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PubMed1985

Modification of opioid ligand binding in the central and the peripheral nervous system by different buffers.

The modification of binding parameters (equilibrium dissociation constant and binding capacity) of three opioid ligands (DADLE, Etorphine and EKC) on bovine adrenal medulla and rat brain membranes have been examined in three buffer systems: Tris-HCl 50 mM, Hepes-NaOH 10 mM and Tes-KOH 10 mM. Major differences of these parameters have been found: Hepes-NaOH provoked a diminution of the apparent number of binding sites, while a concomitant diminution of the KD and Bmax was observed in Tes-KOH buffer. Substitution of counterions in these two buffers produced further changes of binding characteristics: in Hepes buffer we have observed an abolition of 3H DADLE binding, an enhancement of 3H EKC binding and no modification of 3H etorphine binding characteristics. On the contrary an abolition of the specific binding of all three ligands in Tes buffer was found in the bovine adrenal medulla while minor changes were observed in rat brain. It is concluded that, inspite same disadvantages (substitution for bivalent cations and temperature dependence), Tris-HCl is the buffer of choice for the analysis of opioid binding site interactions.

Adrenal Medulla↗

PubMed1987

ATP-dependent and ATP-independent calcium buffers determined in isolated squid axoplasm by axoplasmic dilution using calcium-selective electrodes.

Ca2+ buffering in axoplasm extruded from the squid giant axon was studied by axoplasmic dilution and Ca2+-selective electrodes. ATP-dependent Ca2+ buffering was lost when metabolism and endogenous ATP production were inhibited by axoplasmic dilution. Under these conditions ('low ATP' axoplasmic suspensions) Ca2+ activity (aCa) increased spontaneously. This increase was prevented by ATP addition. Addition of a Ca2+ load to axoplasmic suspensions following ATP addition ('normal ATP' axoplasmic suspensions) did not increase aCa. When a Ca2+ load was added to low ATP axoplasmic suspensions the value of aCa was only 5% of that expected from the Ca2+ load in a non-Ca2+ buffered system. Addition of the Ca2+ ionophore A23187 to Ca2+-loaded normal ATP axoplasmic suspensions increased aCa to a value 5% of that expected in a non-Ca2+ buffered system. Addition of A23187 or KCN to low ATP axoplasmic suspensions did not increase aCa above that produced by axoplasmic dilution. These data indicate the presence of ATP-dependent and ATP-independent Ca2+ buffering mechanisms in the squid axoplasm and that the ATP-dependent Ca2+ buffering was mediated by membrane-limited organelles presumably the mitochondria and endoplasmic reticulum.

Adenosine Triphosphate↗

PubMed1978

Initial activity and inactivation of alkaline phosphatase in different lots of buffer.

Alkaline phosphatase activities were determined in six lots of 2-amino-2-methyl-1-propanol (AMP) and in six lots of diethanolamine (DEA) buffers without preincubation of the sample. There appeared to be differences between the lot numbers in both cases, resulting in a variation in initial activity. When serum samples are preincubated with buffer a loss of activity was observed in 4 out of the 6 AMP buffers. Four human isoenzymes showed varying inactivation during preincubation with AMP buffer. No loss of activity was observed when the preincubation was done with the six DEA buffers. These results indicate that the purity of the commercially-available buffers is quite unsatisfactory.

Alkaline Phosphatase↗

PubMed1979

Influence of zinc ions addition to different lots of 2-amino-2-methyl-1-propanol (AMP) buffer on the alkaline phosphatase activities.

The influence of Zn2+ on the inactivation of alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1 3.1] in serum during preincubation with 2-amino-2-methyl-1-propanol (AMP) buffers was investigated. Addition of Zn2+ to the buffer before preincubation increases the enzyme activity. An optimum Zn2+ concentration different for each lot of AMP buffer can be found, at which the enzyme activities are restored to a level equal to activities measured without preincubation. There is a relation between the inactivating properties of the different AMP buffers and the amount of Zn2+ needed to prevent this inactivation. Since Zn2+ chelating substituted diamines are held responsible for the inactivation by removing Zn2+ from the enzyme, we assume that the addition of Zn2+ to the buffer prevents this removal. As Zn2+ itself is an inhibitor of the enzyme, the addition of both too much or too little Zn2+ results in lower enzyme activities after preincubation with AMP buffer.

Alkaline Phosphatase↗

PubMed1987

Effects of buffer composition on water stability of alkylating agents. The example of N-ethyl-N-nitrosourea.

The half-life of N-ethyl-N-nitrosourea (ENU) in water solution was measured at pH 6.1, 6.8 and 7.3 using buffer systems characterized by differences in quality and quantity of the component species. The decay of the concentration of ENU as a function of time was detected both indirectly, by measuring its residual capability of inducing alkali-labile sites in CHO DNA, and directly, by measuring its residual concentration by means of 1H-nmr spectroscopy. Under controlled pH, temperature, and buffering conditions, the rate of hydrolytic loss of ENU measured by the biological and chemical experiments compared quite well, whereas at constant pH and temperature, it was substantially dependent upon the concentration of the buffer. This was attributed to the occurrence of general base catalysis promoted by the nucleophilic anions present in the buffer systems. This conclusion, largely supported by analysis of previously reported data concerning the kinetics of ENU in water, suggests that buffering conditions may play a role in determining the actual levels of reactive substrates in in vitro assays and, therefore, that different buffering conditions may lead to poorly reproducible results.

Alkylating Agents↗

PubMed1978

Intracellular buffering.

In the quantification of buffering the distinct roles of bicarbonate and of other buffers must be taken into account. The determination of the total non-bicarbonate buffer value, betaa, in intact tissues is complicated by active pH regulation and by heterogeneity of cytoplasm with respect to betaa, while heterogeneity with respect to pH in vivo could cause errors in estimates made with homogenates. Available estimates of betaa are discussed, as are the individual contributions of proteins, dipeptides and phosphates. A high betaa is appropriate in cells which sometimes have high rates of glycolysis, or which buffer extracellular fluid, but non-protein buffer concentrations can be well below the limits imposed by osmolarity, perhaps because buffering can upset ionic gradients.

Acid-Base Equilibrium↗

PubMed1988

Calcium regulation by and buffer capacity of molluscan neurons during calcium transients.

The properties of Ca-regulation and -buffering of physiological levels of Ca-transients were examined in the soma of Archidoris monteryensis neurons. The rate of recovery from a Ca-transient was examined with two experimental protocols; in one the pulse duration was kept constant and its amplitude was varied, and in the other the duration was varied while the amplitude was kept constant. These experiments revealed that the recovery from a Ca-transient was approximately a first order process and the apparent first order rate constant was dependent on the duration of Ca-influx. The calcium buffer capacity of the cytoplasm was determined by an indirect method which utilised measured amounts of intracellular EGTA to reduce transient changes in free calcium. An equation for the cytoplasmic buffer capacity was derived on the assumption that the capacities of exogenous and endogenous Ca buffers summate linearly. The resting cytoplasmic Ca buffer capacity was 45.2 microM/delta pCa, when it was assumed that the incoming Ca diffuses a distance of 10 microns into the cytoplasm. For a diffusion distance of 5 microns it was 34.5 microM/delta pCa. In both cases, the buffer capacity increased with an increase in the size of Ca transient.

Animals↗

PubMedDate not supplied

pH gradients generated by polyprotic buffers. II. Experimental validation.

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.

Anions↗

PubMed1991

Physico-chemical properties of amphoteric, isoelectric, macroreticulate buffers.

We report here the properties of a new family of resins possessing an amphoteric character and able to strongly buffer at their pI values. They have been adopted as carriers for growth of cells in tissue culture and for hydroponics (Righetti et al. 1991; J. Biotechnol. 17, 169-176) but it is to be expected that such resins could have interesting chromatographic applications. It has been found that such beads [made by incorporating a pK 6.2 weak acrylamido base and a pK 4.6 weak acrylamido acid in a 2:1 ratio (thus with a pI of 6.2) into a neutral polyacrylamide backbone], independently from their initial conditioning (acid- or base-washed), spontaneously seek their equilibrium position (pI value) upon washing off excess titrant. Thus, upon potentiometric titration, they are seen to buffer in both directions of the pH scale (contrary to the behaviour of a pure carboxyl or a pure amino surface, which will exhibit only unidirectional buffering power). From the behaviour of these amphoteric beads when polymerized in the absence or in the presence of salts (0.2 M NaCl), it is hypothesized that, for exerting buffering power, both the buffering ion and its counterion must be incorporated non-randomly in the chain, but as a couple or in close proximity. Upon random incorporation of the two ions, buffering power is lost.

Buffers↗

PubMed1992

Kinetics of diffusion in a spherical cell. I. No solute buffering.

Realistic neuron models that involve effects of concentration changes of second messengers on ion channels must include processes such as diffusion and solute buffering. These processes, which span a wide range of spatial and temporal scales, may impose a severe computational burden. In this paper and its companion, we examine the kinetics of diffusion and present methods for stimulating it accurately and efficiency. The problem of calcium diffusion in a spherical cell is used as a device to demonstrate the practical application of our analysis. However, the scope of these papers is not limited to this problem. The same analysis that we apply and concerns that we raise are germane to the spread of any second messenger, and can be adapted to other geometries. The focus of this paper is the simplest case: diffusion in the absence of solute buffering. This analysis also applies whenever buffering is so fast that it is instantaneous compared to diffusion, or so slow that concentration gradients have dissipated before substantial buffering takes place. The second paper investigates the more difficult situation where diffusion and buffering occur at comparable rates. In the absence of buffering, concentration changes produced by diffusion can be fit by an infinite series of exponential terms. We show how to design a model with N + 1 compartments that fits the N slowest terms of this series exactly in a shell just inside the cell membrane.

Animals↗

PubMed1995

Influence of Hepes- and CO2/HCO(3-)-buffer on Ca2+ transients induced by TRH and elevated K+ in rat pituitary GH4C1 cells.

The influence of two buffer systems (Hepes and CO2/HCO3-) on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH and by elevated K+ were studied in single, and small clusters of, clonal rat pituitary GH4C1 cells using Fura 2. The steady-state level of [Ca2+]i was virtually identical in Hepes and CO2/HCO3-. In both buffers, addition of TRH induced a transient increase in [Ca2+]i which attained a significantly higher peak in Hepes (357 +/- 43 nM) when compared with values measured in the presence of CO2/HCO3- (184 +/- 21 nM). In Hepes, the basal IP3-level was higher than in CO2/HCO3-. The TRH-evoked increase in IP3 was higher in magnitude in Hepes than in CO2/HCO3-, although the stimulated/basal ratio was not different between the two buffers. The buffer composition had no effect on the specific binding of 3H-TRH to the cells. Furthermore, the amplitude of the increase in [Ca2+]i evoked by 50 mM K+ was identical in both buffers. TRH and K+ had no effect on pHi in either buffer. The present results indicate that HCO3- has an influence on TRH-induced Ca2+ transient, at least in part by modifying the TRH-evoked production of IP3.

Animals↗

PubMed1995

Buffer composition mediates a switch between cooperative and independent binding of an initiator protein to DNA.

The regulation of many biological processes, including DNA replication, is frequently achieved by protein-protein interactions, as well as protein-DNA interactions. Multiple protein-binding sites are often involved. For example, the replication of plasmid R6K involves binding of the initiator protein pi to seven 22-bp direct repeats (DR) in the gamma origin of replication (gamma ori). A mutant protein pi S87N has been isolated, that in Tris.borate buffer (TB) binds cooperatively to seven DR, whereas wild-type (wt) pi binds independently [Filutowicz et al., Nucleic Acids Res. 22 (1994) 4211-4215]. Surprisingly, we found that wt pi can also bind cooperatively when Tris.acetate (TA), Tris.succinate or Tris.glutamate buffers are used instead of TB. The cooperative binding of the wt pi protein was also observed in the TB buffer at high concentrations of Na2EDTA. These results suggest that pi may be able to assume two functionally distinct conformations as a result of either mutation or buffer composition. Moreover, we found that the mode of pi binding is determined not by the composition of the buffer in which the reaction was assembled, but by the composition of the electrophoresis buffer. We discuss the general implications of these findings.

Acetates↗

PubMed2006

Comparison of biochemical parameters of Muscovy drake semen diluted and stored at 4 degrees C in three buffers.

A comparison study of biochemical parameters of semen from Muscovy drakes diluted and stored at 4 degrees C in three buffers--IMV-buffer (France), HIA-1 and AU (Bulgaria) was carried out. The ejaculates were collected twice a week from ten 1-year-old Muscovy drakes using laying Muscovy females as teaser. Semen was diluted immediately, respectively, with IMV-buffer, HIA-1 and AU, and cold-stored (4 degrees C) for 1, 3 and 6h. The intensities of oxygen uptake at the third hour in semen diluted, respectively, with IMV-buffer (200+/-1.6 nAO/10(9) spermcellsmin), with HIA (224+/-44 nAO/10(9) spermcellsmin) and with AU (238+/-48 nAO/10(9) spermcellsmin) were highly significant in comparison with neat semen (75+/-0.7 nAO/10(9)spermcellsmin). The observed intensity of fructolysis was highest when using AU, followed by HIA-1 and IMV-buffer. During the first hour of storage the level of pyruvic acid was significantly lower in semen diluted with Bulgarian extenders, and this stability for AU referred to the entire period. For lactic acid, the differences were not statistical significant. Our investigations do not show significant differences concerning the dynamics of inorganic phosphate and total lipids after dilution with all tested extenders. On the contrary, high increase of cholesterol efflux from spermatozoa to seminal plasma-diluents were obtained after 6 h of storage. All extenders, IMV-buffer (France), HIA-1 and AU (Bulgaria) for diluting and short time storage of semen from Muscovy drakes at 4 degrees C maintain the necessary comfort of energy metabolism of the spermatozoa.

Animals↗

PubMed2004

Role of bicarbonate/CO2 buffer in the initiation of vesicle-mediated calcification: mechanisms of aortic calcification related to atherosclerosis.

Calcifying vesicles play an important role in the mechanism of aortic calcification induced by dietary cholesterol interventions. This study was initiated to test the hypothesis that alterations in the ratio of bicarbonate/CO2, which is a main physiological buffer, could affect vesicle-mediated calcification. Using rabbits as a model, in vitro calcification of vesicles isolated from aortas was performed to study the effect of the bicarbonate buffer on the mineralization process. When Tris buffer was initially used to maintain pH of the media, ATP-dependent vesicle calcification increased with pH of calcifying media. By replacing Tris with physiological bicarbonate/CO2 buffer, ATP-dependent vesicle calcification increased rapidly with increased ratios of bicarbonate/CO2. The increase appears to be a result of elevated levels of pH through the alteration in the ratios of bicarbonate/CO2. The effect of the physiological concentration of bicarbonate (30 mM) on pH of calcifying media was remarkable since 50 mM of Tris buffer at pH 7.6 failed to prevent a rapid rise in pH under atmospheric CO2. The effect of bicarbonate and CO2 on vesicle calcification was dependent on the ratio of the surface area to the volume of calcifying media, since the ratio profoundly affects the exchange rate between the gas and liquid phases of CO2. Although the pathological conditions that alter the pH remain unknown, it is conceivable that blockage in the supply of blood CO2 to the media by intimal thickening in the lesions could contribute to focal calcification. We conclude that bicarbonate buffer could provide a dynamic and rapid transitional increase in pH of extracellular fluids, thereby creating a favorable condition for the initiation of vesicle-mediated calcification under pathological conditions.

Adenosine Triphosphate↗

PubMed2005

Optimization of a model of red blood cells for the study of anti-oxidant drugs, in terms of concentration of oxidant and phosphate buffer.

The aggression of erythrocytes by an oxidative stress induces hemolysis. This paper aims to valid a model of erythrocytes in terms of composition of the phosphate buffer solution and of concentration of a well-known oxidant, AAPH. Three compositions of phosphate buffer solution are mixed with three concentrations of oxidant. The influence of these two parameters on hemolysis is independently studied by a variance analysis and a Kruskal-Wallis test when ANOVA is not available. The hemolysis rate increases with time at fixed oxidant concentration, but is not influenced by the composition of the buffer solution. The highest hemolysis rate, 90%, was only measured within 2 h with the highest oxidant concentration. If we retain this concentration of oxidant, the lower concentration of the buffer can by eliminated by a significant less hemolysis and the highest concentration of the buffer can by chosen in regard of the better precision for a similar hemolysis compared to the mean buffer. We hope to study the effect of anti-oxidant agent with such a model of erythrocytes.

Antioxidants↗

PubMed2006

Modulation of neuronal excitability by intracellular calcium buffering: from spiking to bursting.

We have investigated the detailed regulation of neuronal firing pattern by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch-clamp recording in acute slice. Theoretical results show that a high calcium buffer concentration alters the characteristic regular firing of cerebellar granule cells and that a transition to various modes of oscillations occurs, including bursting. Using bifurcation analysis, we show that this transition from spiking to bursting is a consequence of the major slowdown of calcium dynamics. Patch-clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal dramatic alterations in their excitability as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit all bursting behaviors predicted by the model whereas bursting is never observed in low buffering. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing patterns leading to generation of complex patterns and therefore that calcium-binding proteins may play a critical role in the non-synaptic plasticity and information processing in the central nervous system.

Animals↗

PubMed2006

Retention of ionisable compounds on high-performance liquid chromatography XVI. Estimation of retention with acetonitrile/water mobile phases from aqueous buffer pH and analyte pKa.

In agreement with our previous studies and those of other authors, it is shown that much better fits of retention time as a function of pH are obtained for acid-base analytes when pH is measured in the mobile phase, than when pH is measured in the aqueous buffer when buffers of different nature are used. However, in some instances it may be more practical to measure the pH in the aqueous buffer before addition of the organic modifier. Thus, an open methodology is presented that allows prediction of chromatographic retention of acid-base analytes from the pH measured in the aqueous buffer. The model presented estimates the pH of the buffer and the pKa of the analyte in a particular acetonitrile/water mobile phase from the pH and pKa values in water. The retention of the analyte can be easily estimated, at a buffer pH close to the solute pKa, from these values and from the retentions of the pure acidic and basic forms of the analyte. Since in many instances, the analyte pKa values in water are not known, the methodology has been also tested by using Internet software, at reach of many chemists, which calculates analyte pKa values from chemical structure. The approach is successfully tested for some pharmaceutical drugs.

Acetonitriles↗

PubMed2004

Potassium buffering in the central nervous system.

Rapid changes in extracellular K+ concentration ([K+](o)) in the mammalian CNS are counteracted by simple passive diffusion as well as by cellular mechanisms of K+ clearance. Buffering of [K+](o) can occur via glial or neuronal uptake of K+ ions through transporters or K+-selective channels. The best studied mechanism for [K+](o) buffering in the brain is called K+ spatial buffering, wherein the glial syncytium disperses local extracellular K+ increases by transferring K+ ions from sites of elevated [K+](o) to those with lower [K+](o). In recent years, K+ spatial buffering has been implicated or directly demonstrated by a variety of experimental approaches including electrophysiological and optical methods. A specialized form of spatial buffering named K+ siphoning takes place in the vertebrate retina, where glial Muller cells express inwardly rectifying K+ channels (Kir channels) positioned in the membrane domains near to the vitreous humor and blood vessels. This highly compartmentalized distribution of Kir channels in retinal glia directs K+ ions from the synaptic layers to the vitreous humor and blood vessels. Here, we review the principal mechanisms of [K+](o) buffering in the CNS and recent molecular studies on the structure and functions of glial Kir channels. We also discuss intriguing new data that suggest a close physical and functional relationship between Kir and water channels in glial cells.

Animals↗