Separation of chiral compounds by capillary electrophoresis

This review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.     

Isolation of ultrapure supercoiled plasmid-DNA using preparative electrophoresis

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) system for the isolation of high-purity supercoiled plasmid-DNA is described. This method should prove suitable for the isolation of large DNA molecules, either plasmid or linear DNA, that is required for the production of transgenic animals, for instance. The efficiency of the method is illustrated by the isolation of the gene for the green fluorescent protein, cloned into a mammalian expression vector and used for transfection of eukaryotic cells.     

Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.     

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.     

Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry

A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.     

Biochemistry of Vibrio cholerae virulence: purification of cholera enterotoxin by preparative disc electrophoresis

Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxin-antigen as well as its biological activity were determined at various setps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase.     

Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis

Anthranilic acid (AA) has attracted considerable attention as one of the L-tryptophan-kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of AA has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Serum and cerebrospinal fluid (CSF) AA concentrations in different animal species were measured. CSF AA concentrations in rabbits were 1.1 +/- 0.1 nmol/liter, which were 5. 7-33.0 times lower than those in other species studied. Serum AA concentrations, however, were slightly higher in rabbits than in other species. In contrast, the concentrations of L-kynurenine (L-KYN) in both serum and CSF were substantially higher in rabbits than in other species. Tissue kynurenine pathway enzymes, indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase, kynurenine 3-hydroxylase, and kynureninase were determined in rabbits, rats, gerbils, and mice. These enzymes varied among species, especially lung IDO activities in rabbits were 146-516 times higher than those found in other species, but rabbit liver kynurenine 3-hydroxylase activities were lower by one order of magnitude than those of the other species tested. Furthermore, brain kynurenine 3-hydroxylasae activities were 12.3-23.2 times higher in gerbils than those in the other species tested. In addition, AA concentrations in serum following intravenous administration of L-KYN (5 mg/kg) were also measured in rabbits. AA levels peaked sharply within 5 min after administration and decreased in a time-dependent manner. At 5 min after administration, CSF L-KYN and AA concentrations were also increased by 1.76- and 2.56-fold, respectively, compared with basal levels. Increased AA concentrations in CSF following L-KYN administration may reflect the entry of AA into the CSF after conversion to AA in systemic tissue and/or the local synthesis of AA from L-KYN in the CNS.     

Protein profile characterization of bacterial lysates by capillary electrophoresis

Persistent inhibition of bacterial growth, called postantibiotic effect (PAE), after a short exposure to a new carbapenem, meropenem, was determined in different strains of the Enterobacteriaceae family. Capillary electrophoresis (CE), as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to study the outer membrane protein (OMP) profiles before and after meropenem treatment. CE proved to be suitable for the characterization of the OMP profiles of bacteria. Significant changes in the electrophoretic patterns were observed, showing the consequential effect of meropenem on bacteria.     

Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.     

Separation of viable from radiation-induced apoptotic lymphocytes by free-flow electrophoresis

A human lymphocyte population undergoing apoptosis in vitro due to gamma-irradiation was fractionated by free-flow electrophoresis in triethanolamine--Na-acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.     

Use of preformed cavities in rabbits for the quantitation of leukocyte chemotaxis caused by bacterial lipopolysaccharides

Wound chambers implanted subcutaneously in rabbits proved suitable for measurements of leukocyte chemotaxis. Injection of bacterial lipopolysaccharide (LPS) in the chambers the sixth day after implantation was followed by a marked increase of polymorphonuclear leukocytes in the wound chamber fluid, the number of which was dependent on the time after application of LPS. Up to a certain amount of LPS, the concentration of leukocytes in the chamber fluid was dose-dependent. The histopathological appearance of the granulation tissue lining the chamber wall one day after the injection of LPS from Veillonella revealed aggregation of blood cells plugging the lumina of small vessels and many eosinophilic leukocytes.     

Separation of 24 dansylamino acids by capillary electrophoresis with a non-ionic surfactant

The separation of 24 dansylamino acids was investigated by capillary electrophoresis with an additive of micelles of a non-ionic surfactant, Tween 20. Although two pairs of peaks, norvaline and methionine derivatives, and didansyltyrosine and solvent (methanol), did not show good resolution, other dansylamino acids were well separated within 70 min using 100 mM Tween 20 and pH 2.40. The theoretical plate numbers calculated for dansylamino acids were 28,000-111,000 with a 19-cm capillary column.     

Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis

A disadvantage of genotyping bacterial strains by pulsed-field gel electrophoresis is that the procedure requires up to 6 days to complete. We modified a standard pulsed-field gel electrophoresis method (B.E. Murray, K.V. Singh, J.D. Health, B.R. Sharma, and G.M. Weinstock, J.Clin. Microbiol. 28:2059-2063, 1990) so that it could be completed in less than 3 days. We successfully applied this method to the analysis of a variety of gram-positive and gram-negative bacteria.     

Separation of proteolytic enzymes originating from Antarctic krill (Euphausia superba) by capillary electrophoresis

The viscosity-adjustable property of F127 block copolymer PEO99PPO69PEO99, PEO and PPO being poly(ethylene oxide) and poly(propylene oxide), respectively, was found to be useful for the development of automated capillary electrophoresis (CE). The polymer solution can form a gel-like structure with sieving ability and can also serve as a dynamic coating material, thereby effectively suppressing the electroosmotic flow induced by the ionization of the silanol group on the quartz capillary inner wall. When applied to CE as a separation medium, F127 block copolymer can provide the advantages of high separation resolution, easy injection and replacement of the triblock copolymer solution and convenient capillary column treatment. High reproducibility of DNA electrophoretic migration time in CE by replenishing F127 solution in acid-washed capillary tubings can be achieved. The relative standard deviation of the DNA migration time is less than 2%. In the investigation of F127 concentration and temperature effects on the performance of DNA separation in CE, we have found that the DNA electrophoretic migration behavior in the F127 gel-like solution cannot be described by any one of the existing models.     

Separation of aromatic acids, DOPA, and methyl-DOPA by capillary electrophoresis with dendrimers as buffer additives

The underlying disease of a candidate for lung transplantation, especially advanced pulmonary fibrosis, can cause particular and dramatic difficulties. Pulmonary fibrosis is the end-result of a variety of pathological diseases and their associated processes. This article summarizes the diagnosis and management of some of the more common causes of fibrosis, outlines their natural histories and treatment outcomes, and describes the trade-off of pulmonary fibrosis for lung transplantation. Four main categories of end-stage fibrosis are discussed: idiopathic pulmonary fibrosis, sarcoidosis, pulmonary fibrosis from systemic diseases or drugs, and occupational- or environmental-related pulmonary fibrosis. Each group will be covered systematically and the options and indications for lung transplantation will be addressed.     

纸上高压电泳分离-荧光光度法测定钇镧镥

以柠檬酸为电解液,纸上高压电泳法分离稀土元素钇、镧、镥,分离后的稀土离子在纸上与桑色素(morin)形成络合物,用426 nm激发光照射,纸上络合物产生荧光,在494 nm处测定荧光强度。Y3+,La3+和Lu3+的质量浓度分别在0.1~30 ng/μL,0.5~50 ng/μL和0.15~40 ng/μL范围内与荧光强度成良好的线性关系;检出限分别为0.03,0.15,0.045 ng/μL。方法试液用量少,操作简单,已成功用于分离、测定合成试样中钇、镧、镥。     

Characterization of proteins from human cerebrospinal fluid by a combination of preparative two-dimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To purify and characterize low-abundance proteins in complex biological mixtures, we used a novel strategy that combined preparative two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative 2D-LPE is based on the same isoelectric focusing and gel electrophoresis principles as the widely used analytical 2D gel electrophoresis, except that analytes remain in solution throughout separation. This novel approach shows many improvements compared to analytical 2D gel electrophoresis for the separation of proteins in biological fluids. For example, larger volumes/amounts of samples can be loaded, yielding sufficient amounts of low-abundance proteins for further characterization. Since proteins remain in liquid phase during the entire procedure, extra steps such as electroelution, extraction, or transfer to membranes from the gels prior to mass spectrometric analysis are obviated. We report the usefulness of 2D-LPE combined with MALDI-TOF MS for the purification and characterization of cystatin C and beta-2 microglobulin in human cerebrospinal fluid. This method should be applicable to a wide range of biological fluids, such as cerebrospinal fluid, serum, tissue extracts, cell media, whole cells, and bacterial lysates.