Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

疏水相互作用色谱法分离大豆凝集素

采用疏水相互作用色谱法（ＨＩＣ）,以苯基琼脂糖凝胶ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ为固定相,硫酸铵－磷酸盐缓冲溶液（ｐＨ６．８）为流动相,从大豆匀浆液中分离出大豆凝集素（ＳＢＡ）。     

L-asparaginase fromErwinia carotovora

A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.     

Human lymphocyte sorting by gravitational field-flow fractionation

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.     

High-performance catalytic chromatography. The adapter approach

A sequence-specific DNA that binds EcoRI endonuclease was immobilized on glycidioloxypropyl-silica and Sepharose by cyanogen bromide (CNBr)-activated coupling. Elution of bound enzyme by conventional affinity strategies (increase of salt concentration) or by catalysis-induced elution (adding a Mg2+ cofactor required for catalysis) was compared. Greater yield and fold-purification was obtained with catalysis-induced elution for both DNA-silica and DNA-Sepharose columns, and silica gives higher performance than Sepharose. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed primarily a single band for EcoRI endonuclease for catalysis-induced elution from DNA-silica columns. Since catalysis-induced elution decreases the lifetime of DNA affinity columns, an alternative approach for preparing re-usable DNA columns was also developed. In this approach, a single stranded adapter DNA sequence is first coupled to silica or Sepharose and then annealed with another DNA sequence that contains a complementary, single stranded tail and the duplex binding site for EcoRI endonuclease. After use, replacing the hydrolyzed DNA regenerates the column. For this adapter approach, Sepharose gives better purity than silica and comparable yields and catalytic based elution gave the highest purity and yield, regardless of support. Substrate DNA with either a tail (for annealing to the column) at one end or both ends were compared and the former gave higher purity. Finally, enzyme binding to the substrate in solution ("trapping") or on a pre-bound substrate column was compared and trapping gave higher yield and similar purity to the alternative. Thus, trapping with a single tailed substrate oligonucleotide on a Sepharose adapter column and using catalytic elution gave the highest performance.     

Chromatography of E. coli tRNA on 5–20 μm Agarose Beads

Abstract

Escherichia coli transfer RNAs have been separated by chromatography on 5–20 μm beads of divinylsulfone-crosslinked agarose with the use of isocratic elution combined with a negative salt gradient. For example, tRNA^Leu was resolved into six isoacceptor species and tRNA^Ser into four. Leucine-charged tRNA's were eluted after their corresponding uncharged isoacceptor species. By optimizing flow rate, column length and elution buffer the fractionation of tRNA could be performed within 3 hours.     

Fractionation of styrene-methyl methacrylate copolymer

Styrene-methyl methacrylate copolymer was fractionated by the column elution and coacervate extraction techniques. Different solvent-nonsolvent combinations were used; both batchwise and continuous column elution fractionations were carried out. Successful resolution of fractions according to molecular weight was achieved. The effect of the solvent-precipitant pair on the fractionation efficiency, as tested by gel permeation chromatography, is discussed. The results of continuous column elution depend significantly on the elution rate.     

Group separation of trivalent actinides and lanthanides by tertiary pyridine-type anion-exchange resin embedded in silica beads

The separation of trivalent actinides and lanthanides was studied by using newly developed tertiary pyridine-type anion-exchange resin embedded in silica beads. Chromatographic elution experiments were carried out by using a packed column of the new resin and methanol-hydrochloric acid solution as an effluent. We confirmed that the actinides were eluted well from the elution bands of lanthanides. Actinides and lanthanides were eluted according to the reverse order of their atomic number.     

Fractionation Of Bovine Lutropin With The Aid Of Anion-Exchange High Performance Liquid ChromatographY

Abstract

Two bovine lutropin standard preparations (NIH-LH-B9, NIAMDD-bLH4) were applied on a recently developed anion-exchange column (Protein Pak DEAE 5PW,Waters Associates). The content of the eluted fractions was examined by a homologous radioimmunoassay of lutropin. In order to compare this fractionation with other independent methods, the two lutropin preparations were applied to SDS polyacrylamide gel eletrophoresis. The results indicate that the elution with a iris-hydrochloric acid buffer pH 6.5 allows within 2 minutes a convenient separation of the most LH immuno-reactive components of these preparations. Analysis on gel elect-rophoresis indicates that after fractionation on this anion-exchange column, some stained bands were removed from NIH-LH-B9 but not from NIAMDD-bLH-4.     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

Affinity Chromatography of Mushroom Tyrosinase

Abstract

The purification by column chromatography of a phenol-oxidizing enzyme, mushroom tyrosinase, was investigated using solid phase adsorbents designed to have specific affinity for the enzyme. Sepharose 4B, aminophenyl-bearing porous glass, and p-aminobenzylcellulose were chemically modified to introduce phenolic, catecholic, or benzoic groups on the polymer surface. The resulting preparations were tested for their effectiveness in separating tyrosinase from an impure protein mixture. The phenolic and benzoic polymers displayed no specific affinity for tyrosinase. Aminophenyl glass, with or without an attached phenolic group, adsorbed appreciable quantities of protein nonspecif-ically, thus complicating studies of its tyrosinase affinity properties. Dopamine, a dihydroxyphenyl derivative, was bound to Sepharose and was found to be effective in retaining tyrosinase at pH 5.5; elution of the enzyme by washing at pH 8.8 resulted in its purification by a factor of 10 to 14. Enzymatic oxidation of the adsorbent limited the number of purification cycles which could be carried out on a single column.     

Transferrin immunoextraction for determination of carbohydrate‐deficient transferrin in human serum by capillary zone electrophoresis

CZE‐based assays for carbohydrate‐deficient transferrin (CDT) in which serum is mixed with an Fe(III) ion‐containing solution prior to analysis are effective approaches for the determination of CDT in patient samples. Sera of patients with progressed diseases, however, are prone to interferences comigrating with transferrin (Tf) that prevent the proper determination of CDT by CZE in these samples. The need of a simple and economic approach to immunoextract Tf from human serum prompted us to investigate the use of a laboratory‐made anti‐Tf spin column containing polyclonal rabbit anti‐human Tf antibodies linked to Sepharose 4 Fast Flow beads. This article reports extraction column manufacturing and column characterization with sera having normal and elevated CDT levels. The developed procedure was applied to a number of relevant hepatology and dialysis patient samples and could thereby be shown to represent an effective method for extraction and concentration of all Tf isoforms. Furthermore, lipemic sera were delipidated using a mixture of diisopropyl ether and butanol prior to immunoextraction. CDT could unambiguously be determined in all pretreated samples.     

High‐density polyethylene fractionation with supercritical propane

During the development of column extraction techniques, two methods of separation were identified. The first method is based on altering polymer solubility by varying the ratio of solvent in a solvent/nonsolvent mixture at a constant temperature above the polymer melting point (gradient solvent elution fractionation). This method fractionates polymers according to molecular weight. The second method is based on altering polymer solubility by varying solvent temperature (temperature rising elution fractionation—TREF). TREF fractionates semicrystalline polymers with respect to their crystallizability, independently of molecular weight effects. In the present article, supercritical propane will be used to fractionate a high‐density polyethylene sample by molecular weight and short chain branching. The main advantage of supercritical fluid fractionation is that large polymer fractions with narrow molecular weight distributions (isothermal fractionation) or narrow short chain branching distributions (isobaric fractionation) can be obtained without using hazardous organic chlorinated solvents. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 553–560, 1999     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.     

预衍生化-气相色谱-质谱法测定水中酸性除草剂

向水样中逐滴加硫酸(1+2)调节至pH<2,用二氯甲烷先后提取3次。合并的提取液用无水酸性硫酸钠脱水,并蒸缩至约1 mL后用吹氮法蒸干。加入丙酮溶解残渣后,在催化剂碳酸钾存在下用五氟苄基溴进行衍生化,溶于正己烷中的衍生产物用极性硅胶柱进行纯化。先用甲苯-正己烷(1+6)混合溶剂淋洗净化柱以除去衍生产物中的干扰副产物,然后用甲苯-正己烷(9+1)从净化柱上将目标化合物洗脱,所得洗脱液经浓缩并加入八氟联苯作内标后供气相色谱-质谱分析。气相色谱中用HP-5MS毛细管色谱柱进行分离,质谱分析中用负化学离子源及选择离子扫描(SIM)。此方法对测定的14种酸性除草剂的检出限(3S/N)均小于10 ng.L-1。对所述14种除草剂做了回收试验,结果在66.0%~117.0%之间,测定7次的相对标准偏差均小于9%。     

Preparation of an ion-exchangeable polymer bead wrapped with bilayer membrane structures for high performance liquid chromatography

We synthesized a chromatographic packing material that has a non-covalently attached dihexadecyl phosphate (DHP) bilayer membrane structure on a CA08S, a nonporous-type cationic polymer bead with a diameter ranging from 11 to 14 μm. Confocal fluorescence microscopic and differential scanning calorimetric analyses of the DHP-CA08S complex revealed that the DHP bilayer membrane structures were formed on the surface of the CA08S polymer beads. When the functionality of the DHP-CA08S complex was evaluated in the ion-exchange HPLC of proteins, the retention behavior of the proteins on the DHP-CA08S complex column totally mirrored the anionic property of the DHP bilayer membrane surface, not the cationic property of the CA08S bead. Methylene blue (MB) was eluted from the DHP-CA08S complex column in the isocratic elution mode, but not at all from a CK08S column, a styrene-divinylbenzene based cation-exchange polymer. When the column temperature was elevated from 50 to 60 °C, the peak shape of MB on the DHP-CA08S complex column became fairly sharp without a change in its peak area, which mirrored the characteristic phase transition of the DHP bilayer membrane formed on the DHP-CA08S complex.     

Separation and identification of highly fluorescent compounds derived from trans-resveratrol in the leaves of Vitis vinifera infected by Plasmopara viticola

A method for identification of highly fluorescent compounds in vine leaves infected by Plasmopara viticola was developed using reversed phase liquid chromatography with simultaneous diode array and fluorometric detection. Fluorescent compounds were extracted from leaves with a methanol-water mixture (70:30). Separation by HPLC was performed using a C(18) column and gradient elution with water-acetonitrile mixtures (20-80% of acetonitrile). The main unknown fluorescent compound was identified by line spectral comparison with a standard obtained by UV photoisomerization of trans-resveratrol glucoside, and its structure was confirmed by liquid chromatography-mass spectrometry. Identification and structural elucidation of the fluorescent compound in the leaves of Vitis vinifera allows early detection of Plasmopara viticola invasion.     

Agarose-polyaldehyde microsphere beads: Synthesis and biomedical applications

Polyaldehyde microspheres, polyglutaraldehyde (PGL), and polyacrolein (PA) were synthesized by polymerizing glutaraldehyde and acrolein in the presence of an appropriate surfactant. The microspheres with average diameter of 0.2 micron were used for the specific labeling of human red blood cells (RBC) and mouse lymphocytes. The "naked" microspheres were encapsulated with agarose and formed agarose-polyaldehyde microsphere beads in sizes ranging from 50 microns up to 1 cm. The encapsulated beads, with diameters ranging from 50 to 150 microns were used as insoluble adsorbents for affinity purification of antibodies. Beads with diameters varied from 150 to 250 microns were used for cell fractionation purposes (mouse B splenocytes from T splenocytes). Uniform beads of 1 mm diameter were designed for hemoperfusion purposes. As a model, the removal in vitro of anti-BSA from immunized goat whole blood was studied.