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Biomolecules Feb 2019The assessment of weak acid membrane permeability () frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpH, in...
The assessment of weak acid membrane permeability () frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpH, in response to a sudden augmentation of external acid concentration. However, ΔpH may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpH, as we show here. The uniexponential time constant (τ) of ΔpH(t) is an invalid measure of as Arrhenius plots of and τ reveal different activation energies for acid influx. We calculate by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a of 30.8 ± 3.5 μm/s for formic acid for 1,2-dioleoyl--glycero-3-phosphocholine (DOPC) vesicles.
Topics: Buffers; Formates; Hydrogen-Ion Concentration; Phosphatidylcholines; Unilamellar Liposomes
PubMed: 30781892
DOI: 10.3390/biom9020063 -
New Biotechnology Sep 2023The preparation of buffer solutions used in the biopharmaceutical industry is typically performed manually by the addition of one or multiple buffering reagents to...
The preparation of buffer solutions used in the biopharmaceutical industry is typically performed manually by the addition of one or multiple buffering reagents to water. Recently, the adaptation of powder feeders for continuous solid feeding was demonstrated for continuous buffer preparation. However, the intrinsic characteristics of powders can change the stability of the process, due to the hygroscopic nature of some substances and humidity-induced caking and compaction behavior, but there is no simple and easy methodology available for predicting this behavior for buffer species. To predict which buffering reagents are suitable without special precautions and investigate their behavior, force displacement measurements were conducted with a customized rheometer over 18 h. While most of the eight investigated buffering reagents indicated uniform compaction, especially sodium acetate and dipotassium hydrogen phosphate (KHPO) showed a significant increase in yield stress after 2 h. Experiments conducted with a 3D printed miniaturized screw conveyor confirmed the increased yield stress measurements by visible compaction and failure of the feeding. By taking additional precautions and adjusting the design of the hopper, we demonstrated a highly linear profile of all buffering reagents over a duration of 12 and 24 h. We showed that force displacement measurements accurately predict the behavior of buffer components in continuous feeding devices for continuous buffer preparation and are a valuable tool to identify buffer components that need special precautions. Stable, precise feeding of all tested buffer components was demonstrated, highlighting the importance of identifying buffers that need a specialized setup with a rapid methodology.
Topics: Powders; Buffers
PubMed: 37230177
DOI: 10.1016/j.nbt.2023.05.003 -
Journal of the American Chemical Society Nov 2021There are many open questions regarding the supramolecular properties of ions in water, a fact that has ramifications within any field of study involving buffered...
There are many open questions regarding the supramolecular properties of ions in water, a fact that has ramifications within any field of study involving buffered solutions. Indeed, as Pielak has noted (Buffers, Especially the Good Kind, , , in press. DOI:10.1021/acs.biochem.1c00200) buffers were conceived of with little regard to their supramolecular properties. But there is a difficulty here; the mathematical models supramolecular chemists use for affinity determinations do not account for screening. As a result, there is uncertainty as to the magnitude of any screening effect and how this compares to competitive salt/buffer binding. Here we use a tetra-cation cavitand to compare halide affinities obtained using a traditional unscreened model and a screened (Debye-Hückel) model. The rule of thumb that emerges is that if ionic strength is changed by >1 order of magnitude─either during a titration or if a comparison is sought between two different buffered solutions─screening should be considered. We also build a competitive mathematical model showing that binding attenuation in buffer is largely due to competitive binding to the host by said buffer. For the system at hand, we find that the effect of competition is approximately twice that of the effect of screening (∼ at 25 °C). Thus, for strong binders it is less important to account for screening than it is to account for competitive complexation, but for weaker binders both effects should be considered. We anticipate these results will help supramolecular chemists unravel the properties of buffers and so help guide studies of biomacromolecules.
Topics: Binding, Competitive; Buffers; Cations; Hydrogen Bonding; Osmolar Concentration; Salts; Water
PubMed: 34704751
DOI: 10.1021/jacs.1c08457 -
Biophysical Journal Jan 2014Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to...
Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl2 or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.
Topics: Buffers; DNA; Diffusion; Magnesium; Microspheres; Motion; Viscosity
PubMed: 24461015
DOI: 10.1016/j.bpj.2013.11.4501 -
European Journal of Biochemistry Aug 1980Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = square root 1-q2 is fulfilled. In this relation...
Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = square root 1-q2 is fulfilled. In this relation L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load, i.e. the irreversible ATP-utilizing processes in the cell, and q the degree of coupling of oxidative phosphorylation driven by respiration. Since during short time intervals L11 and q are constant whereas L33 fluctuates in the cell, oxidative phosphorylation would only rarely operate at optimal efficiency due to violation of conductance matching. This paper demonstrates that the reversible ATP-utilizing reaction catalyzed by adenylate kinase can effectively compensate deviations from conductance matching in the presence of a fluctuating L33 and hence allows oxidative phosphorylation to operate at optimal efficiency in the cell. Since the adenylate kinase reaction was found to buffer a thermodynamic potential, i.e. the phosphate potential, this finding was generalized to the concept of thermodynamic buffering. The thermodynamic buffering ability of the adenylate kinase reaction was demonstrated by experiments with incubated rat-liver mitochondria. Considerations of changes introduced in the entropy production by the adenylate kinase reaction allowed to establish the theoretical framework for thermodynamic buffering. The ability of thermodynamic buffering to compensate deviations from conductance matching in the presence of fluctuating loads was demonstrated by computer simulations. The possibility of other reversible ATP-utilizing reactions, like the ones catalyzed by creatine kinase and arginine kinase, to contribute to thermodynamic buffering is discussed. Finally, the comparison of the theoretically calculated steady-stae cytosolic adenine nucleotide concentrations with experimental data from perfused livers demonstrated that in livers from fed rats conductance matching is fulfilled on a time average and that the degree of coupling corresponded to qpec = 0.97 permitting the most economic maintenance of a maximal output power of oxidative phosphorylation. For the case of livers from starved rats this analysis suggested that the degree of coupling corresponded to qfec = 0.95, permitting the most economic maintenance of a maximal net rate of ATP synthesis at optimal efficiency of oxidative phosphorylation.
Topics: Adenine Nucleotides; Buffers; Enzymes; Hexokinase; Kinetics; Mathematics; Mitochondria; Oxidative Phosphorylation; Thermodynamics
PubMed: 7408880
DOI: 10.1111/j.1432-1033.1980.tb04791.x -
Lactate- or bicarbonate-buffered solutions in continuous extracorporeal renal replacement therapies.Kidney International. Supplement Nov 1999Continuous renal replacement therapies (CRRTs) are well accepted for critically ill patients with acute renal failure (ARF). Today, daily fluid exchange in CRRT reaches... (Review)
Review
BACKGROUND
Continuous renal replacement therapies (CRRTs) are well accepted for critically ill patients with acute renal failure (ARF). Today, daily fluid exchange in CRRT reaches 30 to 40 liter and more. Therefore, the composition of the substitution/dialysate fluid, often primarily developed either for intermittent treatment or for peritoneal dialysis, becomes more relevant. Lactate (30 to 45 mmol/liter) is frequently used as the buffer because of the high stability of this substance. However, lactate is thought to have negative effects on metabolic and hemodynamic parameters.
METHODS
Published data for different substitution fluids are presented with respect to acidosis and lactate concentration, uremia, and hemodynamic and metabolic alterations.
RESULTS
Only a few studies compare substitution fluids with different buffers. Uremia and acidosis (pH, base excess) were sufficiently controlled during CRRT with an exchange volume of in average 30 liters using either buffer. If patients with severe liver failure and lactic acidosis were excluded, no difference in hemodynamic and metabolic parameters between the solutions occurred. The plasma lactate concentration was elevated during lactate use in some cases, but lactate levels remained within normal limits in patients without liver impairment. The bicarbonate concentration in the solutions should exceed 35 to 40 mmol/liter, as in some cases the buffer capacity of the solutions was inadequate. In patients with severe liver failure or lactic acidosis, solutions with lactate buffer were shown not to be indicated.
CONCLUSION
In patients with reduced lactate metabolism, for example, concomitant severe liver failure, after liver transplantation or in lactic acidosis, bicarbonate-buffered solutions should be used. In nearly all other cases of critically ill patients with ARF, lactate-buffered solutions may be used as well as bicarbonate solutions.
Topics: Acute Kidney Injury; Bicarbonates; Buffers; Critical Illness; Hemodialysis Solutions; Humans; Lactates; Renal Replacement Therapy
PubMed: 10560802
DOI: No ID Found -
Journal of Biological Inorganic... Mar 2022The interaction between Eu(III) ion and different pH buffers, popular in biology and biochemistry, viz. HEPES, PIPES, MES, MOPS, and TRIS, has been studied by solution...
The interaction between Eu(III) ion and different pH buffers, popular in biology and biochemistry, viz. HEPES, PIPES, MES, MOPS, and TRIS, has been studied by solution nuclear magnetic resonance spectroscopy (NMR) and time-resolved laser-induced fluorescence spectroscopy (TRLFS) techniques. The Good's buffers reveal non-negligible interaction with Eu(III) as determined from their complex stability constants, where the sites of interaction are the morpholine and piperazine nitrogen atoms, respectively. In contrast, TRIS buffer shows practically no affinity towards Eu(III). Therefore, when investigating lanthanides, TRIS buffer should be preferred over Good's buffers.
Topics: Buffers; Europium; Hydrogen-Ion Concentration; Ions; Lanthanoid Series Elements; Tromethamine
PubMed: 35150337
DOI: 10.1007/s00775-022-01930-x -
Selection of In Vivo Predictive Dissolution Media Using Drug Substance and Physiological Properties.The AAPS Journal Jan 2020The rate and extent of drug dissolution in the gastrointestinal (GI) tract are highly dependent upon drug physicochemical properties and GI fluid properties. Biorelevant...
The rate and extent of drug dissolution in the gastrointestinal (GI) tract are highly dependent upon drug physicochemical properties and GI fluid properties. Biorelevant dissolution media (BDM), which aim to facilitate in vitro prediction of in vivo dissolution performance, have evolved with our understanding of GI physiology. However, BDM with a variety of properties and compositions are available, making the choice of dissolution medium challenging. In this tutorial, we describe a simple and quantitative methodology for selecting practical, yet physiologically relevant BDM representative of fasted humans for evaluating dissolution of immediate release formulations. Specifically, this methodology describes selection of pH, buffer species, and concentration and evaluates the importance of including bile salts and phospholipids in the BDM based upon drug substance log D, pK, and intrinsic solubility. The methodology is based upon a mechanistic understanding of how three main factors affect dissolution, including (1) drug ionization at gastrointestinal pH, (2) alteration of surface pH by charged drug species, and (3) drug solubilization in mixed lipidic aggregates comprising bile salts and phospholipids. Assessment of this methodology through testing and comparison with literature reports showed that the recommendations correctly identified when a biorelevant buffer capacity or the addition of bile salts and phospholipids to the medium would appreciably change the drug dissolution profile. This methodology can enable informed decisions about when a time, complexity, and/or cost-saving buffer is expected to lead to physiologically meaningful in vitro dissolution testing, versus when a more complex buffer would be required.
Topics: Bile Acids and Salts; Buffers; Drug Compounding; Fasting; Gastric Juice; Humans; Hydrogen-Ion Concentration; Intestinal Secretions; Pharmaceutical Preparations; Phospholipids; Solubility; Surface Properties
PubMed: 31989343
DOI: 10.1208/s12248-020-0417-8 -
Blood Transfusion = Trasfusione Del... Jan 2014It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is...
BACKGROUND
It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase.
MATERIALS AND METHODS
We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes' ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency.
RESULTS
The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline.
CONCLUSION
These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.
Topics: Bacterial Proteins; Bacteroides fragilis; Blood Group Antigens; Buffers; Erythrocytes; Glucose; Humans; alpha-Galactosidase; alpha-N-Acetylgalactosaminidase
PubMed: 24333060
DOI: 10.2450/2013.0023-13 -
Journal of Chromatography. A Dec 2020Buffer preparation and storage requires a significant facility footprint in large scale bioprocessing and together with the costs of supply chain management can have a...
Buffer preparation and storage requires a significant facility footprint in large scale bioprocessing and together with the costs of supply chain management can have a substantial economic impact. In-line buffer mixing in chromatography is commonly performed by blending different buffer solutions using at least two pumps and a static or dynamic mixer. We developed a device for an in-line gradient delivery of buffering agents directly from solids to be applied for chromatographic separation processes. A solid feeding device with a screw conveyor and a hold tank for the solids was designed and a miniaturized system was 3D printed. The coefficient of variation for the precision of the solid feeding of 5 different buffering agents was below 5% even for very small solid flow rates necessary for lab-scale chromatography. Stability was demonstrated by a constant linear solid feed at a very low dosing rate of 0.05 g.min over 24 hours. We demonstrated the suitability for chromatography by directly connecting the system to a standard chromatography workstation for protein chromatography. The solids were fed into a miniaturized continuously stirred tank reactor connected to an ÄKTA purification system. The performance of the in-line gradient delivery of buffering agents directly from solids was compared to conventional in-line buffer mixing. We were able to achieve highly linear gradients for elution using only one pump of a chromatographic system, generating the gradient by the direct addition of solids avoiding the necessity of additional pumps and hold tanks. By direct conditioning of buffers and the addition of solids a simple, just in time, at site preparation of buffers was possible. The design of the feeding unit for solid addition for buffer preparation is easily scalable and adaptable to work with or as a replacement for already existing in-line dilution or conditioning units.
Topics: Buffers; Chromatography; Proteins
PubMed: 33181354
DOI: 10.1016/j.chroma.2020.461663