Sequential Elution Research Articles

Lipid quantitation in formalin-fixed liver sections.

We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.

Isolation and primary structure of a neuropeptide hormone from Heliothis zea with hypertrehalosemic and adipokinetic activities

A neuropeptide was isolated from the corpora cardiaca of the corn earworm moth Heliothis zea, and purified by sequential gradient elution in three reversed phase-high performance liquid chromatographic steps. The primary structure, pGlu-Leu-Thr-Phe-Ser-Ser-Gly-Trp-Gly-Asn-NH2 was determined by automated gas-phase Edman degradation of the peptide deblocked with pyroglutamate aminopeptidase, and confirmed by fast atom bombardment mass spectrometry. The hormone was synthesized and the natural and synthetic peptides had identical chromatographic and spectroscopic properties. Both natural and synthetic hormones caused the elevation of trehalose and lipid levels in the hemolymph of adult H. zea males.

Anion-Exchange Separation of Nd and the Transplutonium Elements in Spent Nuclear Fuels for Burn-up Determination

Abstract Three methods of anion-exchange separation of Nd, Am, and Cm from spent nuclear fuel samples for destructive burn-up measurements with nitric acid-methyl alcohol mixed media have been developed. The first method was developed for the routine analysis of Nd by isotope dilution mass spectrometry to separate Nd from Am and Cm as well as from major fission products, U and Pu, on a conventional anion-exchange resin with 1 M HNO3−90% CH3OH and 0.5 M HNO3−80% CH3OH media at ambient temperature. The second method quantitatively separates Nd, Am, and Cm using a fine resin and a 0.5 M HNO3−80% CH3OH medium at a considerably slower flow rate at ambient temperature. The third method is a more rapid separation: the preliminary separation of irradiated U at elevated pressure is followed by the sequential elutions of Nd, Am, and Cm at elevated temperature and pressure

151 AFFINITY CHROMATOGRAPHY ON AMP-SEPHAROSE 4R FOR THE SEPARATION OF “HIGH Km” AND “LOW Km” 5‘-NUCLEOTIDASES AND OTHER AMP METABOLIZING ENZYMES

AMP-Sepharose 4B has been widely used as a general ligand affinity chromatopraphy for purification of AMP deaminase, 5′-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AftP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a sinple run. Using 0.5 M MaCl, 10 mU ATP and 5 mU adenosine as elating agents, it was possible to separate on AMP-Sepharose column AMP deaminase, “high Km” and “low Km” 5′-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, “hiph Km” and “low Km” 5′-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and “high Km” 5′-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 umoles/min/mg protein of AMP deaminase, “high Km” 5′-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

Interfacial tension behavior and ESR properties of asphaltenes derived from heavy oil

Asphaltenes extracted from Lloydminster heavy oil (from Saskatchewan, Canada) were separated by sequential elution solvent chromatography (SESC) using ten different organic solvents. The fractions so obtained were dissolved in toluene and then examined for their interfacial tension (IFT) behavior against 0.1 wt.% sodium hydroxide solution. It was found that only about 40% (by weight) of the original asphaltenes contained species which were capable of lowering IFT against the alkaline solution. Multi-functional phenolic and other polar compounds were considered to be the components most likely responsible for the observed interfacial activity. Electron spin resonance measurements showed that the individual fractions contained varying concentrations of free radicals and VO 2+ ions. However, no direct correlation between the content of either of these two species and the interfacial tension could be detected.

A α and A β class II I-A determinants of antigen-specific T-helper factor and its antigen-nonbinding chain

Antigen-specific T-helper factor (ThF) of CBA (H-2 k) origin in the picryl (TNP) contact sensitivity system ( M r 60–70 kDa) was reduced with dithiothreitol under mild conditions. Affinity chromatography on antigen yielded an antigen-binding chain ( M r 20–30 kDa) and an antigen-nonbinding chain ( M r 40–50 kDa). Both chains were glycoproteins and were bound by lentil lectin. Affinity chromatography on anti-I-A monoclonal antibodies showed that I-A determinants occurred on the complete molecule and on the antigen-nonbinding, but not on the antigen-binding, chain. In contrast, five different monoclonal antibodies to I-E α failed to absorb ThF. Moreover, the complete molecule and the I-A + antigen-nonbinding chains had determinants of the alpha and beta chains of I-A and conformational determinants which are based on both chains. Sequential absorption and elution showed that A α and A β determinants occurred on the same molecular complex. These data suggest a minimal model of ThF as a two-chain disulfide-bonded structure with an antigen-binding chain and a separate I-A + antigen-nonbinding chain which behaves as a single unit in phosphate-buffered saline and has elements of both A α and A β.

High performance liquid chromatography of glycoconjugates

AbstractStationary phases were prepared for the analytical and micropreparative high performance affinity chromatography of carbohydrates and glycoproteins by covalent attachment of lectins and certain biogenic amines to the surface of microparticulate, macroporous diol‐silica. Columns packed with immobilized concanavalin A or wheat germ agglutinin afforded rapid separation of both glycoproteins and low molecular weight carbohydrates. The combined use of the two lectins in a mixed bed column or in two columns in series by sequential elution with the appropriate haptenic sugars facilitated the separation of mixtures having a broad range of complex carbohydrates. Chromatographic experiments for the study of the interaction of certain biogenic amines with immobilized glycoproteins used as the stationary phase led to the development of siliceous bonded phases with serotonin, tryptamine or phenylpropanolamine ligates. The sorbents thus obtained were mildly hydrophobic and cationic in contact with eluents of pH 8 or less. They were used for the separation of various carbohydrates; in addition they also proved effective for the separation of protein mixtures under conditions similar to those customarily employed in electrostatic or hydrophobic interaction chromatography.

Characterization of pentane-solubles from low rank coal extract

The structural characterization by FT-i.r. and 1H n.m.r. spectroscopy of the pentane-soluble fraction of a toluene extract from a Spanish low rank coal has been performed. The pentane-solubles have been fractionated on deactivated SiO 2 by sequential elution with hexane, benzene and tetrahydrofuran. The structural parameters calculated for these fractions indicate branched and cyclic alkanes on the aromatic units, and the number of aromatic rings and rings containing ring-joining methylene and heteroatoms appears to be between 1 and 5. Benzene and THF eluates showed the presence of hydroxyl and carbonyl groups. A comparison with the pentane-solubles from BF 3-phenol depolymerized lignite was made to evaluate the influence of this process on the nature of the soluble products. The pentane solubles of lignite extract have a more aliphatic and less polar character than the same fraction of chemically depolymerized lignite, which contains a maximum of four aliphatic carbon atoms in the average molecule of its benzene and tetrahydrofuran eluates.

Mutagenic and clastogenic activity of direct-acting components from air pollutants of the Silesian industrial region

Sequential elution solvent chromatography (SESC) developed by Farcasiu for characterization of coal liquids was used for the fractionation of benzene extracts of airborne particulate pollutants. Mutagenic and clastogenic activity of SESC fractions was determined by the Salmonella/microsome test and the assay for V79 cell chromosomal aberrations (CAs), respectively. Five out of 8 obtained fractions showed differentiated, direct and indirect mutagenic activity. Selected ‘direct’ fractions, examined by the rodent cell chromosome aberration test, also gave a clastogenic response that increased with prolonged treatment time. The SESC system combined with 2 biological assays, the Ames test and the CAs test, seems to be a useful method for examination of genotoxic components of environmental pollutants.

Automated and Manual In-Line Solid-Phase Sample Preparation for High-Performance Liquid Chromatography Analysis of Bittering Substances in Worts and Beers

The feasibility of in-line solid-phase treatment of samples (5–50 μl) of stout, ale, and lager beers prior to their injection for high-performance liquid chromatographic measurement of their contents of iso-α-acids was established using manual column-switching. Samples were injected directly onto a short (1 cm) precolumn packed with octadecyl-silica and were purged free from interfering substances by sequential step elution with solvents of decreasing polarity. The analytes were then recovered from the precolumn by elution with the chromatographic mobile phase and transferred to an analytical column. Different sample pretreatments using sequential step elution were then developed using fully automated column switching equipment. The protocols used were shown to give accurate and precise measurements of iso-α-acids and accomplished satisfactory “cleanup” of the beer samples. Automated column-switching methodology was then developed to analyze simultaneously the iso-α-acids, α-acids, and β-acids in wort samples, using a precolumn and two analytical columns. Column switching allowed satisfactory analyses of wort samples to be performed in one-third of the time taken by conventional isocratic elution. An electrochemical detector was shown to be more than 50 times more sensitive for the measurement of α-acids and β-acids than an ultraviolet detector. The methods were applied to the measurement of iso-α-acids in beers bittered either with hops or pre-isomerized hop extracts. In addition, the course of α-acid isomerization was monitored in kettles boiled at atmospheric pressure.

Construction of an ion-exchange amino acid analyser kit for use with high-performance liquid chromatography apparatus

A conventional ion-exchange amino acid analyser kit has been simply and economically constructed for use with existing high-performance liquid chromatography (HPLC) apparatus. Sequential elution conditions are fully programmable by virtue of a 32K BBC microcomputer interfaced with an elution buffer selection valve and a thermostatically controlled column. Post-column derivatization with o-phthalaldehyde—2-mercaptoethanol reagent enables fluorimetric detection at the picomole level. The system enables sensitive amino acid analysis of complex mixtures to be carried out by clinical and research laboratories who already possess HPLC apparatus but whose sample turnover does not merit purchase of a dedicated fully automated analyser.

Human beta-adrenergic receptors. Simultaneous purification of beta 1- and beta 2-adrenergic-receptor peptides.

Beta-Adrenergic receptors from basal membranes of human placenta were purified from digitonin extracts by sequential rounds of affinity chromatography, hydrophobic chromatography, ion-exchange chromatography and steric-exclusion h.p.l.c. Basal membranes display both beta 1- and beta 2-adrenergic receptors, in the ratio 65:35. Affinity chromatography, hydrophobic chromatography on heptylamine-Sepharose and ion-exchange chromatography on DEAE-Sephacel removed most of the contaminating proteins, and final purification of the receptor to apparent homogeneity was achieved by steric-exclusion h.p.l.c. The purified receptors showed Mr 67000 on SDS/polyacrylamide-gel electrophoresis under reducing conditions. Specific binding of radioligand to the purified beta-adrenergic receptors displayed stereoselectivity, and the agonist competition profiles demonstrated the presence of both beta 1- and beta 2-receptors. By using the subtype-selective ligands CGP-20712A (beta 1-selective) and ICI-118,551 (beta 2-selective), the purified Mr-67000 species was shown to be composed of equivalent amounts of beta 1- and beta 2-adrenergic receptors. Affinity chromatography on Sepharose-alprenolol and sequential elution with 1 microM-CGP-20712A followed by 100 microM(-)-alprenolol permitted beta 1-adrenergic receptors to be resolved from the mixture of beta 1-/beta 2-adrenergic receptors. The pharmacologically distinct human beta 1 and beta 2-adrenergic receptors are shown to be structurally very similar peptides.

A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs.

A hybridoma secreting a monoclonal antibody (H-20) that recognizes the 2,2,7-trimethylguanosine(m3G)-containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G-containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess of the cross-reactive nucleoside 7-methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein-A--Sepharose immunoprecipitation, mAb H-20 crossreacts with intact m7G cap structures. In particular we could show that non-denatured m7G-capped SP6/beta-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RNAs did not react. Thus the monoclonal antibody H-20 should have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G-capped transcripts.

Identification and characterization of the dihydropyridine-binding subunit of the skeletal muscle dihydropyridine receptor.

Photoaffinity labeling of isolated triads and purified dihydropyridine receptor with [3H]azidopine and (+)-[3H]PN200-110 has been used to identify and characterize the dihydropyridine-binding subunit of the 1,4-dihydropyridine receptor of rabbit skeletal muscle. The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads contains four protein subunits of 175,000, 170,000, 52,000, and 32,000 Da (Leung, A., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). Photoaffinity labeling of isolated triads with [3H]azidopine resulted in specific and covalent incorporation of [3H]azidopine into only the 170,000-Da subunit of the dihydropyridine receptor and not into the 175,000-Da glycoprotein subunit of the receptor. The [3H]azidopine-labeled 170,000-Da subunit was separated from the 175,000-Da glycoprotein subunit by sequential elution from a wheat germ agglutinin-Sepharose column with 1% sodium dodecyl sulfate followed by 200 mM N-acetylglucosamine. Photoaffinity labeling of purified dihydropyridine receptor with [3H]azidopine or (+)-[3H]PN200-110 also resulted in the specific and covalent incorporation of either ligand into only the 170,000-Da subunit. Therefore, our results show that the dihydropyridine-binding subunit of the skeletal muscle 1,4-dihydropyridine receptor is the 170,000-Da subunit and not the 175,000-Da glycoprotein subunit.

Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.

A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After extraction of rat liver membranes with Triton X-100, the enzyme was found to exist in two molecular weight forms of markedly differing size, separable on Sephadex G-200. The low Mr form was separated from the high Mr form on columns of CM-Sephadex and hydroxylapatite, and was further purified by sequential elutions with NaCl, UDP-GlcNAc, and EDTA from the 5-mercuri-UDP-GlcNAc affinity adsorbant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified low Mr form under reducing conditions revealed two protein bands of Mr 48,000 and 43,000. The purified enzyme catalyzes the transfer of N-acetylglucosamine from UDP-GlcNAc to the compound: (Formula: see text) The high Mr form of the enzyme, which eluted in the void volume of Sephadex G-200, was resistant to a number of treatments in attempts to reduce its molecular weight. These results suggest that the high Mr form of the enzyme may represent either a complex which normally exists in Golgi membranes as a result of strong protein-protein interactions or a protein with one or more "anchor" segments.

46] Vicia villosa lectins

The seeds of Vicia villosa (V. Villosa), commonly known as hairy winter vetch, contain a family of tetrameric lectins composed of two different subunits (designated A and B) with distinct carbohydrate-binding activities. The chapter describes various assay methods used, which include erythrocyte agglutination and I-labeled lectin binding and hapten inhibition. For the purpose of purification, the N-acetylgalactosamine-binding lectins from V. villosa are isolated from a seed extract by adsorption to a porcine blood-group substance affinity column and sequential elution with different concentrations of N-acetylgalactosamine. The B4, A4, and A2B2 lectins are not purified to homogeneity by affinity chromatography and must be purified further by anionexchange chromatography. The chapter describes the purification procedure and properties of the lectins.

Direct liquefaction of different lignocellulosics and their constituents: 1. Fractionation, elemental composition

Oils have been produced thermochemically from the following lignocellulosic materials by applying constant hydrogenolytic conditions in an aqueous phase with palladium as catalyst : spruce and birch wood, spruce and birch holocellulose, cellulose, pine bark, spruce and bagasse organosolv lignins, and birch Willstätter lignin. The liquid products were separated into water-, acetone- and dichloromethane-soluble (oil) fractions. The highest oil yield was obtained from spruce organosolv lignin (64%) and the lowest from carbohydrates and pine bark (20–31%). The oils were separated into neutral, strongly acidic and weakly acidic fractions by a liquid/liquid extraction procedure. Sequential elution by solvent chromatography was used for additional comparative characterization of the oils. Calorific values of the feedstocks and oils were calculated based upon the data of elemental analysis.

Separation and preconcentration of the rare-earth elements and yttrium from geological materials by ion-exchange and sequential acid elution

The abundance of rare-earth elements (REE) and yttrium in geological materials is generally low, and most samples contain elements that interfere in the determination of the REE and Y, so a separation and/or preconcentration step is often necessary. This is often achieved by ion-exchange chromatography with either nitric or hydrochloric acid. It is advantageous, however, to use both acids sequentially. The final solution thus obtained contains only the REE and Y, with minor amounts of Al, Ba, Ca, Sc, Sr and Ti. Elements that potentially interfere, such as Be, Co, Cr, Fe, Mn, Th, U, V and Zr, are virtually eliminated. Inductively-coupled argon plasma atomic-emission spectroscopy can then be used for a final precise and accurate measurement. The method can also be used with other instrumental methods of analysis.

Rapid Method for the Quantitative Extraction and Simultaneous Class Separation of Milk Lipids

A method is described for the isolation of lipids from milk, cream, or buttermilk. Lipid isolation is accomplished by solvent elution of a column containing a mixture of the milk product, anhydrous sodium sulfate, and Celite 545. Total lipids are isolated by elution with a 90:10 mixture of dichloromethane:methanol. Alternatively, lipids may be separated into a neutral lipid fraction by sequential elution of the column with dichloromethane and a polar fraction by elution with a 90:10 mixture of dichloromethane:methanol. The proposed method was compared with the traditional Roese-Gottlieb method for a series of milk samples with fat content ranging from<1 to 37%. Fat determinations for a series of five milk types by the dry column and Roese-Gottlieb methods were significantly different for whole raw milk and buttermilk, while means for phospholipids were significantly different for all milk types tested except buttermilk.

Regular coal structure and conversion severity

In two separate coal conversion studies, one dealing with CO-water conversions of Illinois No. 6 coal, and the other with H 2S addition to conventional tetralin conversions of the lower-rank Wyodak 2, data have been collected over a range of conversion levels. For both studies the degrees of conversion were increased by increasing the reducing power of the medium, while the conversion temperature was kept constant at 400 °C for all runs. The conversion levels for the CO-water runs ranged from 29 to 60%, while the tetralin-H 2S runs yielded conversions from 44 to 67%. Sequential elution solvent chromatography and field ionization mass spectrometry were used for analyses of toluene-soluble fractions from the CO-water runs and THF-soluble fractions from the H 2S-tetralin runs, respectively. The analyses showed that the fraction compositions remained constant with increasing degrees of conversion. This finding suggests that there may be some gross regularity in the structure of coal. Moreover, the results here are in contrast to work reported earlier, in which increased conversion levels, reached through greater conversion temperatures, yielded products with successively increased condensation and aromatization. It is thus suggested that severity be considered in terms of two components. Thermal severity reflects the temperature/time component of conversion chemistry, and derives from simple Arrhenius behaviour of competing reactions, while reduction severity reflects only on the reducing power of the system.