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PubMed2000

Stacking and discontinuous buffers in capillary zone electrophoresis.

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.

Buffers↗
PubMed2000

Competitive binding of a Tris run buffer with chiral crown ether in chiral capillary electrophoresis.

In capillary electrophoresis of primary amine racemates using (+)-(18-crown-6)-tetracarboxylic acid (18C6H4) as a chiral selector, chiral recognition emanates from the differences in the complex formation between 18C6H4 and the two protonated amine enantiomers. The presence of buffer constituents such as tris(hydroxymethyl)aminomethane (Tris) or Na+, capable of forming complexes with 18C6H4, is thus detrimental to the chiral separation of primary amines. Such a competitive binding of buffer constituents was studied by comparing the electrophoretic mobilities of racemic analytes obtained in Tris/citric acid and triethylamine/citric acid buffers. We developed a simple fitting method to determine the competitive binding constant and applied it to the Tris buffer system. The competitive binding constant of Tris with 18C6H4 obtained at pH 3.0 was 27 +/- 4.

Anti-Infective Agents↗
PubMed1975

The effects of various fixative-buffer combinations on lung fine structure.

A variety of fixatives, buffers and fixation procedures were compared in rat and squirrel monkey lung in an attempt to preserve optimally both the cytologic details of pulmonary parenchyma as well as the acellular avelolar lining layer. In initial experiments utilizing the fixative of Ito and Karnovsky ('68), an electron-dense deposit was observed on the alveolar surface. Experiments were carried out in an attempt to determine what component of this fixative was responsible for the reaction product observed. In addition,immersion fixation of tissue blocks was compared to the whole lung fixation method of Kikkawa ('70). Kikkawa ('70) achieved excellent preservation of the acellular alveolar lining layer by such a fixation technique. In all lungs examined, whenever a phosphate buffer was utilized with primary aldehyde fixation, an electron-dense precipitate was observed on the luminal surfaces of the type I and II pulmonary epithelial cells. Additional sites of reaction product were pinocytotic vesicles of the type I cells and membranous arrays within the alveolar lumen. Such deposits were never observed when a sodium cacodylate buffer was used. No such granules were observed in areas of lung where the acellular alveolar lining layer had been preserved. The implications of these findings with regard to lung histochemical procedures and the possible relationship of these phosphate buffer-dependent granules to the surfactant system are discussed.

Animals↗
PubMed1981

The effect of buffer molarity on axonal exposure and axoaxonal apposition in the rat molar pulp.

Axons in the rat molar pulp have been examined morphometrically to determine axonal size and the degree of axonal exposure and axoaxonal apposition in tissue fixed by perfusion using 2% glutaraldehyde in cacodylate buffers ranging in molarity form 0.025 M to 0.4 M. Between 31.2% and 45.0% of the axons were incompletely ensheathed. This proportion of axons exposed was linearly related to the buffer molarity (P less than 0.05) and was approximately double that found in more central axons. Between 32.3% and 45.0% of the axons were in contact with other axons. This proportion was not linearly related to buffer molarity but was least ten times higher than that observed in more centrally positioned nerve fibers in the inferior alveolar nerves. Increasing buffer molarity reduced the size of the axons, a relationship not found in the more central axons. It is suggested that axonal exposure and axoaxonal apposition are constant, significant features of pulpal nerve fibers that may be related to the onset and spread of nociceptive activity. The permeability properties of pulpal axons may differ from those of more centrally placed axons.

Animals↗
PubMed1989

Conformation of bombesin in buffer and in the presence of lysolecithin micelles: NMR, CD, and fluorescence studies.

The conformation of the tetradecapeptide hormone bombesin has been studied in buffer and in the presence of lysolecithin micelles, using static and dynamic fluorescence, CD, and one- and two-dimensional nmr. The results obtained show that in buffer bombesin is present in an extended flexible chain, with no evidence for any ordered secondary structure. A marked change in the CD spectrum is observed changing from buffer to the lipid suspension. Concomitantly, the 1H-nmr spectrum of bombesin, in a D2O lipid dispersion, shows the persistence of resonances due to exchangeable protons and in similar conditions the fluorescence intensity increases. We think therefore that these results strongly support the hypothesis that bombesin interacts with the lipid phase, assuming ordered secondary structure. Finally, the marked dependence of tryptophan fluorescence quantum efficiency and order parameter from the hormone concentration in the presence of lysolecithin but not in buffer leads to the conclusion that bombesin can associate into the lipid matrix.

Bombesin↗
PubMed1992

Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.

The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 microM) displayed only plus-ended growth. The elongation velocity was 7.8 microns/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 microns/min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 microM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 microM) were assayed in ultrafiltrates (MW cut-off less than 30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 microns/min for 26 min before a catastrophe and rapid shortening at 11.8 microns/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.

Animals↗
PubMedDate not supplied

Immobilized buffers for isoelectric focusing: from gradient gels to membranes.

Some major improvements to a pH gradient simulator for isoelectric focusing in immobilized buffers are here described. They allow creation of graphs (giving the profiles of pH gradient, deviation from ideal shape, buffering power and ionic strength) with up to 200 increments (which, in a 1 pH unit span means mapping of pH intervals at barely 0.005 pH increments per step). In addition, for preparative purification protocols, utilizing multicompartment electrolyzers with isoelectric, buffering membranes, this modified program allows easy calculation of the precise pI value at any point along the pH gradient used for the analytical assessment of the pI values of the proteins to be purified. At the desired pH value, reached by moving the cursor along the pH gradient, a simple clicking of the mouse button allows the display of the instant Immobiline composition responsible for that pH value. The molarity ratio of the various Immobilines present at this precise point of the pH scale in fact gives the composition (with the accompanying value of buffering power and ionic strength) of the membrane to be utilized as a pH-stat in the preparative scale run. This is the only method that allows reproducible and valid scaling up from the analytical to the preparative scale.

Buffers↗
PubMed1998

Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers.

A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25 degrees C) high speed separations (10-12 min analysis time at 800 V/cm) could only be elicited in 20 microm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH=pI=2.77) in the presence of 7 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 microm ID), ensuring a five-times higher sensitivity. The gliadin patterns thus obtained are species-specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.

Buffers↗
PubMed2003

Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers.

This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.

Bread↗
PubMed2006

Loading capacity of carrier ampholytes--based buffers in capillary electrophoresis.

Narrow pH cuts of carrier ampholytes (CAs), originally designed for IEF, have been used as BGEs in CE. Their physicochemical properties, rather high buffering capacity and low conductivity, allow very efficient protein separations under high electric field strength. Due to their isoelectric properties, CA BGEs are expected to present a low ionic concentration and consequently a low loading capacity. In this study, we developed a simple method that allows the estimation of the loading capacity of a UV-absorbing BGE by CE. We first characterized in terms of loading capacity, classical ammediol-chromate UV-absorbing BGEs and a 10 mM histidine solution, a classical isoelectric buffer. Then, the loading capacity of four different CA-based BGEs has been assessed. Experimental results have shown that the CA-based buffers were presenting a rather high loading capacity, comparable to classical buffer ones and far higher than the one of the 10 mM histidine solution.

Buffers↗
PubMedDate not supplied

Cryosurvival of human spermatozoa frozen in eight different buffer systems.

The present study was conducted to ascertain optimal cryoconditions for human spermatozoa by comparing the relative cryoprotective efficiency of eight buffer systems and assessing various cryovials and thaw rates under two freeze rates. Spermatozoa that were cryopreserved in one of four zwitterion buffers (TES-Tris-citrate-egg yolk-glycerol; TES-Tris-citrate-I) maintained higher progressive motility at 0, 1, 2, and 4 hours post-thaw as compared to cells frozen in glycerol only, citrate-egg yolk-glycerol and TES-Tris-citrate-egg yolk without glycerol (TES-Tris-citrate-III; P less than 0.01). Freezing in TES-Tris-citrate-I also resulted in spermatozoa that penetrated the furthest distance through cervical mucus and possessed the highest percent live spermatozoa when compared to other cryoprotective media. Spermatozoa were analyzed for their ability to penetrate zona-free hamster ova and no difference was found between buffers when the assay was corrected for progressive motility. After removal of seminal plasma/buffers and incubation for 2 hours in BWW, TES-Tris-citrate-II and TES-Tris-citrate-milk showed the greatest sperm longevity (P less than 0.05). Pooled semen was extended in TES-Tris-citrate-I and frozen in straws or ampoules in static N2 vapor or in pellets on dry ice. Thaw bath temperatures ranged from 0 to 37 C. Post-thaw progressive motility and cervical mucus penetration were similar in all treatment groups. In conclusion, the present results indicate the use of TES-Tris-citrate-I for cryopreservation of human spermatozoa. With this optimal cryoprotective buffer, the containers and thaw rates used have little effect on human sperm cryosurvival.

Buffers↗
PubMed2005

Amyloid-beta aggregates formed at polar-nonpolar interfaces differ from amyloid-beta protofibrils produced in aqueous buffers.

The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.

Amyloid beta-Peptides↗
PubMed1981

Dissolution kinetics of carboxylic acids II: effect of buffers.

The dissolution behavior of 2-naphthoic acid from rotating compressed disks into aqueous buffered solutions of constant ionic strength (mu = 0.5 with potassium chloride) at 25 degrees was investigated. A model was developed for the flux of a solid monoprotic carboxylic acid in aqueous buffered solutions as a function of the solution pH and the physicochemical properties of the buffer. The model assumes a diffusion layer-controlled mass transport process and simple, instantaneously established reaction equilibrium between all reactive species (acids and bases) across the diffusion layer. Using intrinsic solubilities, pKa values, and diffusion coefficients, the model accurately predicts the dissolution of 2-naphthoic acid as a function of the bulk solution composition. The concentration profiles of all species across the diffusion layer are generated for each buffer concentration and bulk solution pH, including the pH profile within the microclimate of the diffusion layer and the pH at the solid-solution boundary.

Buffers↗
PubMed1979

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly.

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES--1 m M EGTA--1 mM MgCl2--0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 is raised to 10 mM, reassembly occurs. The results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 microM--10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.

Biological Transport↗
PubMed2005

Sodium glycylglycine as effective electrolyte run buffer for ascorbic and uric acid separation by CZE: a comparison with two other CE assays.

A new CE method for ascorbic acid (AA) and uric acid (UA) detection in human plasma has been developed. Analytes were resolved in less than 4 min by employing sodium glycylglycine (Glygly) as electrolyte run buffer at pH 8.0. Using the diode array detector ability to measure multiple wavelengths simultaneously, detection was optimized by monitoring the run at 262 nm for AA and at 288 nm for UA. Electrophoretic parameters such as resolution, migration times, efficiency, and peak areas of this new method were compared to those obtained by the two CE assays described in literature, in which the analytes separation was achieved by using sodium borate (that allows faster migration times but poor resolution) or tricine (with the highest resolution but elevated migration times) as electrolyte run buffer. Sodium Glygly allows to obtain the same good resolution given by the tricine buffer but with the faster analysis times of the sodium borate run buffer. Ascorbate and urate levels were measured in 35 healthy volunteers by the three methods and the obtained data were compared by three different statistical tests (mountain plot, Passing-Bablok regression, and Bland-Altman test) in order to verify the accuracy of our proposed method.

Ascorbic Acid↗
PubMed2006

Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC of folates with conventional and volatile buffers under highly aqueous conditions.

Applicability of several alkyl-bonded silica stationary phases was tested for gradient RP-HPLC of folates under highly aqueous conditions. High retention of folates was achieved on alternative phases with enhanced polarity and classical phases with higher carbon content. Phases exhibiting polar secondary interactions were found to provide better selectivity for late-eluting folates, whereas selectivity for early-eluting folates was mostly dependent on hydrophobic interactions. Best selectivity in phosphate buffered mobile phase was achieved on polar-endcapped silica phases (Aquasil C18 and HyPurity Aquastar) followed by alternative Atlantis dC18. Classical phases exhibited poorer separation of 10-formyl-folic acid and 5-formyl-tetrahydrofolate, but it could be considerably improved by increasing the buffer pH. Strong secondary interactions of ion-exchange character on polar-embedded phases resulted in marked peak deterioration, loss of recovery and dramatic changes in retention behaviour for early- and late-eluting folates when changing the mobile phase composition and pH. Therefore, polar-embedded phases such as HyPurity Advance were found to be unsuitable for separating folates. Stationary phases exhibited peak deterioration when using volatile buffer of low ionic strength. Better results were obtained with classical phases, whereas alternative phases showed not only peak deterioration but also a decrease in recovery and poorer selectivity due to increased secondary interactions in volatile buffer.

Buffers↗
PubMed1997

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Buffers↗
PubMed2001

Differential effect of buffer on the spin trapping of nitric oxide by iron chelates.

Nitric oxide synthase (NOS) generates nitric oxide (NO*) by the oxidation of l-arginine. Spin trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO* in real time and at its site of generation. The spin trapping of NO* from isolated NOS I oxidation of L-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO-Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO* and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO-Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at spin trapping NO* from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at spin trapping NO* but their maximal efficiency may be affected by experimental conditions.

Animals↗