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PubMed2005

Hydrophobic interaction chromatography: harnessing multivalent protein-surface interactions for purification procedures.

Hydrophobic interaction chromatography (HIC) is one of the basic purification procedures in the biosciences. However, because of its complexity, it has not gained the same foothold in the methodological repertoire of protein chemistry as has affinity chromatography or ion exchange chromatography. This is mainly a result of the lack of a general optimization procedure for the reversible adsorption and elution of a novel protein to be purified. Further problems arise from the fact that most commercial hydrophobic adsorbents are inadequate for an ideal performance in downstream processing procedures, because these media are too hydrophobic and elution of proteins in their native state is often impossible. Therefore, as in the 1970s, a bioscientist of today has to be capable of synthesizing a small library of hydrophobic gels from which he or she can then select and optimize the ideal matrix for their special needs. In addition, a general optimization method employing the critical hydrophobicity concept has now been devised that should allow the application of HIC methodology to many hitherto unpurified proteins. In this chapter, the reader is first introduced to the theoretical background (multivalence, negative cooperativity, adsorption hysteresis) of the binding of protein ligands to hydrophobic supports, so that they will be capable of independently adapting HIC to a novel protein. Then a simple nontoxic method is described for the synthesis of HIC-gel libraries consisting of a homologous series of uncharged alkyl-Sepharoses of three chain lengths (butyl, pentyl, and hexyl Sepharose) prepared with different degrees of separation. From this series a critical hydrophobicity gel can then be selected and employed for critical hydrophobicity HIC. A detailed example for the chromatography of human fibrinogen is given that has been employed as a one-step procedure for the purification of fibrinogen from human plasma.

Binding Sites↗
PubMed2005

[Polyethylene glycol-accompanied ion-exchange chromatography to purify recombinant hepatitis B virus surface antigen].

The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.

Animals↗
PubMed2006

[Purification of plastocynin from Ulva pertusa by column chromatography and analysis of its N-terminal amino acid sequence].

Protein plastocyanin from a green alga, Ulva pertusa, has been purified. Samples were homogenized in 0.02 mol/L phosphate buffer (pH 7.2) and then centrifuged to remove debris and subjected to ammonium sulfate fractionation (40%-80% saturation). Ion exchange column chromatography with DEAE-Sepharose Fast Flow and gel filtration column chromatography with Sephadex G-75 were then employed for further purification of plastocyanin. Three peaks, A, B and C, were eluted with 0.01 mol/L phosphate buffer, containing a NaCl linear gradient from 0 to 1.0 mol/L at the flow rate of 32 mL/h through DEAE-Sepharose chromatography. The protein fractions containing the plastocyanin were then purified further with Sephardex G-75 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) is analysis indicates that the protein was purified to homogeneity and its relative molecular mass is 10,000. N-terminal amino acid sequence was used to identify the protein. The protein was transblotted to PVDF membrane and N-terminal amino acid sequence was performed via Edman degradation with an automated amino acid sequencer. The 20 N-terminal amino acid residues are AAIVKLGPDDGSLAFVPSKI, which share 85% homology with the 20 N-terminal amino acid sequence of U. prolifera and U. arasakii, and share 90% homology with the ones of U. pertusa formerly reported.

Chlorophyta↗
PubMedDate not supplied

Effect of preferred binding domains on peptide retention behavior in reversed-phase chromatography: amphipathic alpha-helices.

A nonpolar environment, such as the hydrophobic stationary phase of a reversed-phase chromatographic packing, may induce helical structures in potentially helical molecules. If a molecule becomes helical on binding and contains a preferred binding domain, as in the case of an amphipathic helix, then some residues may not be contributing to the same extent to the overall hydrophobicity of the peptide. Amphipathic alpha-helical structures may play an important role in protein folding and the interaction of amphipathic alpha-helices with a hydrophobic surface during RPC is likely to be a good mimic of their hydrophobic interactions with other hydrophobic regions in the folded protein. We have designed and synthesized two sets of model peptides of 7, 14, 21, 28 and 35 residues having the same composition but different sequences (Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-amide and Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-amide, where n = 1-4). Circular dichroism studies demonstrated that both sets of peptides had a high potential to form alpha-helical structure in a nonpolar environment, one set representing amphipathic alpha-helical structures and the other set representing non-amphipathic alpha-helical structures. Size-exclusion chromatography confirmed that all of the peptides in both sets were monomeric when bound to a reversed-phase matrix and also under the conditions used for circular dichroism measurements. Peptides with the same amino acid composition and similar secondary structure could be separated by reversed-phase chromatography. The difference in retention time between peptides of the same length increased with the peptide chain length, ranging from a difference of 2.9 min on a C8 column for the two 14-residue peptides up to a maximum difference of 7.3 min for the 35-residue peptides. From the observed and predicted retention times of these two sets of peptides during reversed-phase chromatography, we have demonstrated that it is possible not only to predict the retention behavior of amphipathic alpha-helices during reversed-phase chromatography, but also to deduce the presence of amphipathic alpha-helical structure in peptides based upon their retention data. If from studies such as these we are eventually able to predict, from only amino acid sequence information, the secondary structure of a peptide on binding to a hydrophobic matrix, we may be able to extrapolate this predictive facility to the conformation of the same sequence in larger polypeptides or proteins.

Amino Acid Sequence↗
PubMed1989

Application of high performance liquid chromatography to the analysis of some non-volatile coffee components.

High performance liquid chromatography (HPLC) was applied to the analysis of caffeine, trigonelline, nicotinic acid and sucrose in Arabica and Robusta coffee. Green and roasted coffee samples were used in this study and the degradation of sucrose and trigonelline, with the formation of nicotinic acid, was followed during roasting. Caffeine did not undergo significant degradation with only 5.4% being lost under severe roasting. Sucrose was degraded rapidly during processing with light roasting producing a 97% loss and dark roasting degrading it completely. Loss of trigonelline was strongly dependent on the degree of roasting, being higher in the Robusta coffee. Trigonelline degradation was associated with nicotinic acid formation both in the Arabica and Robusta coffees as a consequence of the roasting process. Trigonelline and sucrose were determined simultaneously by partition chromatography and detection with the mass detector. Determination of caffeine was carried out using reversed phase chromatography and nicotinic acid by ion-pair reversed phase chromatography. Detection in both cases was achieved using an ultraviolet detector at 272 nm or 254 nm, respectively. HPLC showed adequate precision and accuracy for routine analyses. In addition, the methods used were more rapid and simple than traditional procedures. HPLC appears to be a suitable technique for quality control in the coffee industry, and for fundamental investigation on the mechanisms involved in the roasting process.

Alkaloids↗
PubMed1989

Protein purification by multidimensional liquid chromatography.

Recently Giddings discussed the prospect of combining two separation mechanisms in such a way that when "a sample is subjected to two displacement processes oriented at right angles to one another" a two-dimensional separation is carried out. In this review I have focused attention on the various ramifications of this concept in terms of combining two or more chromatographic techniques on-line to conduct MDLC for the purpose of purifying proteins. In general, the MDLC approaches discussed here were classified into two major categories. The first category involves the placement of several separation mechanisms in the same chromatographic work space (the chromatographic column). In this case the displacement processes are collinear. It is hoped that these new chromatographic packings and columns will display surface characteristics capable of a wide range of highly discriminating selectivities that can be modulated by mobile-phase changes to a greater extent than the nonspecific chromatographic techniques such as IEC and HIC. The ability to modulate the mobile phase to generate new selectivities is important in expanding the usefulness of these packings in comparison to the very high selectivity of affinity chromatography, which usually has little use outside its initial intended purpose to purify a particular protein. The second category involves the on-line physical coupling of two or more chromatography columns, each packed with a different chromatography material. Again the idea is to create or design a simple self-contained system that is capable of generating a wide range of high selectivities. Indeed, the on-line coupling of two different chromatographic packings for the purification of a single protein represents a line trace through "a discrete independent 2-dimensional system". These systems are highly attractive in large-scale purification, especially when using "on-off" chromatography, which eliminates the need for sophisticated gradient elution hardware. The purification of a single protein from its biological matrix is usually a multidimensional process utilizing several different separation technologies. By nature this leads to lengthy purifications that are frequently labor-intensive and expensive to scale up. It is my belief that future developments in the concept of on-line MDLC techniques involving complex chromatographic materials and column coupling will merge to create significant improvements in the protein purification process.

Chemical Phenomena↗
PubMed1989

High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids.

Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.

Animals↗
PubMed1987

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography.

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.

Binding Sites↗
PubMed1986

Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography.

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-3H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca2+- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70 degrees C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.

Animals↗
PubMed1986

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids.

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.

Animals↗
PubMed1986

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5.

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.

Chromatography, Affinity↗
PubMed1984

The nuclear-coded subunits of yeast cytochrome c oxidase. I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reversed phase high performance liquid chromatography.

Previously, cytochrome c oxidase from the yeast Saccharomyces cerevisiae has been thought to be composed of seven different polypeptide subunits. Four of these are small polypeptides (4,000-15,000 daltons), subunits IV-VII, which are encoded by nuclear DNA. Studies described here reveal the presence of two new polypeptides in this size range. These polypeptides, designated as subunits VIIa and VIII, co-migrate with subunit VII (R.O. Poyton and G. Schatz (1975) J. Biol. Chem. 250, 752-761) on low resolution sodium dodecyl sulfate (SDS) polyacrylamide gels, can be partially resolved on high resolution SDS polyacrylamide gels, and can be completely separated from one another by reversed phase high performance liquid chromatography. In order to determine the sequences of each of these six nuclear-coded polypeptides (subunits IV, V, VI, VII, VIIa, and VIII), we have developed new methods for the large scale purification of the holoenzyme and have employed a new strategy for the isolation of each polypeptide. By using octyl-Sepharose chromatography to isolate holocytochrome c oxidase and by extracting the holoenzyme with aprotic organic solvents and fractionating these extracts by reversed phase high performance liquid chromatography, it is possible to isolate several milligrams of each of these subunits. Each subunit preparation gives a single peak during reversed phase high performance liquid chromatography, a single band during SDS-polyacrylamide gel electrophoresis, a single NH2-terminal sequence, and a unique amino acid composition and tryptic peptide map. Since each purified subunit preparation gives close to a 100% yield of its NH2-terminal amino acid during quantitative Edman degradation, we conclude that no subunit has a blocked NH2 terminus and that no subunit preparation contains either blocked or unblocked contaminating polypeptides. Thus, each consists of a single unique polypeptide species. Together, these results demonstrate that yeast cytochrome c oxidase contains six, rather than four, small subunit polypeptides. Thus, it appears that these polypeptides, in combination with the three polypeptides encoded by mitochondrial DNA, constitute a holoenzyme which contains nine subunits, instead of seven as proposed earlier.

Amino Acid Sequence↗
PubMed1981

Self-association of heparan sulfate. Demonstration of binding by affinity chromatography of free chains on heparan sulfate-substituted agarose gels.

We have developed an affinity chromatography procedure which measures binding of heparan sulfate species to agarose gels substituted with different heparan sulfates. Three major subfractions of bovine lung heparan sulfate (HS2, HS3, and HS4) which differ in sulfate content and hexuronate composition have been used. Association-prone variants of these species (HS2-A, HS3-A, and HS4-A) were prepared by gel chromatography. Free heparan sulfate chains were applied to columns of various heparan sulfate-agaroses which were eluted with a linear guanidine gradient and binding was assessed by measuring the hexuronate content of the effluent. Associating heparan sulfate of a particular subfraction was chiefly bound to gels that were substituted with chains of the same kind, i.e. HS2-A to HS2-A-agarose, HS3-A and HS4-A to HS3-A-agarose, and HS4-A to HS4-A-agarose. N-Desulfation and N-acetylation of HS2-A markedly reduced binding to HS2-A-agarose and periodate oxidation of glucuronate in HS3-A completely abolished binding to HS3-A-agarose. Partially oxidized HS2-A was separated into bound and unbound material by affinity chromatography on HS2-A-agarose. Gel chromatography of these fractions indicated that unbound chains were significantly smaller than bound ones. It is concluded that association between heparan sulfate chains may be quite specific and that the strength of binding is dependent on co-operative interactions between a number of contact zones. The latter may correspond to the N-sulfated and iduronate- and glucuronate-containing segments.

Animals↗
PubMed1984

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.

Animals↗
PubMed1981

[A liquid chromatography technique for routine determination of urinary estriol in human pregnancy (author's transl)].

A liquid chromatography procedure for routine determination of urinary estriol concentrations is described. After enzymic hydrolysis of a 1 ml sample, automatic extraction with chloroform/ethyl acetate, purification by chromatography on a disposable "minicolumn" of AG 1-X2, urinary estrogens are separated with normal phase column and isocratic elution with isooctane/ethyl acetate/methanol/water and the optical density at 280 nm is measured. The specificity is assessed by comparison of liquid chromatography with fluorometry and gas liquid chromatography. The technique is simple, rapid and quite suitable for routine determination.

Chromatography, High Pressure Liquid↗
PubMed1995

Identification of arachidonate epoxides/diols by capillary chromatography-mass spectrometry.

The identification of epoxide regioisomers of arachidonic acid (EETs) as methyl esters is difficult because they coelute during gas chromatography and possess similar mass spectra. In the present study, EETs and their hydrolysis products, dihydroxyeicosatrienoic acids (DHETs), were analyzed as pentafluorobenzyl ester derivatives and their properties were compared to other esters. The four EET regioisomers were not resolved by gas chromatography as pentafluorobenzyl, trimethylsilyl, t-butyldimethylsilyl, or methyl esters. However, after being hydrolyzed to DHETs, three of the four regioisomers were resolved as (bis)-t-butyldimethylsilyl ether, pentafluorobenzyl esters. The fourth regioisomer (5,6-DHET) was resolved after being converted to a delta-lactone. Thus, the EETs could be resolved by capillary gas chromatography once converted to DHETs. Pentafluorobenzyl esters of both EETs and DHETs (15-40 ng) provided diagnostic spectra when analyzed by electron ionization mass spectrometry. The mass spectral interpretations that indicated epoxide and diol positions were validated using synthesized EET/DHET [17,17,18,18-d4, 5,6,8,9,11,12,14,15 d8] standards. Lesser amounts of DHETs (5-150 fg) also indicated molecular weights when analyzed in the negative-ion chemical-ionization mode. In summary, EETs in nanogram quantities were identified as pentafluorobenzyl esters using electron ionization mass spectrometry. EETs in femtogram-to-picogram quantities were also identified after conversion to DHETs and analysis by gas chromatography-mass spectrometry in the negative ion-chemical ionization mode.

Arachidonic Acid↗
PubMed1976

Separation by column chromatography of cells active in delayed-onset hypersensitivities.

Lymph node cells from guinea-pigs contact sensitive to 1-thiocyamo-2,4-dinitrobenzene have been fractionated by affinity chromatography over modified polyacrylamide beads. Cells mediating lymphokine release in response to active sensitizer were depleted only by chromatography over dinitrophyenyl (DNP) containing substrates and could be specifically eluted with DNP-glycine. DNP rosette-forming cells (RFC) were equally well depleted by chromatography using either DNP or trinitrophenyl containing materials but could not be eluted from the columns by DNP-glycine. While the antigen receptors of cells mediating the release of macrophage agglutination factor in response to DNP-containing antigens and of DNP-RFC were found to be hapten-specific, their specificity was shown to differ using chromatography over trinitrophenyl containing polyacrylamide.

Animals↗
PubMed1993

[Separation and identifying features of the cardiac aglycones and glycosides of Nerium oleander L. flowers by thin-layer chromatography].

The present work was aimed at extracting some cardiotonic glycosides from Nerium oleander L. (N.o.L.) flowers, free from phytosterols and their esters and at resolving themselves by thick- and thin-layer chromatography. This work may contribute to the scientific recognition of N.o.L. drugs in the field of forensic medicine. MATERIALS AND METHODS. Apparatus. Kieselgel F254 thin-layer (250 mu) and Kieselgel thick-layer (2 mm) glass plates were used. MATERIALS. Chromatographic solvents were used; detectors were p-toluene sulphonic acid (PTSA) and antimony trichloride (SbCl3). STANDARDS. The following standards were used: gitoxigenin, oleandrin, digitoxin, and stigmasterol. Standard solutions were prepared at a M-3 concentration. The solvent system is described in Table I. EXTRACT. The extract was prepared using a mixture of solvents (hexane-diethylether-acetic acis, 50:50:1 v/v). With this mixture we noted that, in thin-layer chromatography, the used standards remained at the origin, while phytosterols and their esters migrated. White flowers of N.o.L. were air dried in the shade at room temperature, powered, and macerated in hexane-diethylether acetic acid for 24 hours. After removal of the phytosterols the remacerating powder was further percolated using a chloroform-methanol mixture (1:1.7 v/v) in order to extract the aglycone and the cardiac glycosides. As the medium's thin-layer chromatogram showed the absence of corresponding spots to test samples, the chloroform-methanol media were combined and evaporated under vacuum. The residue was redissolved in the chloroform-methanol mixture. The obtained extract concentration consisted of 5 grammes of dried flowers in 1 milliliter of solution, called extract E (Fig. 1). RESULTS. The results are reported in Figures 2 and 4 and in Table II (see text for explanation of symbols). Extract E (Fig. 2) and the F1, F2, and F3 concentrated fractions were examined by thin-layer chromatography (Fig. 1 and 3). Only the F3 fraction revealed compounds with the same chromatographic path and colours (detected by PTSA and SbCL3) as the reference standards gitoxigenin, oleandrin, and digitoxin. This finding was confirmed by using five different solvent-systems (Fig. 4). DISCUSSION AND CONCLUSION. The present study shows that the cardiac steroids and other constituents of Nerium oleander's flowers are separated in three fractions by thick-layer chromatography free from phytosterols and their esters. This result may have useful implications in the fields of analytical toxicology and forensic medicine and potential application in clinical practice given that cytotoxic and antileukemic activities of the extracts from plants containing cardiac glucosides have been reported.

Cardiac Glycosides↗