Affinity chromatography without immobilized ligands; A new method for studying macromolecular interactions using high-performance liquid chromatography

A new method for studying macromolecular interactions was devised. The principle is based on affinity chromatography with a mobile zone of affinity ligand instead of a column with immobilized ligand. In this method, the difference in migration velocities between the moving zone of affinity ligand and a sample in a conventional gel-permeation column is utilized. The fast migrating zone (A zone), which is later injected into the column, and the slow migrating zone (B zone), which is injected beforehand, interfere with each other at the passing point in the column if A and B interact such as A + B ? AB. The zone interference deforms each elution profile of A and B, because the complex AB has a migration velocity different from the others. The elution profiles in zone-interference chromatography are calculated by computer simulation in the framework of the plate theory. The binding constant is calculated from the peak shift of elution volume of B in the zone-interference chromatogram. The interaction between single-strand DNA (A zone) and ribonuclease A (B zone) was studied.     

Verrucomicrobia Are Candidates for Polysaccharide-Degrading Bacterioplankton in an Arctic Fjord of Svalbard

In Arctic marine bacterial communities, members of the phylum Verrucomicrobia are consistently detected, although not typically abundant, in 16S rRNA gene clone libraries and pyrotag surveys of the marine water column and in sediments. In an Arctic fjord (Smeerenburgfjord) of Svalbard, members of the Verrucomicrobia, together with Flavobacteria and smaller proportions of Alpha- and Gammaproteobacteria, constituted the most frequently detected bacterioplankton community members in 16S rRNA gene-based clone library analyses of the water column. Parallel measurements in the water column of the activities of six endo-acting polysaccharide hydrolases showed that chondroitin sulfate, laminarin, and xylan hydrolysis accounted for most of the activity. Several Verrucomicrobia water column phylotypes were affiliated with previously sequenced, glycoside hydrolase-rich genomes of individual Verrucomicrobia cells that bound fluorescently labeled laminarin and xylan and therefore constituted candidates for laminarin and xylan hydrolysis. In sediments, the bacterial community was dominated by different lineages of Verrucomicrobia, Bacteroidetes, and Proteobacteria but also included members of multiple phylum-level lineages not observed in the water column. This community hydrolyzed laminarin, xylan, chondroitin sulfate, and three additional polysaccharide substrates at high rates. Comparisons with data from the same fjord in the previous summer showed that the bacterial community in Smeerenburgfjord changed in composition, most conspicuously in the changing detection frequency of Verrucomicrobia in the water column. Nonetheless, in both years the community hydrolyzed the same polysaccharide substrates.     

Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa

The internal oxygen status of seagrass tissues, which is believed to play an important role in events of seagrass die-off, is partly determined by the rates of gas exchange between leaves and water column. In this study, we examined whether water column flow velocity has an effect on gas exchange, and hence on internal oxygen partial pressures (pO₂) in the Mediterranean seagrass, Cymodocea nodosa. We measured the internal pO₂ in the horizontal rhizomes of C. nodosa in darkness at different mainstream flow velocities, combined with different levels of water column oxygen pO₂ using an experimental flume in the laboratory. Flow velocity clearly had an effect on the internal oxygen status. In stagnant, but fully aerated water the mean internal pO₂ was 6.9 kPa, corresponding to about 30% of air saturation. The internal pO₂ increased with increasing flow velocity reaching saturation of around 12.2 kPa (60% of air saturation) at flow velocities ≥7 cm s⁻¹. Flow had a relatively larger influence on internal pO₂ at lower water column oxygen concentrations. By extrapolating linear relationships between internal and water column pO₂ in this experimental setup, rhizomes would become anoxic at a water column oxygen pO₂ of 4–4.5 kPa (∼20% of air saturation) in flowing water, but already at 6.4 kPa (∼30% of air saturation) in stagnant water. Water flow may play an important role for seagrass performance and survival in areas with poor water column oxygen conditions and may, in general, be of importance for the distribution of submerged rooted plants.     

Purification and immobilization of NAD+ synthetase from Escherichia coli

The enzyme NAD⁺synthetase [deamido-NAD⁺: ammonia ligase (AMP-forming), EC 6.3.1.5] is used for the preparation of 2 μmol isotopically labelled [¹³N]NAD⁺, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high yield synthesis of [¹³N]NAD⁺, the NAD⁺synthetase is immobilized on porous glass beads and packed in a column. The NAD⁺synthetase was obtained from Escherichia coli. Different strains were tested; the cell culture technique was optimized. A new, high yield purification was applied. A screening of different immobilization techniques was done. The selected immobilization method was further optimized to increase the enzymatic activity of the enzyme-loaded glass beads. The latter were packed into a glass column. The kinetic properties of this column were investigated and optimized.     

Spectral Characterization of the Photoreducible b-Type Cytochrome of Dictyostelium discoideum

Irradiation of a soluble extract from broken cells of Dictyostelium discoideum causes the photoreduction of a b-type cytochrome. The cytochrome b can be separated from cytochrome c, which is also present in the extract, by column chromatography on Brushite, but the cytochrome b is no longer sensitive to light after separation on the column. Low temperature spectroscopy shows that reduced form of the photoreducible cytochrome b has a Soret band at 423 nm and a split α band with maxima at 558 and 551 nm similar to the b-type cytochrome in complex II of beef heart mitochondria.     

Global distribution and diversity of marine Verrucomicrobia

Verrucomicrobia is a bacterial phylum that is commonly detected in soil, but little is known about the distribution and diversity of this phylum in the marine environment. To address this, we analyzed the marine microbial community composition in 506 samples from the International Census of Marine Microbes as well as 11 coastal samples taken from the California Current. These samples from both the water column and sediments covered a wide range of environmental conditions. Verrucomicrobia were present in 98% of the analyzed samples, and thus appeared nearly ubiquitous in the ocean. Based on the occurrence of amplified 16S ribosomal RNA sequences, Verrucomicrobia constituted on average 2% of the water column and 1.4% of the sediment bacterial communities. The diversity of Verrucomicrobia displayed a biogeography at multiple taxonomic levels and thus, specific lineages appeared to have clear habitat preference. We found that subdivision 1 and 4 generally dominated marine bacterial communities, whereas subdivision 2 was more frequent in low salinity waters. Within the subdivisions, Verrucomicrobia community composition were significantly different in the water column compared with sediment as well as within the water column along gradients of salinity, temperature, nitrate, depth and overall water column depth. Although we still know little about the ecophysiology of Verrucomicrobia lineages, the ubiquity of this phylum suggests that it may be important for the biogeochemical cycle of carbon in the ocean.     

Dynamic model of a nitrifying fixed bed column: Simulation of the biomass distribution of Nitrosomonas and Nitrobacter and of transient behaviour of the column

A dynamic model for a fixed bed nitrifying column with recirculation of the liquid and gas phases was developed. Liquid RTD experiments demonstrated that the liquid phase was perfectly mixed inside the column. Hete- rogeneity of biomass distribution on the solid phase (beads) was represented by an N-tanks in series model, and a back-mixing term was set to account for the well-mixed liquid phase throughout the column. In autotrophic conditions, competition for oxygen is the cause of the spatial segregation of the two species. Nitrosomonas is concentrated on beads at the bottom of the bed whereas Nitrobacter is more widely distributed. This is consistent with biomass distribution results reported by Cox et al. [17] in a nitrifying fixed bed column. Nitrification takes place at the bottom of the column, always in oxygen gas-liquid mass transfer limiting conditions. Nevertheless, considering the whole process, nitrification is complete (>98% of NH₃ oxidised) and there is no oxygen limitation (the outlet dissolved oxygen concentration is not limiting). The dynamic behaviour of the column, in conditions set up to avoid biofilm diffusion limitation, was simulated for different NH₃-load variations and oxygen shutdowns. The simulated behaviour of the column can be compared to results reported by Bazin et al. [16]. This confirms that the output transient nitrite peaks are higher when changes in the process conditions produce a rearrangement of biomass distribution in the fixed bed.     

Electrical properties of the body wall of Hydra

If the hydrostatic pressure in the enteron of Hydra is made more than 2–4 mm of water greater than the outside pressure, the animal becomes distended, indicating that the normal enteron pressure is less than this. Positive enteron pressure attenuates the spontaneous, negative-going electrical spikes across the body wall, which are called contraction pulses (CP''s) because of their relation to column contraction. Pressure has little effect on the transepithelial resting potential. The low frequency electrical impedance of the column is nonlinear. The impedance tends to increase as the transepithelial potential is made more negative. The nonlinearity has both initial and delayed components. The DC impedance of the column near the resting potential averages 100 kohms (approximately 5 kohms-cm²). The phase between transepithelial potential and imposed sinusoidal current approaches -90° with increasing current frequency. Bode plots of the column impedance and the phase lag indicate that the column has three or more time constants. CP''s show several unusual features: (a) their amplitude and frequency are essentially independent of the transepithelial potential when the latter is altered by imposed current; (b) there is practically no change in column impedance during CP firing; (c) when the transepithelial potential is clamped at zero, CP''s continue to appear spontaneously as current spikes. These features are consistent with the hypothesis that the CP-generating membrane forms but a small fraction of the total transverse impedance of the column.     

Preparation of chlorophyll a, chlorophyll b and bacteriochlorophyll a by means of column chromatography with diethylaminoethylcellulose

Chlorophyll a, chlorophyll b and bacteriochlorophyll a were prepared by means of column chromatography with Sephadex LH-20 and diethylaminoethylcellulose. This method provides purified preparations of chlorophylls in about 3 h.To prepare chlorophyll a, blue-green or red algae were used as the starting material. Chlorophyll a was extracted with 90% aqueous acetone from cells of blue-green algae, Anabaena variabilis, Anacystis nidulans and Tolypothrix tenuis, and with 90% aqueous methanol from thalli of a red alga, Porphyra yezoensis. Chlorophyll a was collected as precipitates by adding dioxane and water to the extract according to the method of Iriyama et al. [6]. The crude chlorophyll a preparation was applied to a Sephadex LH-20 column with chloroform as the eluent and then to a DEAE-cellulose column with a chloroform/methanol mixture (49 : 1, v/v) as the eluent. Analysis with thin layer chromatography revealed that the chlorophyll a preparation contained no detectable contaminants.Bacteriochlorophyll a was prepared in a similar manner from purple photosynthetic bacteria, Rhodopseudomonas spheroides and Chromatium vinosum.In order to prepare chlorophyll b, chloroplasts of spinach leaves were used as the starting material. A mixture of chlorophylls a and b was obtained in the same way as described for the preparation of chlorophyll a from the blue-green algae. To separate chlorophyll b from chlorophyll a, the mixture was applied to a diethylaminoethylcellulose column which was developed with a hexane/2-propanol mixture (5 : 2, v/v).     

Analysis of histidine-containing dipeptides, polyamines, and related amino acids by high-performance liquid chromatography: application to guinea pig brain.

A procedure is described for coupling wheat germ agglutinin to cyanogen bromide-activated Sepharose to yield a lectin affinity column of high capacity. Covalent linkage of the lectin to the insoluble matrix is carried out in the presence of a mixture of β-1,4-linked N-acetylglucosamine oligosaccharides prepared from chitin. The lectin-affinity column specifically recognizes glycoproteins containing N-acetylglucosamine residues with the capacity of binding 0.6–1.0 mg of ovomucoid per milliliter of gel. The affinity column is stable (as determined by ovomucoid binding) and shows little loss in binding capacity or specificity after repeated usage. Important characteristics for the use of this column to purify glycoproteins are described.     

Pygidial gland chemistry and potential alarm-recruitment function in column foraging,but not solitary,Nearctic Messor harvesting ants (Hymenoptera: Formicidae: Myrmicinae)

We investigated the role of the pygidial gland on foraging behavior in two ecologically dominant column foraging Nearctic harvesting ants (Messor pergandei and Messor andrei). Using chemical analyses and behavioral tests, we show that n-tridecane is the major biologically active compound of pygidial gland secretions in both species, and that this chemical functions as a powerful alarm-recruitment pheromone. Another major compound of pygidial gland contents is benzaldehyde; this substance does not release behavioral reactions in M. pergandei workers but might function as a defensive secretion. Six solitary foraging Nearctic Messor and two column foraging Palearctic Messor species, did not have large pygidial gland reservoirs.     

Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl-N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [¹⁴COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.     

New internal standards for basic amino acid analyses

Eight derivatives of cysteine and penicillamine with 2- and 4-vinyl-pyridine, p-nitrobenzyl bromide, and p-nitrostyrene were evaluated as potential internal standards for the short and long (physiological) basic columns in amino acid analysis by ion-exchange chromatography. S-β-(4-pyridylethyl)-dl-penicillamine (4-PEP) was found to have an advantage over the previously proposed S-β-(4-pyridylethyl)-l-cysteine (4-PEC) since the elution position of 4-PEP on the short basic column is insensitive to minor changes in pH of the eluting citrate buffer. 4-PEP was found to be stable to acid hydrolysis as used for proteins and its recovery from protein hydrolysates was unaffected by the presence of starch during hydrolysis. However, an extra 14 min is required to elute 4-PEP on the short column.Of the eight compounds studied, six appcar suitable as internal standards on the physiological (long) column. These elute in widely differing positions between histidine and arginine, thus offering a choice of internal standards for special analysis on the basic long column.     

Purification of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes.

Glyoxalase II, a specific glutathione thiolesterase, has been purified 9100-fold from rat erythrocytes using a purification scheme which employs Affi-Gel blue as a hydrophobic affinity column and also employs a glutathione-affinity column prepared by coupling S-(p-chlorophenacyl)glutathione to Affi-Gel 202. This procedure offers a convenient method for the preparation of highly purified glyoxalase II. Also described is a convenient method for the preparation of S-lactoyl-glutathione, a substrate for glyoxalase II.     

Xanthan batch fermentations: compared performances of a bubble column and a stirred tank fermentor

Fermentations of Xanthomonas campestris, NRRL B-1459, were carried out in a bubble column fermentor (BCF) and in a stirred tank fermentor (STF) to allow comparison of representative variables measured during the microbial growth and the gum production. The microbial growth phase was described by a logistic rate equation where maximum cell concentration was provided by nitrogenous compounds balance. The average value of the maximum specific growth rate was higher in the bubble column (μ _M =0.5 h^?1) than in the stirred reactor (μ _M =0.4 h^?1). The upper values of xanthan yield (Y _g-x =0.65 kg xanthan/kg glucose; Y _{O 2?x} xanthan/kg oxygen) and specific production rate (q _x =0.26 kg xanthan/kg biomass · h) were measured when the oxygen transfer coefficient was kept up above 80 h^?1 in the STF fermentor. In the bubble column the fermentation achieved in the same culture medium lasts two times longer than in the stirred aerated tank; this was attributed to the low value of the oxygen transfer coefficient (K _L a =20 h^?1) at the beginning of the gum synthesis phase. The results obtained in the stirred tank were the basis to estimate the optimal biomass concentration which enables to achieve a culture in non-limiting oxygen transfer conditions. Nevertheless, the transfer characteristics were more homogeneous in the bubble column than in the stirred tank where dead stagnant zones were observed. This is of primary importance when establishing fermentation kinetics models.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Effective Purification of Bioproducts by Fast Flow Affinity Chromatography

The performance of affinity adsorbents, such as HiPAC, Toyopearl, and Sepharose 4B coupled with protein A and anti-BSA, was compared. The HiPAC column, which is packed with rigid porous silica particles of 30 μm diameter, showed the highest value of D_pore/r² because of the small particle diameter and rigidity of the adsorbent particles. Further, it was operable at higher flow rates than the others. Thus, experimental data and calculation on the purification process show that fast and efficient purification is possible by use of the HiPAC column.     

The capitate-flowered epiphytic spiranthinae (orchidaceae) and a new genus from Paraguay

The Eurystyles Alliance is reviewed with regard to column characters, and a new genus, Synanthes, is described. Three genera, Eurystyles, Pseudoeurystyles, and Synanthes, compose the alliance of capitate-flowerd epiphytic Spiranthinae.