Fractionation techniques in a hydro-organic environment. I. Sulfolane as a solvent for hydrophobic proteins

Sulfolane (thiophene, tethrahydro-1,1-dioxide), at concentrations of 4 M or above, is an efficient solubilizing agent for water-insoluble proteins (e.g., zein or globin chains). In comparison with urea, it appears indefinitely stable in aqueous solutions and does not chemically modify proteins upon storage. Moreover, it favors protein structure, i.e., it increases their alpha-helix content, while urea decreases it. Sulfolane is compatible with electrophoretic techniques (it only slightly reduces polyacrylamide polymerization efficiency and it does not interfere with protein and peptide detection methods) and with chromatographic methods (it has negligible A280 nm). With hydrophilic proteins, sulfolane behaves as a mild denaturant and precipitates them at concentrations between 5 and 7 M.     

Polyacrylamide gel electrophoresis in the differentiation of Taenia (cestoda) by total protein

Bursey C. C., Mckenzie J. A. & Burt M. D. B. 1980. Polyacrylamide gel electrophoresis in the differentiation of Taenia (Cestoda) by total protein. International Journal for Parasitology10: 167–174, Step-gradient polyacrylamide gels were used to examine total protein in 3 taeniid species. Preliminary investigations revealed that band patterns of different parts of the taeniid strobila were basically identical; hence, individual worms were useful for analysis regardless of their state of development when collected. Using Hymenolepis diminula as a control, the band pattern for each of Taenia taeniaeformis, T. macrocystis and T. pisiformis was found to be species specific with marked differences. The procedure was adapted for use under the more adverse conditions present in field laboratories. Small quantities of tapeworm material used in analysis also allows specific differentiation of single proglottids of Taenia spontaneously expelled from live hosts.     

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.     

Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: I. optimization of enzyme environment

Biocatalytic transesterification of methylmethacrylate is possible in many different solvents. The reaction rate is readily controlled by variation in solvent physical properties. The reaction proceeds better in hydrophobic solvents, and activity can be restored in hydrophilic solvents by the addition of water. We have now demonstrated that supercritical carbon dioxide is not a good solvent for the reaction between 2-ethlhexanol and methylmethacrylate. It apperars that the supercritical carbon dioxide may either alter the pH of the microaqueous environment associated with the protein or reversibly form covalent complexes with free amine groups on the surface of the enzyme. Although supercritical carbon dioxide is a poor solvent for acrylate transesterification, many other supercritical fluids (ethane, ethylene, sulfur hexafluoride, and fluoroform) are better than most conventional solvents. In supercritical ethane it is possible to control the activity of the enzyme by changing pressure, and the enzyme appears to follow Michaelis-Menten Kinetics. We find that sulfur hexafluoride, the first anhydrous inorganic solvent in which biocatalytic activity has been reported, is a better solvent than any conventional or supercritical organic fluid tested.     

The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate, a zwitterionic detergent, has been shown to exhibit superior membrane protein solubilizing characteristics as compared to nonionic detergents. Replacement of NP-40 with CHAPS in isoelectric focusing of rainbow trout liver microsomes has increased resolution markedly. The two-dimensional electrophoretic system described will allow effective resolution of up to 300 micrograms of crude microsomal protein. CHAPS exhibits no effect on the stability or type of pH gradient when compared to NP-40 during isoelectric focusing in the presence of urea.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74–4.43 and have molecular weights ranging from 55 000–72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S₁ and S₂ which show partial identity. The results do not necessarily support. Juneja et al.s (1979) contention of two closely linked α1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.     

Use of cationic detergents for polyacrylamide gel electrophoresis in multiphasic buffer systems

An improved system for polyacrylamide gel electrophoresis in the presence of cationic detergents, cetyltrimethylammonium bromide and cetylpyridinium chloride, respectively, is described. An acidic discontinuous buffer system generated according to the theory of multiphasic zone electrophoresis developed by T. M. Jovin (1973, Biochemistry 12, 871-904) was used. It was optimized with respect to the operational conditions and to the desirable range of relative mobility values for the proteins that have molecular weights from 16,500 to 90,300. Also presented is a procedure for the elimination of interference from cationic detergents frequently encountered during staining of gels. The electrophoretic system was suitable for fractionating a wide variety of proteins. The technique can also be used to provide an alternative estimate of molecular weight. To fully account for accurate estimations, the Ferguson relationship between mobility and gel concentration and the relation of molecular weight to mobility at a single gel concentration were both considered. Examples reported in this paper include the separation and/or molecular weight determination of several common proteins, histones, and microfibrillar and myofibrillar proteins. The results suggest that electrophoresis in the presence of cationic detergents offers the same degree of reliability in analysis of most proteins as is provided by the anionic detergent sodium dodecyl sulfate electrophoresis.     

High-throughput Three-dimensional Gel Electrophoresis for Versatile Utilities： A Stacked Slice-gel System for Separation and Reactions （4SR）

Introduction The completion of the Human Genome Project has triggered large-scale screening of genomes (1) and proteomes (2) in aims to find out candidate genes related to diseases (3), perform expression analyses at the mRNA level (4) or at the protein level (5), discover new drugs (6), and analyze molecular in- teractions (7). For such purposes, technologies han- dling a tiny amount of samples should be developed, of which the importance has already been described as the ambient analyte th…     

Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector for capillary electrophoresis

Vancomycin is one of a family of related macrocyclic glycopeptide antibiotics that were discovered by scientists at the Eli Lilly Company in the 1950s. It has been used to treat severe staphylococcal infections, particularly when bacterial resistance to other antibiotics has developed. Vancomycin is a naturally occurring chiral compound and has a number of stereogenic centers. Furthermore, it contains a variety of functionalities that are known to be useful for enantioselective interactions (e.g., hydrogen bonding groups, hydrophobic pockets, aromatic groups, amide linkages, etc.). The physicochemical properties of vancomycin, including its stability in solution, are discussed as they pertain to capillary electrophoresis. Over 100 racemates were resolved including many nonsteroidal antiinflammatory drugs, antineoplastic compounds and N-derivatized amino acids. Many of these compounds had very high resolution factors. Optimization and the effect of different experimental parameters on the enantioselective separations are discussed. © 1994 Wiley-Liss, Inc.     

Glycosidases in organic solvents: II. Transgalactosylation catalysed by polyethylene glycol-modified β-galactosidase

β- -Galactoside galactohydrolase (E.C. 3.2.1.23) was chemically modified with 1,1′-carbonyldiimidazole-activated polyethylene glycols (MW 2,000, 8,000, and 20,000). The modified β-galactosidases had over 50% of amino groups coupled to polyethylene glycol but retained over 50% of the original activity. The hydrophobically modified enzymes were soluble in chlorinated organic solvents in which transferase activity has been demonstrated. The transferase activity, its dependency on water content, and the thermostability of all three modified enzymes were compared.     

Development and validation of method for analysis of some ototoxic solvents in saliva matrix by headspace gas chromatography/mass spectrometry

The aim of this study was to develop an analytical method to monitor the saliva matrix for ototoxic solvents absorption: the method is based on headspace gas chromatography/mass spectrometry and represents an alternative biological monitoring for investigating low exposure to hazardous ototoxic solvents. Simultaneous determination of toluene, ethylbenzene, xylenes and styrene has been carried out and the method has been optimized for both instrumental parameters and samples treatment. Chromatographic conditions have been set in order to obtain a good separation of xylene isomers due to the interest in p-xylene as ototoxic one. Method validation has been performed on standards spiked in blank saliva by using two internal standards (2-fluorotoluene and deuterated styrene-d₈). This method showed the possibility to detect the target compounds with a linear dynamic range of at least a 2 orders of magnitude characterized by a linear determination coefficient (r²) greater than 0.999. The limit of detection (LOD) ranged between 0.19 ng/mL (styrene) and 0.54 ng/mL (m-xylene) and the lower limit of quantification (LLOQ) ranged between 0.64 ng/mL (styrene) and 1.8 ng/mL (m-xylene). The method achieved good accuracy (from 99 to 105%) and precision for both intra- and inter-assay (relative standard deviation ranging from 1.7 to 13.8%) for all six compounds concerned. The repeatability was improved by adding sodium sulphate to the matrix. Saliva samples resulted stable for at least 7 days after collection, if stored in headspace vials, at the temperature of 4 °C. An evaluation of the main sources of uncertainty of the method is also included: expanded uncertainties ranges between 10 and 16% for all of the target compounds. In summary, the headspace gas chromatography/mass spectrometry method is a highly sensitive, versatile and flexible technique for the biological monitoring of exposure to ototoxic solvents by saliva analysis.     

Ixodes ricinus: the potential of two-dimensional gel electrophoresis as a tool for studying host-vector-pathogen interactions

Ixodes ricinus is a three-host tick, with three active instars. For moulting to occur the tick has to find a host where it can take a blood meal. Throughout feeding I. ricinus can be infected or infect the host with different pathogens, e.g., Tick-Borne Encephalitis virus or Borrelia burgdorferi. The host-vector-pathogen interaction is very complex, making a detailed study difficult. Here we analyse the potential of two-dimensional gel electrophoresis (2DE) to study the host-vector-pathogen interaction. We examined 20 nymphs, which as larvae parasitised either mouse or hen. After moulting, they were kept alive for up to 30 weeks, to analyse whether tick ageing influenced host determination, and for comparison of the 2D-gels. Even though the number of proteins in the gel decreased during ageing, some proteins of the host determination persisted for all 30 weeks. We also discovered persisting proteins in relation to nymphs. These findings showed that 2DE is suitable as a tool for studying host-vector-pathogen interactions.     

Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis

Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.     

Increased serum bile acids as a possible biomarker of hepatotoxicity in Brazilian workers exposed to solvents in car repainting shops

Abstract

The objective was to evaluate total serum bile acids (SBA) as a biological marker of hepatotoxicity in car painters exposed to organic solvents and to compare their performance with classic biochemical parameters of liver function. SBA were analysed in a selected group of workers (n=57) occupationally exposed to a mixture of organic solvents and in a control group (n=51). In addition, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) were determined in the two groups. Urinary hippuric acid was measured in all samples. Statistical analysis of the data revealed a significant increase in the concentration of SBA, AST, ALP and TB in exposed workers compared with controls (Mann–Whitney, p≤0.05). However, SBA was the parameter most frequently altered in exposed workers and showed higher significance between the two groups (chi-square test) compared with the upper limit of the reference range (8?µmol?l^?1). In conclusion, SBA can be considered to be a sensitive parameter of hepatotoxicity induced by organic solvents than the traditional tests and it can be used as an biological marker of subclinical liver injury.     

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved. Here we propose an approach toward standardizing mitochondrial 2D PAGE data in support of a globally relevant proteomics consensus.     

Carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis

A carboxymethylated cyclosophoraose (CM-Cys) was synthesized by the chemical modification of neutral Cys, which was isolated from Rhizobium trifolii TA-1. CM-Cys was successfully applied as a novel chiral selector for the separation of some flavonoids including catechin, 3,5,7,3′,4′-pentahydroxyflavanone, hesperidin, hesperetin, isosakuranetin, naringenin, naringin, and eriodictyol. The effects of pH, chiral additive concentration, and temperature on resolution and migration time were also studied.     

Enantioseparation using cyclosophoraoses as a novel chiral additive in capillary electrophoresis

Cyclosophoraoses, cyclic beta-(1-->2)-D-glucans produced by Rhizobium meliloti 2011, were used as a novel chiral additive for the separation of terbutaline, amethopterin, thyroxine and N-acetylphenylalanine enantiomers in aqueous capillary electrophoresis (CE). Enantioseparation took place in the normal- or reversed-polarity mode when a high concentration of neutral (60 mM) or anionic (40 mM) cyclosophoraoses was added to the background electrolyte (BGE).