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Seminars in Nephrology Jul 2019Although students initially learn of ionic buffering in basic chemistry, buffering and acid-base transport in biology often is relegated to specialized classes,... (Review)
Review
Although students initially learn of ionic buffering in basic chemistry, buffering and acid-base transport in biology often is relegated to specialized classes, discussions, or situations. That said, for physiology, nephrology, pulmonology, and anesthesiology, these basic principles often are critically important for mechanistic understanding, medical treatments, and assessing therapy effectiveness. This short introductory perspective focuses on basic chemistry and transport of buffers and acid-base equivalents, provides an outline of basic science acid-base concepts, tools used to monitor intracellular pH, model cellular responses to pH buffer changes, and the more recent development and use of genetically encoded pH-indicators. Examples of newer genetically encoded pH-indicators (pHerry and pHire) are provided, and their use for in vitro, ex vivo, and in vivo experiments are described. The continued use and development of these basic tools provide increasing opportunities for both basic and potentially clinical investigations.
Topics: Acid-Base Equilibrium; Animals; Biological Transport; Buffers; Humans; Hydrogen; Hydrogen-Ion Concentration; Intracellular Fluid
PubMed: 31300088
DOI: 10.1016/j.semnephrol.2019.04.002 -
Journal of Chromatography. A Apr 2023Buffer management for biopharmaceutical purification processes include buffer preparation, storage of buffers and restocking the buffers when needed. This is usually...
Buffer management for biopharmaceutical purification processes include buffer preparation, storage of buffers and restocking the buffers when needed. This is usually performed manually by the operators for small scale operations. However, buffer management can become a bottleneck when running integrated continuous purification processes for prolonged times, even at small scale. To address this issue, a buffer management system for the application in continuous lab-scale bioprocessing is presented in this paper. For this purpose, an ÄKTA™ explorer chromatography system was reconfigured to perform the buffer formulation. The system formulated all buffers from stock solutions and water according to pre-specified recipes. A digital twin of the physical system was introduced in the research software Orbit, written in python. Orbit was also used for full automation and control of the buffer system, which could run independently without operator input and handle buffer management for one or several connected buffer-consuming purification systems. The developed buffer management system performed automatic monitoring of buffer volumes, buffer order handling as well as buffer preparation and delivery. To demonstrate the capability of the developed system, it was integrated with a continuous downstream process and supplied all 9 required buffers to the process equipment during a 10-day operation. The buffer management system processed 55 orders and delivered 38 L of buffers, corresponding to 20% of its capacity. The pH and conductivity profiles observed during the purification steps were consistent across the cycles. The deviation in conductivity and pH from the measured average value was within ±0.89% in conductivity and ±0.045 in pH, well within the typical specification for buffer release, indicating that the prepared buffers had the correct composition. The operation of the developed buffer management system was robust and fully automated, and provides one solution to the buffer management bottleneck on lab scale for integrated continuous downstream bioprocessing.
Topics: Buffers; Chromatography; Automation; Water
PubMed: 37015183
DOI: 10.1016/j.chroma.2023.463942 -
Physiological Reviews Oct 2023Calcium signaling underlies much of physiology. Almost all the Ca in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels... (Review)
Review
Calcium signaling underlies much of physiology. Almost all the Ca in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca buffers include small molecules and proteins, and experimentally Ca indicators will also buffer calcium. The chemistry of interactions between Ca and buffers determines the extent and speed of Ca binding. The physiological effects of Ca buffers are determined by the kinetics with which they bind Ca and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca, the Ca concentration, and whether Ca ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca signals as well as changes of Ca concentration in organelles. It can also facilitate Ca diffusion inside the cell. Ca buffering affects synaptic transmission, muscle contraction, Ca transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.
Topics: Humans; Calcium; Buffers; Cytoplasm; Heart; Synaptic Transmission; Calcium Signaling
PubMed: 37326298
DOI: 10.1152/physrev.00042.2022 -
Science (New York, N.Y.) Jan 2020
Topics: Buffers; Noise
PubMed: 31974233
DOI: 10.1126/science.aba0446 -
Current Protocols in Human Genetics May 2001This appendix describes the preparation of selected bacterial media and of buffers and reagents used in the manipulation of nucleic acids and proteins. Recipes for cell...
This appendix describes the preparation of selected bacterial media and of buffers and reagents used in the manipulation of nucleic acids and proteins. Recipes for cell culture media and reagents are located elsewhere in the manual. RECIPES: Acids, concentrated stock solutions; Ammonium acetate, 10 M; Ammonium hydroxide, concentrated stock solution; ATP, 100 mM; BCIP, 5% (w/v); BSA (bovine serum albumin), 10% (100 mg/ml); Denhardt solution, 100x; dNTPs: dATP, dTTP, dCTP, and dGTP; DTT, 1 M; EDTA, 0.5 M (pH 8.0); Ethidium bromide solution; Formamide loading buffer, 2x; Gel loading buffer, 6x; HBSS (Hanks balanced salt solution); HCl, 1 M; HEPES-buffered saline, 2x; KCl, 1 M; LB medium; LB plates; Loading buffer; 2-ME, (2-mercaptoethanol)50 mM; MgCl(2), 1 M; MgSO(4), 1 M; NaCl, 5 M; NaOH, 10 M; NBT (nitroblue tetrazolium chloride), 5% (w/v); PCR amplification buffer, 10x; Phosphate-buffered saline (PBS), pH approximately 7.3; Potassium acetate buffer, 0.1 M; Potassium phosphate buffer, 0.1 M; RNase a stock solution (DNase-free), 2 mg/ml; SDS, 20%; SOC medium; Sodium acetate, 3 M; Sodium acetate buffer, 0.1 M; Sodium phosphate buffer, 0.1 M; SSC (sodium chloride/sodium citrate), 20x; SSPE (sodium chloride/sodium phosphate/EDTA), 20x; T4 DNA ligase buffer, 10x; TAE buffer, 50x; TBE buffer, 10x; TBS (Tris-buffered saline); TCA (trichloroacetic acid), 100% (w/v); TE buffer; Terrific broth (TB); TrisCl, 1 M; TY medium, 2x; Urea loading buffer, 2x.
Topics: Buffers; Genetic Techniques; Genetics, Medical; Humans; Solutions
PubMed: 18428217
DOI: 10.1002/0471142905.hga02ds26 -
European Biophysics Journal : EBJ May 2021The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological...
The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1 day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.
Topics: Buffers; Dynamic Light Scattering; Fluorometry; Protein Stability; Proteins
PubMed: 33554291
DOI: 10.1007/s00249-021-01497-6 -
Phosphate buffer interferes dissolution of prazosin hydrochloride in compendial dissolution testing.Drug Metabolism and Pharmacokinetics Aug 2023The purpose of this study was to elucidate the lack of supersaturation behavior in the dissolution profile of prazosin hydrochloride (PRZ-HCl) in the compendial...
The purpose of this study was to elucidate the lack of supersaturation behavior in the dissolution profile of prazosin hydrochloride (PRZ-HCl) in the compendial dissolution test. The equilibrium solubility was measured by a shake-flask method. Dissolution tests were performed by a compendial paddle method with a phosphate buffer solution (pH 6.8, 50 mM phosphate). The solid form of the residual particles was identified by Raman spectroscopy. In the pH range below 6.5, the equilibrium solubility in phosphate buffer was lower than that in the unbuffered solutions (pH adjusted by HCl and NaOH). Raman spectra showed that the residual solid was a phosphate salt of PRZ. In the pH range above 6.5, the pH-solubility profiles in the phosphate buffer solutions and the unbuffered solutions were the same. The residual solid was a PRZ freebase (PRZ-FB). In the dissolution test, PRZ-HCl particles first changed to a phosphate salt within 5 min, then gradually changed to PRZ-FB after several hours. Since the intestinal fluid is buffered by the bicarbonate system in vivo, the dissolution behavior in vivo may not be properly evaluated using a phosphate buffer solution. For drugs with a low phosphate solubility product, it is necessary to consider this aspect.
Topics: Buffers; Hydrogen-Ion Concentration; Solubility; Bicarbonates; Phosphates
PubMed: 37393739
DOI: 10.1016/j.dmpk.2023.100519 -
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 -
Cell Calcium Sep 1996When the two-solution approach is used to make CaEGTA buffers, the most critical requirement is that the CaEGTA stock solution contain equimolar quantities of calcium... (Review)
Review
When the two-solution approach is used to make CaEGTA buffers, the most critical requirement is that the CaEGTA stock solution contain equimolar quantities of calcium and chelator. The impact on [Ca2+] of errors in the molar ratio is magnified at high buffer ratios, so stoichiometric imbalance is most readily detected there. In this paper we examine some of the properties of calcium buffer systems that illustrate these principles, and describe a simple and sensitive method for detecting and correcting stoichiometric imbalance in the CaEGTA stock solution. The method derives its high sensitivity from the fact that the CaEGTA stock solution itself represents a calcium buffer system with a very high buffer ratio.
Topics: Buffers; Calcium; Egtazic Acid
PubMed: 8894269
DOI: 10.1016/s0143-4160(96)90028-7 -
Journal of Food Science Apr 2020The pH of most acid food products depends on undefined and complex buffering of ingredients but is critically important for regulatory purposes and food safety. Our...
The pH of most acid food products depends on undefined and complex buffering of ingredients but is critically important for regulatory purposes and food safety. Our objective was to define the buffer capacity (BC) of ingredients in salad dressing products. Ingredients of salad dressings were titrated individually and in combination using concentrations typical of dressing products. Titration curves from pH 2 to 12 were generated with sodium hydroxide and hydrochloric acid, which were then used to generate BC curves. A matrix of concentration and pK values for a series of monoprotic buffers approximated the pH of each ingredient. Some buffer series required anion or cation corrections for accurate pH prediction, possibly due to the presence of salts of acid or bases. Most buffers had BC values less than 10-fold the BC of acetic acid (0.25 β) typically in dressing formulations and had little influence on the final product pH of the dressings tested. Unexpectedly, we found that sugars in dressing formulations, including sucrose or corn syrup, exhibited buffering at pH values greater than 11 (0.035 β and 0.059 β, respectively), which was likely due to weakly acidic hydroxyl groups on the sugar molecules. However, the concentration and pK for buffers above pH 11 or below pH 2 were difficult to quantify due to the BC of water. The BC data may help to quantify the effects of salad dressing ingredients on the final product pH and benefit regulatory agencies and manufacturers in assessing product pH and safety. PRACTICAL APPLICATION: Buffer capacity data for salad dressing ingredients may help determine the influence ingredient addition will have on the final pH of a salad dressing product. The addition of low acid ingredients with little or no buffering may not significantly alter pH. The modeling method may be useful for regulatory purposes to estimate the effects of low acid ingredients on pH changes for food safety and may also be useful for product development of acid and acidified foods.
Topics: Acetic Acid; Buffers; Condiments; Food Ingredients; Hydrogen-Ion Concentration; Salts
PubMed: 32198767
DOI: 10.1111/1750-3841.15018