Gel Permeation Column Research Articles

Application of short column gel permeation in the study of protein-protein interactions

A simple and rapid procedure based on the gel filtration principle is described together with its applicability to the study of protein-protein interactions including subunit-subunit and enzyme-enzyme interactions. Using this procedure, it is shown that phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) interact with a stoichiometry of one PGK molecule combining with one monomeric subunit of GPDH. This interaction has been observed with both enzymes being from the same, as well as from different, species. The K d values for rabbit muscle PGK and porcine muscle GPDH complex and that for the rabbit muscle PGK and yeast GPDH complex are found to be (4.5 ± 2.0) × 10 −7 M and (6.5 ± 1.7) × 10 −7 M, respectively. The specificity of bienzyme association is stronger when enzymes are from the same species than when they are from different species.

Quantitative Analysis of Collagen and Collagen Subtypes I, III, and V in Human Pancreatic Cancer, Tumor-Associated Chronic Pancreatitis, and Alcoholic Chronic Pancreatitis

The collagen content in human pancreatic cancer tissue, tissue of tumor-associated chronic pancreatitis (TACP), and normal pancreatic tissue was determined in 14 patients with pancreatic cancer by measuring the amount of 4-hydroxyproline. Four patients with alcoholic chronic pancreatitis (AlCP) were also analyzed. The mean collagen content in both pancreatic cancer tissue and TACP tissue was approximately threefold higher than in normal pancreatic tissue. Cyanogen bromide peptides of type I, III, and V collagens from invasive ductal carcinomatous tissue of the pancreas and from TACP tissue of eight patients were analyzed sequentially using high-performance liquid chromatography with ion-exchange and gel-permeation columns. No difference in the proportion of type I, III, and V collagens was detected between pancreatic cancer tissue and TACP tissue. The mean collagen content in AlCP tissue was significantly lower than that in TACP tissue, but no difference in the proportion of type I, III, and V collagens was detected between these two tissues. These results indicate a similar quantity and distribution pattern of fibrillar collagen in human pancreatic cancer and TACP.

Polymer-Surfactant Interactions Studied by Phase Behavior, GPC, and NMR

Osmotic effects are important both in static phase behavior and dynamic gel permeation chromatography (GPC) analysis of polymer surfactant systems. Besides affecting the salinity for the II(-)/III phase transition in microemulsions, the water content in the middle phase will decrease when nonassociative, water-soluble polymers are added. The same systems will in a GPC column, using the surfactant solution as eluent, separate both due to osmosis and size exclusion. Association between polyethylene oxide and anionic ethoxylated surfactants disappears if more than three ethylene oxide (EO) groups are added to the surfactant head group. The phase separation of micelles and polymer into separate aqueous phases in the II(-) state occurred at lower salinity when the number of EO groups in the surfactant increased. The phase separation could qualitatively be modeled within a simple Flory-Huggins approach. With NMR self-diffusion measurements it was possible to study if added polymer influenced the structure of the surfactant aggregates.

The Effect of Temperature on GPC for the Separation of PCBs from Transformer Oil and Subsequent Analysis by GC-MSD

The analysis of PCBs often involves lengthy and expensive cleanup procedures to remove interferences associated with environmental sample matrices. Gel permeation chromatography (GPC) has proven to be a useful tool in removing many of these interferences from environmental samples, especially from difficult matrices such as oils, lipids and sediments. This paper describes the effect of temperature upon the GPC column in separating PCBs from transformer oil and its implication on GC-MS analysis.

Separation of juvenile hormone metabolites with a silica-based gel permeation column.

Metabolites of juvenile hormone (JH) III, JH acid, JH diol and JH acid diol in an aqueous solution were separated by gel permeation chromatography using an UltraSpherogel SEC 2000 column. Each metabolite was eluted in an inclusion volume in the order JH acid diol, JH acid and JH diol. Although JH was the last compound eluted, it was co-eluted with a JH-binding protein (JHBP) when JHBP was present in the solution. Using this method, in vivo and in vitro JH catabolism studies were performed in the fifth stadium larvae of Bombyx mori.

Magnetic Nanoparticles Produced in Spontaneous Cationic-Anionic Vesicles: Room Temperature Synthesis and Characterization

Unilamellar vesicles formed spontaneously by forming solutions of single-tailed cationic and anionic surfactants [cetyltrimethylammonium bromide (CTAB) and dodecylbenzenesulfonic acid (HDBS), respectively] have been used as reactors for the direct, room temperature synthesis of nanometer-sized magnetic particles within their internal cores. A micellar anionic surfactant solution was titrated slowly into a micellar cationic surfactant solution containing ferrous chloride, forming defect-free unilamellar vesicles that contained the reactant ferrous ions. CTAB/HDBS molar ratios of 13/7 and 6/4 and ferrous chloride concentrations of 0.1 and 0.05 M were used. Extravesicular ferrous ions were replaced with sodium ions by passing this suspension through a gel permeation chromatography column saturated with isotonic sodium chloride solution. Addition of sodium hydroxide to the extravesicular region caused hydroxyl ions to permeate into the vesicles and react with the available ferrous ions to form the product. Powder X-ray diffraction of the particulate sample showed the intense peaks of magnetite or γ-ferrite. Bragg peak broadening was characteristic of crystallites of roughly the same diameter as particles measured by transmission electron microscopy, indicating that each particle was monocrystalline. All of the intense peaks were distinctly asymmetric. Magnetization measurements showed that the particles were superparamagnetic. Using the Langevin function folded with the particle size distribution to model the magnetization of the sample in response to an applied magnetic field yielded "magnetic" diameter distributions that were consistently smaller than particle size distributions obtained from transmission electron microscopy. The magnetic size is consistent with the existence of a magnetically anomalous surface layer on each particle, as has been reported with other ferrites. The asymmetric diffraction peaks may be explained with a model that permits surface relaxation, and it is possible that the two phenomena are linked.

Photophysical properties of nitrogen substituted fullerenes

By arc vaporization of graphite in the presence of pyrrole vapor, products are formed containing nitrogen atoms substituted in the C60 and C70 cage accompanied by other carbonaceous clusters. Fractions which are rich in nitrogen were isolated by high performance liquid chromatography using a gel permeation column and characterized by optical absorption, fluorescence, transient absorption, and time-resolved fluorescence measurements. Fluorescence spectra are attributed to intrinsic excitation in the nitrogen doped species as well as to separated charges in the fragmented fullerenes. Transient absorption spectra at time delays of 20 ns following excitation of the ground state molecules are attributed to delayed fluorescence. The average fluorescence lifetime of these molecules were determined to be 4 ns. These data, together with earlier studies from this laboratory of the mass spectra, absorption and fluorescence of fractions isolated by column chromatography, add further credence to the existence of stable nitrogen doped fullerenes.

Stage-specific Isoforms of Complex II (Succinate-Ubiquinone Oxidoreductase) in Mitochondria from the Parasitic Nematode, Ascaris suum

Complex II from mitochondria of the adult parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron transport observed in these organelles. In contrast, mitochondria isolated from free living second stage larvae (L2) of A. suum show much lower fumarate reductase activity than those from adults, whereas succinate dehydrogenase activities of mitochondria in both stages are comparable. In the present study, biochemical and antigenic properties of the partially purified enzymes from both larval and adult mitochondria were compared. Larval complex II eluted from the DEAE-Cellulofine column chromatography at a lower salt concentration than adult enzyme, whereas the apparent molecular size of both enzyme complexes estimated by gel permeation column chromatography was the same. The fumarate reductase activity of larval complex II was less than 3% of that of adult enzyme, and the Km values for substrates were significantly different between the two complexes. The flavoprotein subunit of larval complex II could be distinguished from that of adult complex II by two-dimensional gel electrophoresis and peptide mapping. The antibody against the smallest subunit (small subunit of cytochrome b558) of the adult enzyme did not cross-react with that of the larval enzyme. These results suggest that larval complex II differs from adult enzyme and is more similar to aerobic mammalian enzymes with low fumarate reductase activity. This is the first direct indication of the two different stage-specific forms of mitochondrial complex II.

Effective production of the hepatitis C virus core antigen having high purity in Escherichia coli

The amino-terminal half of putative nucleocapsid (core) protein (amino acids 1–115) of hepatitis C virus (HCV) was directly overproduced in Escherichia coli under the control of the tac promoter. Overproduction of core antigen was achieved by inserting several target genes and by optimizing the culture conditions, whereas a large amount of directly expressed and purified core antigen has not yet been reported. Although the level of expression was comparable to that of the conventional E. coli fused expression system, our recombinant proteins contain only HCV amino acid sequence. Using recombinant E. coli, overproduced large-scale culture system was achieved in jar-fermenter. A highly purified sample of the expressed protein was obtained by ion-exchange and gel permeation column chromatography in the presence of 8 M urea. From a 3.5 1 culture, ~ 440 mg of recombinant core protein was obtained after a two-step purification procedure. An enzyme-linked immunosorbent assay developed using the highly purified antigen satisfactorily diagnosed hepatitis C.

The catalytic synthesis of polycarbosilane in the presence of boron tributoxide

Synthesis of substantially polycrystalline silicon carbide fibre, which has excellent mechanical properties and heat resistance, has been reported. Such a silicon carbide fibre can be prepared from preceramic polymers [1, 2]. Usually, the synthesis of precursor polycarbosilane (PC) was at high temperature and under high pressure. Then, in the synthesis process, polyborodiphenylsiloxane (PBSO) was used as a reaction catalyst [3-5], which is prepared from the slow, complex reaction between boric acid and diphenyldichlorosilane. In our experiment, polycarbosilane precursor containing titanium was obtained by the reaction between titanium tetrabutoxide [Ti(OBu)4] and liquid polysilane (LPS), which was derived from destructive distillation of polysilane (PS) in nitrogen gas [6]. Therefore, a similar reaction between boron tributoxide [B(OBu)3 ] and LPS was carried out in order to prepare polycarbosilane containing boron. The experimental results show that it seems to be difficult to introduce B(OBu)3 into the molecular structure of polycarbosilane, but the increase of molecular weight of polycarbosilane is accelerated remarkably in the presence of B(OBu)3. Evidently, the troublesome preparation process of PBSO can be eliminated by replacement of PBSO with B(OBu)3 as a catalyst. In the present work, the synthesis of polycarbosilane from LPS and B(OBu)3 according to different weight ratios is reported. The effects of B(OBu)3 on the structure and properties of polycarbosilane products are discussed. B(OBu)3 and LPS derived from destructive distillation in nitrogen gas up to 420 °C were mixed in certain weight ratios in a three-necked flask equipped with a quartz reactor tube, a recycling system and a thermometer. The mixtures were heated slowly in a nitrogen gas flow to a given temperature and the reaction proceeded at this temperature for 1 h, then the products were dissolved in toluene. After filtering and distillation, PC as a yellow solid was obtained. The infrared (IR) spectra of products were measured using a Hitachi 270-30 infrared spectrophotometer. The solid samples were measured by the KBr pellet method and the samples obtained at different t e m p e ~ e s during the reaction process were measured with a fixed-thickness (0.2 ram) cell of KBr in CC14 solution. The munber average molecular weights (Mn) and molecular weight distributions were measured using a Watters-244 liquid chromatograph with Ultrastyragel as the gel permeation chromatography (GPC) column in toluene solution. The melting points (Tm) were obtained with a melting point apparatus (model HMK, Germany). LPS can be regarded as a mixture of cyclic and linear low molecular weight polysilane, and its structure is assumed to be [6]:

Multi Residue Method for Determination of Pesticides Using Luke Method Extraction, Gel Permeation Chromatography and Gas Chromatography

We studied multi residue method of pesticides using Luke Method extraction, gel permeation chromatography (GPC) and dual column GC. A sample was extracted with acetone and filtered. Aliquot of the extracts was extracted with dichloromethane-petroleum ether (1:1) without evaporation of acetone. The extract was dehydrated, evaporated in vacuo, dissolved in dichloromethane-cyclohexane (1:1) and cleaned up by GPC using Bio Beads S-X3 and mobile phase dichloromethane-cyclohexane (1:1). The eluate fraction of pesticides was evaporated in vacuo and dissolved in acetone. The sample was determined with a dual column GC (column DB-1 and DB-1701, or DB-210 and DB-1) equipped with NPD and FPD. Silica gel column fractionation was effective for furthermore clean-up. Recoveries of 65 organic phosphate, carbamate and nitrogen containing pesticides from fortified tomato, lettuce and strawberry ranged 53-122%, except propamocarb, acephate, methamidophos and aldicarb. This multi residue method is fast and convenient to determine pesticide residues in crops.

Chemical Structure of Lipid Bioflocculant Produced byRhodococcus erythropolis

We have identified the chemical structure of the lipid bioflocculant produced by Rhodococcus erythropolis S-1 strain. It was hydrolyzed with alkali and decomposed to sugar and fatty acid fractions. The sugar fraction was identified as a mixture of glucose and trehalose by HPLC and TLC analyses combined with acid and enzyme hydrolyses. The fatty acid fraction was identified as a mixture of many molecular classes of mycolic and mycoleic acids (2-alkyl-3-hydroxy long-chain fatty acids) of which the molecular masses were from 496 to 606 (C32-C40) with GC-MS analysis after methylesterification and trimethylsilylation. A gel permeation column chromatography study suggested that the lipid bioflocculant contained trehalose monomycolate, glucose monomycolate, and trehalose dimycolate. Furthermore, we showed that the lipid bioflocculant can be extracted directly from Rhodococcus erythropolis S-1 cells with organic solvents such as ethanol.

Biosynthesis of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina.

The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)- eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)- eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme.

Evaporative light scattering mass detection for high‐performance liquid chromatographic analysis of sucrose polyester blends in cooking oils

AbstractA high‐performance liquid chromatographic method is described to determine the sucrose polyester (SPE) content in seven blends of cooking oils. Four gel‐permeation chromatography (GPC) columns were used in series with an evaporative light scattering mass detector to separate the SPE from the acylglycerols in the final chromatogram. The SPE fraction was collected off the GPC column and injected onto a reverse‐phase C‐18 column for quantitation with sucrose octaacetate as an internal standard and a gradient of nonaqueous solvents as mobile phase. The chromatograms were interference‐free, with only two sharp peaks appearing. The standards were linear from 500 to 5000 μg/mL with a correlation coefficient of r=0.999. The mean percent recovery (n=9) and standard deviation were 102±6.7. The detector could detect amounts as low as 5 μg SPE.

Calibration of gel-permeation columns in the high-molecular-mass range: fixed human thrombocytes for the estimation of interstitial volume and the haemocyanin of the Vineyard snail Helix pomatia as a molecular mass calibration substance

Human thrombocytes, fixed with formaldehyde, are shown to be a universal indicator of the interstitial volume m gel-permeation chromatography (size-exclusion chromatography) for gels of all molecular mass separation ranges. The fixed thrombocytes are simple to prepare and to handle. Furthermore, native or intramolecularly cross-linked haemocyanin of the Roman snail Helix pomatia is shown to be well suited as a molecular mass calibration substance. We propose 8.8 · 10 6 as its molecular mass for calibration. This haemocyanin can therefore be added to the list of globular protein molecular mass calibration substances in the range of very high molecular masses.

Rat seminiferous tubular culture medium contains a biological factor that inhibits Leydig cell steroidogenesis: its purification and mechanism of action

Seminiferous tubules prepared from adult rats cultured for 48 h in serum-free conditions produce multiple biological factors that modulate Leydig cell steroidogenic function in vitro. Using gel filtration chromatography, it was shown that seminiferous tubular culture medium (STCM) contained at least three inhibitory activities designated AI, AII, and AIII that inhibited testosterone production by purified Leydig cells. The factor that induced AIII activity, designated Leydig cell inhibitor (LCI), was further purified to apparent homogeneity by sequential HPLC using gel permeation, C8-, C18-, C2/C18-reversed-phase, and microbore anion exchange columns. When this batch of purified factor was resolved by SDS-PAGE under reducing conditions, only a single silver stained band with an apparent Mr of 21 000 was detected. Protein sequence analysis using about 100 pmol of purified LCI revealed that its N-terminus was blocked. Incubation of this highly purified factor with Percoll gradient purified Leydig cells induced a dose-dependent inhibition of hCG-stimulated testosterone production. LCI inhibited the basal testosterone production and hCG-stimulated cAMP production by Leydig cell dose-dependently. It also inhibited the forskolin- and cholera toxin-stimulated testosterone and cAMP production but had no apparent effect on the binding of 125I-labeled hCG to LH receptors. These data suggest that this LCI exerts its inhibitory action at steps beyond the LH receptors but prior to the cAMP formation by affecting the adenylate cyclase activity directly or indirectly through inhibition of the stimulatory G-protein (Gs-protein); however, it is also possible that it decreases the coupling of the receptors to the Gs-protein. LCI also inhibited the conversion of exogenously added 22R-hydroxycholesterol, pregnenolone, progesterone, and 17α-hydroxyprogesterone to testosterone. However, it had no effect on the conversion of dehydroepiandrostenedione and androstenedione to testosterone. These data strongly suggest that LCI affects the steroidogenic enzymes metabolizing cholesterol to testosterone, the cytochrome P-450 side-chain cleavage (P-450SCC), and cytochrome P-450 17α-hydroxylase/17,20-lyase (P-450C17). However, it has no effect on the 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) enzyme activities. Based on the results of the present study, it is apparent that this LCI is distinct from other known potent Leydig cells inhibitors such as interleukin-1 (IL-1) and transforming growth factor-β (TGF-β). The LCI appears to involve in the paracrine regulation of Leydig cell function.

Analysis of tocopherols in vegetable oils by high‐performance liquid chromatography: Comparison of fluorescence and evaporative light‐scattering detection

AbstractA comparison of the responses of an evaporative light‐scattering detector (ELSD) and a fluorescence detector for tocopherols in vegetable oils by high‐performance liquid chromatography is presented. The tocopherols were separated from acylglycerols by gel‐permeation chromatography (GPC). The tocopherol fraction was collected off a set of four GPC columns with a mobile phase of methylene chloride before separation on a normal‐phase silica column with a mobile phase of hexane/isopropanol, 99.7∶0.3 (vol/vol). An internal standard of 5,7 dimethyltocol, which was detected by both the ELSD and fluorescence detector, was used to obtain quantitative data. The fluorescence detector was ten times more sensitive than the ELSD. γ‐Tocopherol was the major tocopherol detected in the vegetable oils studied and ranged from 24.1–93.3 mg/100 g. The amounts of tocopherols found in the vegetable oils agreed favorably with the literature values.

The presence of low molecular weight germinative substances in Bacillus cereus T spore extract.

An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. 1 might be a free amino acid such as L-alanine and/or Ca2+ and a Ca(2+)-binding substance.

Identification of the active component in the papilla-regulating extract from barley leaves

A partially purified extract from barley seedlings, referred to as the papilla-regulating extract (PRE), has been reported to induce both the formation of oversize papillae and resistance to penetration by the powdery mildew fungus in inoculated, susceptible barley coleoptiles. Purification and identification of the active component in the PRE were accomplished in this study. Biological activity was monitored by bioassay. The extract, obtained from barley seedlings through autoclaving, was first passed through a C18 reverse phase column. The eluent, in which activity was detected, was next passed through a gel permeation chromatography column packed with Sephadex LH-20 in 50% methanol. The active fractions were chromatographed on a Bio-Gel P-2 column in water. Although xylonate was a major organic component in the active fractions from the P-2 column, authentic xylonate was not biologically active by itself. After rechromatography in the P-2 column, it was determined by ion chromatography and inductively coupled argon plasma atomic emission spectroscopy that the active compound was potassium phosphate. Bioassays using authentic potassium phosphates confirmed this conclusion. The effects of the PRE were also found to be Ca 2+ -mediated. Potassium phosphates in plants may be important in regulating papilla formation and induced resistance.

Proteoglycan Content in Fresh and Cryopreserved Porcine Aortic Tissue

Quantitative analysis of proteoglycans (PGs) revealed that the content of PG material from cryopreserved aorta, measured as uronate-positive material, was similar to that from fresh tissue (440 ± 30 versus 430 ± 7μg/g wet tissue). Gel permeation column chromatography studies suggested that three PG fractions from cryopreserved tissue had molecular weights similar to PG fractions from fresh tissue; K av = 0.13, 0.47 (I), 0.20 (II), and 0.43 (III) from cryopreserved tissue and K av = 0.13, 0.50 (I), 0.23 (II), and 0.40 (III) from fresh tissue. Sequential extraction of tissue with guanidine-HCI (GdnHCI) followed by digestions with collagenase, elastase, and papain also demonstrated that there was no difference between fresh and cryopreserved tissues in the distribution of PGs in the extracts. Transmission electron microscopy analysis revealed less densely packed collagen fibers in cryopreserved tissues compared to fresh tissues. These studies indicate that there is no significant alteration in the content, molecular size, or distribution of PGs in properly cryopreserved tissue.