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PubMed1995

The use of fura-2 for estimating Ca buffers and Ca fluxes.

The compound fura-2 (Grynkiewicz et al., J. Biol. Chem. 260, 3440-3450, 1985) is generally known as an indicator dye for measuring the concentration of free calcium ([Ca2+]) inside living cells. It should be appreciated, however, that this is not what it actually is. More accurately, it is a divalent metal ion chelator which changes its fluorescence properties upon complexation. Thus, [Ca2+] has to be inferred indirectly by means of the law of mass action. As a chelator, fura-2 may influence the quantity of interest, the Ca signal. On the other hand, the chelator action may be used for a number of other purposes, some of them more directly related to its molecular properties: as a chelator, competing with endogenous Ca buffers, it can be used to estimate endogenous buffers and their properties. When present at sufficiently high concentration, such that it outcompetes endogenous buffers, fura-2 reports total Ca changes and is a probe for Ca fluxes across the membrane. Here, theory and methodological considerations of such applications of fura-2 will be summarized and results on Ca buffer and Ca flux measurements derived from various methods will be compared.

Animals↗
PubMed1980

The buffer value of weak acids and bases: origin of the concept, and first mathematical derivation and application to physico-chemical systems. The work of M. Koppel and K. Spiro (1914).

A translation is offered of a paper by M. Koppel and K. Spiro, published in 1914, and entitled: 'On the action of moderators (buffers) in the shift of the acid-base equilibrium of biological fluids'. In this work, which appeared in Vol. 65 of the Biochemische Zeitschrift, the authors introduced the modern concept of buffer value and, for the first time, quantified the buffering power of weak acids and bases, ampholytes, dibasic acids, and mixtures of buffers. The circumstances that surround the eclipse of this trailblazing research have been critically examined, and biographical details have been provided.

Acid-Base Equilibrium↗
PubMed1990

Anticardiolipin antibodies and thrombosis: buffer's influence on the detection and quantitation of anticardiolipin antibody measured by ELISA.

The influence of adult bovine serum (ABS) and fetal calf serum (FCS) on IgG anticardiolipin antibodies (ACA) levels measured by ELISA has been investigated. In control subjects the frequency of the optical density (O.D.) distribution was found to be non-gaussian. The O.D. of the 97th percentile was similar with both buffers, although the distribution of O.D. was statistically different (P less than 0.004). The slope of the dilution curves of two house standards as well as the curves obtained with reference standards were found to be very different depending on the buffer. In 55 consecutive patients with thrombotic events, recurrent fetal loss or auto-immune disorders, ACA positivity was also dependent on the way to express the data and the choice of the buffer: when O.D. values were read with their respective standard curves, IgG ACA positivity was 98% with FCS and 20% with ABS (P less than 0.001), but when the 97th percentile O.D. was considered, 40% were positive with FCS and 51% with ABS (n.s.). Furthermore 10 patients were found to be negative with FCS and positive with ABS and 4 others showed the inverse picture. Levels of positivity were also dependent on the buffer. These differences arise from the solution used to dilute the sera and not of that to block the plates. These methodological problems may explain some conflicting data found in the literature about the prevalence of ACA positivity.

Antibodies↗
PubMed1990

Effect of Tris, HEPES, and TES buffers on binding at mu-, delta-, and kappa-opioid sites in guinea pig brain.

In homogenates of guinea-pig brain minus cerebellum, the delta-binding of 1.5 nM [3H]-[D-Pen2,D-Pen5]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10-75 mM). However, the mu-binding of 1 nM [3H]-[D]Ala2,MePhe4,Gly-ol5]enkephalin or the kappa-binding of 1.5 nM [3H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl2 on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that mu- and kappa-sites, but not delta-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl2 at the kappa-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the kappa-sites of these tissues.

Animals↗
PubMed1987

Isolation of specific IgM monoclonal antibodies by affinity chromatography using alkaline buffers.

Comparative studies on the efficacies of various buffers in eluting absorbent-bound antibodies revealed the denaturative effect of 3 M KSCN on all three IgM antibodies but not on an IgG protein, and the generally weak eluting power of glycine-HCl buffer, pH 2.5, on these monoclonal antibodies. A recently described medium consisting of 50% (v/v) ethylene glycol in an alkaline buffer, pH 10.5, was found to be relatively efficient for elution in all cases. However, subsequent studies on one of the IgM antibodies showed that alkali alone could effect elution, with recovery of active protein improving on increasing the pH, till the maximum (38%) at pH 11.0, after which denaturation occurred. Addition of ethylene glycol to the medium facilitated the elution; however, at pH greater than 10.0, the solvent potentiated the denaturative effect of the medium. Since pH 11.0 was found to be the highest pH in which all three IgM antibodies examined were stable, 0.1 M glycine-NaOH buffer, pH 11.0, may be a useful eluent for IgM (and other) antibodies in general.

Antibodies, Monoclonal↗
PubMed1986

Isoelectric focusing using non-amphoteric buffers in free solution: II. Apparatus and measures of pH stability.

Isoelectric focusing of amino acids or proteins requires a time-invariant pH gradient. The maintenance, in an electric field, of such profiles with simple non-amphoteric buffers is possible in multicompartment cells using well determined concentration profiles. The method for determining these concentration profiles has been proposed in a preceding paper. A multicompartment cell has been built for the purpose of verifying the assumptions made. The technical characteristics of this cell will be described here. The time-stability of pH profiles obtained with sodium acetate/acetic acid buffers has been measured for concentration profiles determined so as to keep constant the transport number of each ion throughout the cell. Measured over a time span of 10 h and with a current density of 96 mA cm-2, the pH shift is, in the worst case, 0.009 pH unit/h. This corresponds to a much better stability than the one obtained with a constant concentration of sodium acetate in all compartments, and justifies the chosen concentration profiles based on the condition of constant transport numbers. The above mentioned method for the computation of the buffer concentration assumed a constant relative mobility of the ions. The experiments have shown the limits of this assumption, i.e. the variation of ionic mobilities with the ionic strength and the temperature diminishes the stability of the pH gradient. However, slight adjustments of the concentration in the end compartments allow some improvement of the stability. If the temperature could be precisely controlled in each compartment, a better pH stability than what has been achieved in these experiments should be reached. Two methods of compensation of the migration of ions in the end compartments (buffer renewal method and external recycling method) have been tested and will be discussed.

Buffers↗
PubMed1987

Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase.

A dog kidney (Na+ + K+)-ATPase preparation also catalyzes K+-independent and K+-activated phosphatase reactions with p-nitrophenyl phosphate as substrate. K+-independent activity increases with declining pH over the range 7.5 to 5.8, whereas the other two activities decrease. The increased K+-independent activity is similar with imidazole, histidine, and several Good buffers, and is thus attributable to free H+, probably by affecting enzyme conformations rather than by changing affinity for Mg2+ or substrate or by H+ occupying specific K+-sites. The decrease in K+-phosphatase and (Na+ + K+)-ATPase activities with pH also occurs similarly with those buffers, and is not due to changes in apparent affinity for substrate or for cation activators. However, the Good buffers Pipes and ADA inhibit the K+-independent phosphatase reaction strongly, the K+-activated reaction moderately, and the (Na+ + K+)-ATPase reaction little; both contain two acidic groups, unlike the other buffers tested. Inhibition of the phosphatase reaction by Pipes is associated with a decreased apparent affinity for K+ and an increased sensitivity to inhibition by Na+ and ADP, consistent with Pipes hindering conformational transitions to the E2 enzyme forms required for phosphatase hydrolytic activity.

Alkanesulfonates↗
PubMed1993

Effect of salt concentration of buffer on the binding of sodium dodecyl sulfate and on the viscosity behavior of the protein polypeptide derived from bovine serum albumin in the presence of the surfactant.

The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).

Buffers↗
PubMed1990

The effect of treatment with buffered lactic acid on microbial decontamination and on shelf life of poultry.

The use of buffered lactic acid systems compared with unbuffered lactic acid solutions enhances the decontaminating effect and increases shelf life of chicken legs. A reduction of about 2 pH units of the chicken skin is obtained by treatment with 10% lactic acid buffer. The buffer keeps the pH of the skin lower than that of untreated legs. Legs treated with 10% lactic acid buffer have a shelf life of 12 days at 6 degrees C, which means an increase of 6 days compared with the shelf life of untreated legs.

Animals↗
PubMed1992

Decontamination with lactic acid/sodium lactate buffer in combination with modified atmosphere packaging effects on the shelf life of fresh poultry.

The effect of the treatment with various concentrations (2%, 5%, 7.5% and 10% w/v) of lactic acid/sodium lactate buffer (pH 3.0) combined with modified atmosphere packaging (MAP) (90% CO2/10% O2) on the shelf life and organoleptic quality of fresh chicken legs stored at 6 degrees C was investigated. The CO2 concentration of all samples packed in modified atmosphere (MA) decreased during the first 3 days of storage, followed by a gradual increase after the third day, while O2 showed a corresponding decrease. The buffering capacity of the buffer systems seem to be sufficient to maintain a low pH of the skin during storage. Legs treated with 2, 5, 7.5, and 10% (w/v) lactic acid/sodium lactate buffer (pH 3.0) combined with MAP have a shelf life at 6 degrees C of 14, 15, 16 and 17 days, respectively. The shelf life when the product was not treated with lactic acid was 1, 2.3 and 4 days shorter, respectively.

Animals↗
PubMed1991

The effect of pH buffering on reducing the pain associated with subcutaneous infiltration of bupivicaine.

The authors propose that pH buffering of bupivicaine with sodium bicarbonate reduces the pain associated with its local subcutaneous infiltration. In a double-blind, prospective study, 62 healthy adult volunteers received a 0.5 mL subcutaneous infiltration of 0.5% buffered bupivicaine into the dorsum of a randomly chosen hand. The pH was adjusted to 7.0 by adding 0.05 mL of sodium bicarbonate (1 mEq/L [corrected]) to 10 mL vials of commercially available bupivicaine (1:200 dilution). The control hand was injected with the same amount of unbuffered agent. Pain was scored after each infiltration using a nonsegmented visual analogue scale. Student's t-test for paired measurements was used to analyze intergroup pain score differences. Forty-three subjects (69%) reported less pain with buffered bupivicaine and only 17 (27%) noted a modest increase: two subjects (3%) reported no difference. The mean pain score for the buffered agent was 22 mm compared with 30 mm for the control. The mean difference (control-experimental) was 8 mm (t = 4.64, df = 61, P less than .001). The authors conclude that the addition of sodium bicarbonate to bupivicaine reduces the pain associated with its local infiltration.

Adult↗
PubMedDate not supplied

Pain comparison of unbuffered versus buffered lidocaine in local wound infiltration.

The purpose of this study was to compare the pain of infiltration between unbuffered lidocaine and buffered lidocaine in a traumatic laceration. Solutions of unbuffered 1% lidocaine and buffered 1% lidocaine were randomly assigned to Site I or Site II of a single laceration for each subject, with the patient serving as self-control. Pain scores were recorded for each site, and an anesthetic preference was determined for each patient. It was found that buffered lidocaine had a preference ratio of 3.0 over unbuffered lidocaine (95% confidence interval, 1.86 to 4.84; P less than 0.0001). It was also found that Site I was preferred more often than Site II regardless of which medication was used. The conclusion is that buffered lidocaine is preferred over unbuffered lidocaine and that the order of injection is an important factor in trials that involve multiple sequential injections in the same patient.

Adult↗
PubMed2004

Reduced mitochondrial buffering of voltage-gated calcium influx in aged rat basal forebrain neurons.

Alterations of neuronal Ca(2+) homeostatic mechanisms could be responsible for many of the cognitive deficits associated with aging in mammals. Mitochondrial participation in Ca(2+) signaling is now recognized as a prominent feature in neuronal physiology. We combined voltage-clamp electrophysiology with Ca(2+)-sensitive ratiometric microfluorimetry and laser scanning confocal microscopy to investigate the participation in Ca(2+) buffering of in situ mitochondria in acutely dissociated basal forebrain neurons from young and aged F344 rats. By pharmacologically blocking mitochondrial Ca(2+) uptake, we determined that mitochondria were not involved in rapid buffering of small Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) in the somatic compartment. For larger Ca(2+) influx, aged mitochondria showed a significant buffering deficit. Evidence obtained with the potentiometric indicator, JC-1, suggests a significantly reduced mitochondrial membrane potential in aged neurons. These results support the interpretation that there is a fundamental difference in the way young and aged neurons buffer Ca(2+), and a corresponding difference in the quality of the Ca(2+) signal experienced by young and aged neurons for different intensities of cytoplasmic Ca(2+) influx.

Aging↗
PubMed2004

Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography.

The effect of changing the buffer at constant low pH in the mobile phase is investigated with respect to the separation of a mixture of basic peptides. Considerably worse peak shapes, leading to poorer resolution of complex peptide mixtures, were obtained when using formic acid favoured in LC-MS applications compared with non volatile phosphate buffers or with trifluoroacetic acid (TFA). Poorer peak shapes were largely attributable to reduced column capacity for the peptides when using mobile phases of low ionic strength, due to the increased mutual repulsion of ions held on the hydrophobic column surface which is facilitated in these buffers. However, ion-pairing between the peptides and additives such as TFA or even phosphate may also lessen mutual repulsion effects, leading to greater column capacity. Overloading effects could be observed when sample masses around only 0.1 microg were injected on to standard size analytical columns in formic acid containing mobile phases; sample masses around only 1.5 microg may cause loss of half the system peak capacity in such mobile phases. Results were broadly comparable (after scaling sample size according to column diameter) on columns of both conventional (4.6 mm i.d.) and capillary (0.075 mm i.d.) dimensions. Ammonium formate may be a useful alternative buffer for some applications due to its higher ionic strength.

Buffers↗
PubMed2006

Improved properties of the non-covalent coating with N,N-didodecyl-N, N-dimethylammonium bromide for the separation of basic proteins by capillary electrophoresis with acidic buffers in 25 microm capillaries.

Capillaries (25microm I.D.) treated with the double-alkyl-chain cationic surfactant N,N-didodecyl-N, N-dimethylammonium bromide (DDAB) in an improved coating procedure were used for separation of four basic proteins in volatile buffers (ammonium acetate and ammonium hydroxyacetate) as well as in a non-volatile buffer (sodium phosphate) at pH 4. The DDAB coating was stable enough to, without recoating, permit consecutive separations of the proteins up to 9h with good precisions in peak areas (RSD=1.1%) and migration times and with high apparent efficiencies (over 1 million theoretical plates/m) in the presence of a strong anodic electroosmosis. Adsorption of the proteins onto the capillary surface, which in previous studies was found to give a certain contribution to zone broadening, was eliminated with the new modified coating method. Complex formation between the proteins and phosphate buffer was studied and confirmed, and it is proposed that slow protein-buffer component interactions are the main contributions to zone broadening in protein separations by CE.

Buffers↗
PubMed2005

Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

Buffers↗
PubMed2004

Oxidative DNA damage induced by HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer in the presence of Au(III).

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)](5+) and [Pd(II)(TMPyP)](4+), where [H(2)(TMPyP)](4+) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.

Buffers↗
PubMed2006

Antibody buffering in the brain.

The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.

Aminobutyrates↗