Influence Of Buffer Composition Research Articles

Aqueous decomposition behavior of solid peroxides: Effect of pH and buffer composition on oxygen and hydrogen peroxide formation

The ability of solid peroxides to provide sustained release of both oxygen and hydrogen peroxide makes them potentially suitable for oxygen release or antibacterial applications. Most recent reports using solid peroxides to augment oxygen levels do so by compounding solid peroxide powders in polymers to retard the aqueous decomposition. Compounds with peroxidase activity may be added to reduce hydrogen peroxide toxicity. Peroxides are rarely pure and are mixed with oxide and themselves decompose to form hydroxides in water. Therefore, even if buffering strategies are used, locally the pH at the surface of aqueously immersed peroxide particles is inevitably alkaline. Since pH affects the decomposition of peroxides and hydrogen peroxide stability, this study compared for the first-time the aqueous decomposition products of hydrogen and inorganic peroxides that are in use or have been used for medical applications of have been evaluated preclinically; calcium peroxide (CaO2), magnesium peroxide (MgO2), zinc peroxide (ZnO2), sodium percarbonate (Na2CO3.1.5H2O2) and hydrogen peroxide (H2O2). Since plasma can be approximated to be carbonate buffered phosphate solution, we maintained pH using carbonate and phosphate buffers and compared results with citrate buffers. For a given peroxide compound, we identified not only a strong effect of pH but also of buffer composition on the extent to which oxygen and hydrogen peroxide formation occurred. The influence of buffer composition was not previously appreciated, thereby establishing in vitro parameters for better design of intentional release of specific decomposition species. Statement of significanceThis paper compares for the first time the aqueous decomposition products oxygen and hydrogen peroxide of solid peroxy compounds of metal cations, (calcium, magnesium, sodium and zinc) across a pH range that could feasibly be found in the body, (pH 5,7, 9) either physiologically or pathologically. We find that in addition to pH, buffer composition is also a critically important factor, making translation from in vitro models challenging. Cytotoxicity was related to hydrogen peroxide release, alkalinity and in the case of zinc peroxide to the cation itself. In vitro and preclinical studies generally report release data from polymer-peroxide composites and rarely compare peroxides with one another. Together our data provide guidance for oxygen and ROS delivery from these inorganic materials.

Aqueous Decomposition Behavior of Solid Peroxides: Effect of pH and Buffer Composition on Oxygen and Hydrogen Peroxide Formation

The ability of solid peroxides to provide sustained release of both oxygen and hydrogen peroxide makes them potentially suitable for oxygen release or antibacterial applications. Most recent reports using solid peroxides to augment oxygen levels do so by compounding solid peroxide powders in polymers to retard the aqueous decomposition. Compounds with peroxidase activity may be added to reduce hydrogen peroxide toxicity. Peroxides are rarely pure and are mixed with oxide and themselves decompose to form hydroxides in water. Therefore, even if buffering strategies are used, locally the pH at the surface of aqueously immersed peroxide particles is inevitably alkaline. Since pH affects the decomposition of peroxides and hydrogen peroxide stability, this study compared for the first-time the aqueous decomposition products of hydrogen and inorganic peroxides that are in use or have been used for medical applications of have been evaluated preclinically; calcium peroxide (CaO2), magnesium peroxide (MgO2), zinc peroxide (ZnO2), sodium percarbonate (Na2CO3.1.5H2O2) and hydrogen peroxide (H2O2). Since plasma can be approximated to be carbonate buffered phosphate solution, we maintained pH using carbonate and phosphate buffers and compared results with citrate buffers. For a given peroxide compound, we identified not only a strong effect of pH but also of buffer composition on the extent to which oxygen and hydrogen peroxide formation occurred. The influence of buffer composition was not previously appreciated, thereby establishing in vitro parameters for better design of intentional release of specific decomposition species.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure.

Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.

Lyophilisation and concentration of chitosan/siRNA polyplexes: Influence of buffer composition, oligonucleotide sequence, and hyaluronic acid coating

Chitosan (CS)/siRNA polyplexes have great therapeutic potential for treating multiple diseases by gene silencing. However, clinical application of this technology requires the development of concentrated, hemocompatible, pH neutral formulations for safe and efficient administration. In this study we evaluate physicochemical properties of chitosan polyplexes in various buffers at increasing ionic strengths, to identify conditions for freeze-drying and rehydration at higher doses of uncoated or hyaluronic acid (HA)-coated polyplexes while maintaining physiological compatibility. Optimized formulations are used to evaluate the impact of the siRNA/oligonucleotide sequence on polyplex physicochemical properties, and to measure their in vitro silencing efficiency, cytotoxicity, and hemocompatibility. Specific oligonucleotide sequences influence polyplex physical properties at low N:P ratios, as well as their stability during freeze-drying. Nanoparticles display greater stability for oligodeoxynucleotides ODN vs siRNA; AT-rich vs GC-rich; and overhangs vs blunt ends. Using this knowledge, various CS/siRNA polyplexes are prepared with and without HA coating, freeze-dried and rehydrated at increased concentrations using reduced rehydration volumes. These polyplexes are non-cytotoxic and preserve silencing activity even after rehydration to 20-fold their initial concentration, while HA-coated polyplexes at pH∼7 also displayed increased hemocompatibility. These concentrated formulations represent a critical step towards clinical development of chitosan-based oligonucleotide intravenous delivery systems.

Influence of Buffer Composition and Calcium Chloride on GdnHCl Denaturation of Bacillus licheniformis α-Amylase

The influence of buffer composition on the conformational stability of native and calciumdepleted Bacillus licheniformis α-amylase (BLA) was investigated against guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence and UV-difference spectroscopy. Differential effect of buffer composition on GdnHCl denaturation of BLA was evident from the magnitude of these spectral signals, which followed the order: sodium phosphate > Tris-HCl > HEPES > MOPS. These effects became more pronounced with calcium-depleted BLA. Sephacryl S-200 gel chromatographic results showed significant BLA aggregation in the presence of 6 M GdnHCl.

A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments

A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A-B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10(-6) cm/s) was determined for atenolol (10.7 ± 1.2), caffeine (17.6 ± 3.1), cimetidine (6.9 ± 0.1), metoprolol (12.6 ± 0.7), theophylline (15.3 ± 1.6) and, ranitidine (3.8 ± 0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r = 0.89). These findings showed a high correlation with human data (r = 0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules.

Influence of buffer composition on the distribution of inkjet printed protein molecules and the resulting spot morphology

Producing high quality protein microarrays on inexpensive substrates like polystyrene is a big challenge in the field of diagnostics. Using a non-contact inkjet printer we have produced microarrays on polystyrene slides for two different biotinylated biomolecules, bovine serum albumin (BSA–biotin) and immunoglobulin-G (IgG–biotin), and studied the influence of buffer (composition and pH) on the spot morphology and signal intensity. Atomic force microscopy revealed the morphological pattern of the (biomolecule) spots printed from phosphate buffer (pH 7.4), phosphate buffered saline (pH 7.4) and carbonate buffer (pH 9.6). The spots showed an irregular crust-like appearance when printed in phosphate buffered saline (pH 7.4), mainly due to the high NaCl content, whereas spots of biomolecules printed in carbonate buffer (pH 9.6) showed a smooth morphology. In addition, the rinsing of these dried spots led to the loss of a considerable fraction of the biomolecules, leaving behind a small fraction that is compatible with the (mono)layer. It was confirmed by confocal laser microscopy that the quality of the spots with respect to the uniformity and distribution of the biomolecules therein was superior when printed in carbonate buffer (pH 9.6) as compared to other buffer systems. Particularly, spotting in PBS yielded spots having a very irregular distribution and morphology.

Influence of buffer composition on the capillary electrophoretic separation of carbon nanoparticles

Carbon nanoparticles obtained from the flame of an oil lamp were examined by means of capillary electrophoresis. The influence of buffer composition on the separation of the mixture of negatively charged carbon nanoparticles was studied by varying buffer selection, pH, and concentration. The electrophoretic pattern was affected by both the co- and counter-ion in the buffer solution, influencing selectivity and peak shape. The capillary electrophoretic separations at different pH revealed species with large electrophoretic mobilities under a wide range of pH. The mobility of selected species in the mixture of nanoparticles showed a strong dependence upon the solution ionic strength. The mobility of these nanoparticles as a function of ionic strength was compared to classical electrokinetic theory, suggesting that under the experimental conditions utilized, the species are small, highly charged particles with appreciable zeta potentials, even at low pH.

Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition

The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 degrees C undermines the claim that DRMs consist of lipid nanodomains that exist in an L(o) (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg(2+), K(+)) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 degrees C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 degrees C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 degrees C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.

The influence of buffer composition on tissue integrity during permeability experiments “in vitro”

A well-balanced incubation saline is necessary for permeability experiments with the rat jejunal tissue in the diffusion chambers. At the same time the investigated substance must be chemically stable and sufficiently soluble in this incubation saline. To investigate whether the absence of some ions in incubation salines influences the tissue viability and integrity or the diffusional characteristics of the epithelial membrane the electrical parameters were monitored and the permeability of fluorescein and acyclovir was evaluated during the experiments in side-by-side diffusion chambers. Our results show that the tissue integrity and viability are seriously impaired when Ca 2+ and Mg 2+-free conditions are applied on both sides of the diffusion chambers, but not when only mucosal or only serosal side is Ca 2+ and Mg 2+-free. Bicarbonate-free incubation salines can also alter the measured apparent permeability coefficients even though the tissue viability and integrity do not change. This change in the apparent permeability is most likely due to a change in the pH of the mucosal surface and can be prevented if the buffer capacity of the incubation saline is increased.

Chromatographic Behavior of Bothrops erythromelas Phospholipase and Other Venom Constituents on Superdex 75

In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50 mM Tris/HCl/150 mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non‐overlapping in the two buffers. Other venom constituents, including bradykinin‐potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.

Towards a Capacitive Enzyme Sensor for Direct Determination of Organophosphorus Pesticides: Fundamental Studies and Aspects of Development

The realisation of a miniaturised potentiometric enzyme biosensor is presented. The biosensor chip utilises the enzyme organophosphorus hydrolase (OPH) for the direct determination of pesticides. The transducer structure of the sensors chip consists of a pH-sensitive capacitive electrolyte-insulator-semiconductor (EIS) structure that reacts towards pH changes caused by the OPH-catalised hydrolysis of the organophosphate compounds. The biosensor is operated versus a conventional Ag/AgCl reference electrode. Measurements were performed in the capacitance/voltage (C/V) and the constant capacitance (ConCap) mode for the two different pesticides paraoxon and parathion. For the development of this new type of biosensor, different immobilisation strategies, influence of buffer composition and concentration, transducer material, detection limit, long-term stability and selectivity have been studied.

The luteinizing hormone receptor: influence of buffer composition on ligand binding and signaling of wild type and mutant receptors

There is evidence that ligand binding to and ligand-mediated signaling by the luteinizing hormone receptor (LHR) are influenced by buffer conditions, including ionic type and strength, an issue that becomes important in comparing functional parameters obtained on receptor mutants under different conditions. In order to study this phenomenon, we performed binding (kinetic and saturation) and signaling studies of human chorionic gonadotropin (hCG) with wild type (wt) LHR and several mutants expressed in COS-7 cells using two common buffer systems. One buffer was of low ionic strength and contained a low concentration of Na +, while the other had a near-physiological concentration of Na +. Emphasis was placed on mutations of two amino acid residues in the hinge region of the ectodomain (E332 and D333). It was found that the buffer of higher ionic strength, primarily from Na +, led to an increase of about 4-fold in the K d of hCG binding to wt and mutant LHRs. The reduced binding affinities were attributable to a comparable reduction in the rate constants of association, with no significant differences in the calculated rate constants of dissociation in the two buffers. Analysis of the signaling properties of these mutants showed that, when corrected for the amount of hCG bound under the conditions of the signaling assay, the maximal ligand-mediated cAMP produced in cells maintained in the buffer of low ionic strength was comparable for wt LHR and the mutants, only the D333A mutant being somewhat elevated. In the buffer of higher ionic strength, however, the response by wt LHR was significantly greater than that of the mutants. These results show that E332 and D333 are important in hormone-mediated signaling, but only in the buffer of higher Na + concentration. In addition to mutants of these two residues, the buffer of higher ionic strength also led to reduced binding to a number of mutants throughout the receptor. Since these mutants included additional replacements in the ectodomain and transmembrane helices 6 and 7, the general nature of the buffer effect on wt and mutant LHRs suggests that electrostatic effects are contributing to ligand binding and/or that the LHR ectodomain may exist in two conformations, one being more accessible to ligand at reduced ionic strength.

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis.

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis

Chemisorption of the Dipeptide Arg-Cys on a Gold Surface and the Selectivity of G-Protein Adsorption

Arginine-l-cysteine dipeptide adsorbates are used in this study as a model system for G-protein-coupled receptors (GPCRs). An arginine-containing model molecule is chosen because the GPCR α2A has been shown to include an arginine-rich region in the G-protein-binding part of the third intracellular loop, and the role of arginines by means of recognition is believed to exceed their positive charge. The dipeptide Arg-Cys is adsorbed to gold surfaces and the peptide monolayers are characterized. These peptide monolayers are then used for G-protein adsorption experiments to study the molecular interaction and binding. The molecular adsorption, orientation, and chemical binding of the peptide to the surface are studied by X-ray photoelectron spectroscopy and infrared reflection−absorption spectroscopy. A chemical shift in the S(2p) core level spectrum of the peptide adsorbate on gold shows that there is a strong molecular surface interaction consistent with a chemical binding of the peptide to the surface through the sulfur atom. With the cysteine part linked to the surface, the arginine part of the molecule is available for further adsorption processes. Monolayers of Arg-Cys, l-cysteine, and cysteamine are used for G-protein adsorption experiments. Adsorption of human serum albumin and human immunoglobulins on the same monolayers are studied for comparison. The analytical tool is surface plasmon resonance. Two different buffers are used for the adsorption studies, and the influence of buffer composition on protein adsorption is discussed.

Separation of diadenosine polyphosphates by capillary electrophoresis

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.

Separation of diadenosine polyphosphates by capillary electrophoresis.

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.