified C-PC from Spirulina, Phormidium, and Lyngbya spp. were 112, 131, and 81 kDa, respectively. SDS-PAGE of pure C-Phycocyanin yielded two bands corresponding to a and b subunits. The results of SDS-PAGE demonstrate the same molecular weight of b subunits (24.4 kDa) for all the three cyanobacterial species, whereas the molecular weight of the a subunit is different for all (17 kDa Spirulina sp., 19.1 kDa Phormidium sp., 15.2 kDa Lyngbya sp.). Thus, the C-Phycocyanin was characterized as (ab) 3 for Spirulina and Phormidium spp., while as (ab) 2 for Lyngbya sp.

Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to homogeneity using Amicon concentration and DEAE cellulose chromatography. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis has been found to be 40 kDa. The Km values for veratryl alcohol and H2O2 for the purified enzyme were 58 and 83 μM, respectively. The calculated kcat value of the purified enzyme using veratryl alcohol as the substrate was 2.5 s−1. The pH and temperature optima of lignin peroxidase have been found to be 2.6 and 24°C, respectively.

- by Kapil Yadav
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- Engineering, Materials Engineering, Technology, Biological Sciences
- by Sanjeev Ghadi
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- Chemical Engineering, Genetics, Ion Exchange Chromatography, Enzyme

The main objective of this review was to describe the physicochemical and nutritional characteristics of twenty selected exotic fruits and the influence of their physiologically active compounds on human health, through scientifically proven information. The review presents the biologically active metabolites derived from exotic fruits (polyphenols, flavonoids, flavanols, tannins, ascorbic acid, anthocyanins, volatile compounds, minerals, and organic acids) and various analytical methods for their detection (elemental analysis, electrophoretic separation by SDS-polyacrylamide gel electrophoresis, and fast protein liquid and ion-exchange chromatography; GC-MS, HPLC/diode array detection (DAD), circular dichroism (CD), differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR), ultraviolet spectroscopy, twoand three-dimensional fluorimetry (2D-FL) and (3D-FL), and antioxidant radical scavenging assays (DPPH, FRAP, CUPRAC, ABTS, and ORAC). The correlation between the polyphenols and other bioactive compounds, and their antioxidant activities was reported for different fruit extracts. During the last two decades our international scientific group investigated in vitro the physicochemical and nutritional characteristics of avocado, dragon fruit, durian, kiwifruit, mango, mangosteen, persimmon and snake fruit, and in vivo their influence on laboratory animals and humans. Supplementation of diets with exotic fruits positively affects plasma lipid profile, antioxidant activity and histological examination of aorta in rats fed cholesterolcontaining diets. The interaction between drugs and serum albumin plays an important role in the distribution and metabolism of drugs. The properties of polyphenol methanol extracts of exotic fruits showed the ability to quench serum albumin by forming the complexes similar with the ones between proteins and pure flavonoids. Our experimental data and a wide range of other investigations are included in this review. In conclusion, it is nessasary to promote a consumption of exotic fruits (a rich source of natural antioxidants) as a supplement to everyday human diet.

- by Shela Gorinstein
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- Chemical Engineering, Biochemistry, Food, Food and Nutrition

Peroxidase (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) is an oxidoreductase enzyme found in many fruits and vegetables. This enzyme was purified from sweet gourd (Cucurbita moschata Lam. Poiret) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Furthermore, optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions were determined as 7.2, 50°C, 0.4 M, 8.0, and 40°C, respectively. The molecular weight (MW) of the enzyme was estimated to be 85 kDa by SDS-PAGE method. The values of Km and Vmax were calculated from the Lineweaver-Burk graph for guaiacol/H2O2 substrate patterns.

- by İlhami Ercan
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- Engineering, Technology, Enzymology, Chromatography

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI < 6) and basic (pI > 8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH ∼6 to ∼9, neutral proteins indeed exhibit nearly zero net charge at pH ∼9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.

- by Michel Eppink and +2
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- Engineering, Technology, Optimization, Ph

A method based on single column ion chromatography with UV detection was developed for purity testing and assay of monosodium olpadronate. The analyte aqueous solution is precipitated with methanol to enhance the impurities/olpadronate molar ratio, thus improving purity determination at trace levels. The resulting solution is injected into a standard chromatographic system with UV detector in indirect mode with a Waters IC Pak HR column using diluted nitric acid as the mobile phase. The method was fully validated according to ICH guidelines for the determination of phosphite, phosphate, chloride and methanesulfonic acid in olpadronate being suitable for purity testing and assay.

Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu 2q . Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products. q

Two ion chromatographic methods have been proposed for the simultaneous determination of caffeine, theobromine and theophylline. The separations were based on isocratic elutions and the determinations on ultraviolet absorbance detections at 274 nm. The detection limits (signal-to-noise ratio 3:1) for the three compounds were all below sub-mg ml À1 level. The methods have been successfully applied to the determination of these three compounds in foods and pharmaceutical preparations, and the average recoveries for various samples ranged from 87% to 103%. In addition, the retention mechanisms of these three compounds on various ion-exchange resins were discussed. The results obtained revealed that ion-exchange chromatography is possibly a good alternative to conventional liquid chromatography for the separation and determination of some water-soluble, neutral organic molecules with a certain hydrophobicity.

A method for the determination of salbutamol in both tablets and syrups is described. It utilizes the reduction of the Folin-Ciocalteau reagent by the phenolic group, monitoring the absorbance of the resulting complex at 760 nm. Results obtained are linear over the range 0–6 μg ml−1 salbutamol. Coloring material was removed by anionexchange chromatography prior to analysis and there was no interference from sucrose, neutral flavorings or the common preservative sodium benzoate. This method appears suitable as a general assay for salbutamol.

- by novelette mcknight
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- Analytical Chemistry, Colorimetry, Quantitative analysis, Dosage Form

Mammalian RNA polymerase I1 transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase 11. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration, was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase 11, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase 11. Transcription of protein-coding genes in higher eukaryotes is carried out by a multienzymatic complex that includes RNA polymerase I1 and several accessory transcription factors. One class of factors consists of DNA-binding proteins that recognize specific promoter or enhancer elements, through which they regulate gene-or tissue-specific transcription by RNA polymerase I1 (reviewed in . Another group termed general transcription factors act through the core promoter elements (TATA box and initiator motif) and are required for basal levels of specific transcription initiation at all class two promoters (for review, see . The mechanisms by which the general transcription factors determine the position and directionality of transcription initiation and the biochemical events by which they trigger the formation of the first phosphodiester bond by RNA polymerase I1 are not yet understood.

- by Juan Inostroza
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- Chromatography, Kinetics, Biological Chemistry, RNA polymerase

Primary Na + transport has been essentially attributed to Na + /K + pump. However, there are functional and biochemical evidences that suggest the existence of a K + -independent, ouabain-insensitive Na + pump, associated to a Na + -ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na + -ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na + -ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg 2+ dependence, specific Na + stimulation, K + independence, ouabain insensitivity and inhibition by furosemide (IC 50 : 0.5 mM) and vanadate (IC 50 : 9.1 μM). IgY antibodies against purified Na + -ATPase did not recognize Na + /K + -ATPase and vice versa. Analysis of purified Na + -ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na + /K + -ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na + /K + -ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na + -ATPase, silencing of atna mRNA specifically suppressed Na + -ATPase α-subunit and ouabain-insensitive Na + -ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na + /K + -ATPase α1-isoform. These structural and biochemical evidences identify the K + -independent, ouabain-insensitive Na + -ATPase as a unique P-type ATPase.

- by Luz_Eduviges Thomas R
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- Chemical Engineering, Biology, Membrane Proteins, Medicine

The application of ion-exchange (IEX) chromatography to protein refolding (IExR) has been successfully proven, as supported by various studies using different model proteins, ion-exchange media and flow configurations. Ion-exchange refolding offers a relatively high degree of process intensification, represented by the possibility of performing protein refolding, product purification and product concentration, in one unit operation. Besides its high degree of process intensification, IExR offers an additional set of key advantages including: spatial isolation of the bound protein molecules and the controllable change in chemical composition using gradients. Despite of the acknowledgement of the former advantages, the lack of mechanistic understanding on how they influence the process performance of the ion-exchange refolding reactor, limits the ability to exploit them in order to optimize the performance of the unit. This paper presents a quantitative analysis that assesses the effect that the spatial isolation and the urea gradient, have on the IExR performance, judged on the basis of the refolding yield (Y N ) and the fractional mass recovery (f Prot,Rec ). Additionally, this work discusses the effect of the protein load, the protein loading state (i.e., native, denatured, denatured and reduced (D&R)) and the adsorbent type on f Prot,Rec . The presented work shows: (1) that the protein load has a direct effect on f Prot,Rec , and the magnitude of this effect depends on the loading state of the protein solution and the adsorbent type; (2) that irrespectively of the type of adsorbent used, the saturation capacity of a denatured protein is less than the native protein and that this difference can be linked to differences in accessible binding surface area; (3) that there is a clear correlation between fractional surface coverage (Â) and f Prot,Rec , indicating that the former could serve as a good descriptor to assess spatial isolation, and (4) that the urea gradient has a direct link with the variations on the refolding yield, and this link can be quantitatively estimated using as descriptor the urea gradient slope ( ). Overall, the information provided in this paper aims at the eventual development of rational design or selection strategies of ion-exchange media for the satisfactory and successful refolding of a target protein.

- by Michel Eppink and +1
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- Engineering, Technology, Quantitative analysis, Adsorption

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.

- by Janus C Krarup
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- Engineering, Technology, Comparative Study, Ph