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PubMed2005

Effects of liquid chromatography mobile phases and buffer salts on phosphorus inductively coupled plasma atomic emission and mass spectrometries utilizing ultrasonic nebulization and membrane desolvation.

Atomic spectrometry, specifically inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) show promise for heteroatom-based detection of pharmaceutical compounds. The combination of ultrasonic nebulization (USN) with membrane desolvation (MD) greatly enhances detection limits with these approaches. Because pharmaceutical analyses often incorporate liquid chromatography, the study herein was performed to examine the effects of solvent composition on the analytical behaviors of these approaches. The target analyte was phosphorus, introduced as phosphomycin. AES response was examined at the 253.7 nm atom line and mass 31 ions were monitored for the MS experiments. With pure aqueous solutions, detection limits of 5 ppb (0.5 ng in 0.1 mL injection volumes) were obtained with ICP-MS. The ICP-AES detection limit was 150 ppb. Solvent compositions were varied from 0 to 80% organic (acetonitrile and methanol) with nine buffers at concentrations typically used in liquid chromatography. In general, solvents and buffers had statistically significant, albeit small, effects on ICP-AES sensitivities. A few exceptions occurred in cases where typical liquid chromatography buffer concentrations produced higher mass loadings on the plasma. Indications are that isocratic separations can be reliably performed. Within reasonable accuracy tolerances, it appears that gradient chromatography can be performed without the need for signal response normalization. Organic solvent and buffer effects were more significant with ICP-MS. Sensitivities varied significantly with different buffers and organic solvent content. In these cases, gradient chromatography will require careful analytical calibration as solvent and buffer content is varied. However, for most buffer and solvent combinations, signal and detection limits are only moderately affected. Isocratic separations and detection are feasible.

Acetonitriles↗
PubMed2006

Impacts of agricultural phosphorus use in catchments on shallow lake water quality: About buffers, time delays and equilibria.

Phosphorus (P) losses caused by intensive agriculture are known to have potentially large negative effects on the water quality of lakes. However, due to the buffering capacity of soils and lake ecosystems, such effects may appear long after intensive agriculture started. Here we present the study of a coupled shallow lake catchment model, which allows a glimpse of the magnitude of these buffer-related time delays. Results show that the buffering capacity of the lake water was negligible whereas buffering in the lake sediment postponed the final lake equilibrium for several decades. The surface soil layer in contact with runoff water was accountable for a delay of 5-50 years. The most important buffer, however, was the percolation soil layer that may cause a delay of 150-1700 years depending on agricultural P surplus levels. Although the buffers could postpone final lake equilibria for a considerable time, current and target agricultural surplus levels eventually led to very turbid conditions with total P concentrations of 2.0 and 0.6 mg L(-1) respectively. To secure permanent clear water states the current agricultural P surplus of 15 kg P ha(-1) yr(-1) should drop to 0.7 kg P ha(-1) yr(-1). We present several simple equations that can be used to estimate the sustainable P surplus levels, buffer related time delays and equilibrium P concentrations in other catchment-lake systems.

Agriculture↗
PubMed1986

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.

Animals↗
PubMed1990

Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties.

We have developed a calcium diffusion model for a spherical neuron which incorporates calcium influx and extrusion through the plasma membrane as well as three calcium buffer systems with different capacities, mobilities, and kinetics. The model allows us to calculate the concentration of any of the species involved at all locations in the cell and can be used to account for experimental data obtained with high-speed Ca imaging techniques. The influence of several factors on the Ca2+ transients is studied. The relationship between peak [Ca2+]i and calcium load is shown to be nonlinear and to depend on buffer characteristics. The time course of the Ca2+ signals is also shown to be dependent on buffer properties. In particular, buffer mobility strongly determines the size and time course of Ca2+ signals in the cell interior. The model predicts that the presence of exogenous buffer, such as fura-2, modifies the Ca2+ transients to a variable extent depending on its proportion relative to the natural, intrinsic buffers. The conclusions about natural calcium buffer properties that can be derived from Ca imaging experiments are discussed.

Animals↗
PubMed1994

Effects of rapid buffers on Ca2+ diffusion and Ca2+ oscillations.

Based on realistic mechanisms of Ca2+ buffering that include both stationary and mobile buffers, we derive and investigate models of Ca2+ diffusion in the presence of rapid buffers. We obtain a single transport equation for Ca2+ that contains the effects caused by both stationary and mobile buffers. For stationary buffers alone, we obtain an expression for the effective diffusion constant of Ca2+ that depends on local Ca2+ concentrations. Mobile buffers, such as fura-2, BAPTA, or small endogenous proteins, give rise to a transport equation that is no longer strictly diffusive. Calculations are presented to show that these effects can modify greatly the manner and rate at which Ca2+ diffuses in cells, and we compare these results with recent measurements by Allbritton et al. (1992). As a prelude to work on Ca2+ waves, we use a simplified version of our model of the activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane to illustrate the way in which Ca2+ buffering can affect both the amplitude and existence of Ca2+ oscillations.

Animals↗
PubMed1996

Validity of the rapid buffering approximation near a point source of calcium ions.

In the presence of rapid buffers the full reaction-diffusion equations describing Ca2+ transport can be reduced using the rapid buffering approximation to a single transport equation for [Ca2+]. Here we simulate the full and reduced equations, exploring the conditions necessary for the validity of the rapid buffering approximation for an isolated Ca2+ channel or a cluster of channels. Using a point source and performing numerical simulations of different durations, we quantify the error of the rapid buffering approximation as a function of buffer and source parameters as well as the time and spatial scale set by the resolution of confocal microscopic measurements. We carry out simulations of Ca2+ "sparks" and "puffs," both with and without the indicator dye Ca2+ Green-1, and find that the rapid buffering approximation is excellent. These calculations also show that the traditional calculation of [Ca2+] from a fluorescence signal may grossly underestimate the true value of [Ca2+] near a source. Finally, we use the full model to simulate the transient Ca2+ domain near the pore of an open Ca2+ channel in a cell dialyzed with millimolar concentrations of 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid or EGTA. In this regime, where the rapid buffering approximation is poor. Neher's equation for the steady-state Ca2+ profile is shown to be a reliable approximation adjacent to the pore.

Biophysical Phenomena↗
PubMed2001

Influence of buffer zone concentrations on efficiency in partial filling micellar electrokinetic chromatography.

The potential of counter pressure-moderated partial filling micellar electrokinetic chromatography (PF-MEKC) was investigated in this work. Plate numbers of homologous omega-phenylalcohols were measured in a two-plug PF-MEKC system varying the concentrations and hence the ionic strengths of the background buffer compared to the sodium dodecyl sulfate-containing separation buffer and the counter pressure on the cathodic buffer reservoir. It was observed that plate numbers are strongly influenced by both the buffer concentrations and the counter pressure. Highest plate numbers were obtained with a buffer system where the concentrations are adjusted such that the electroosmotic flow velocities in both zones are equal. Differences in the local electroosmotic flow velocities of the zones caused by different buffer concentrations are responsible for tremendously reduced plate numbers. The efficiency drop is explained in several models by the formation of an intersegmental pressure which produces a parabolically shaped laminar flow component in both zones. Thus, the electroosmotic plug-like flow profile is distorted and the efficiency is reduced. The effect of counter pressure on efficiency turned out to be very complex in dependence on the buffer system applied.

Buffers↗
PubMed1997

Evaluation of phytic acid as a buffer additive for the separation of proteins in capillary electrophoresis.

The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular. Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.

Buffers↗
PubMed1997

Generation of tryptic maps of alpha- and beta-globin chains by capillary electrophoresis in isoelectric buffers.

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH = pI = 2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 microns I.D. and 900 V/cm in 50 microns I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of alpha- and beta-globin chains from human adult hemoglobin. In the case of beta-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.

Adult↗
PubMed2003

A new role for cardioplegic buffering: should acidosis or calcium accumulation be counteracted to salvage jeopardized hearts?

OBJECTIVES: Thirty minutes of unprotected ischemia produced a jeopardized heart that was treated with a blood cardioplegic solution containing the natural erythrocyte and protein buffers. Cardioplegic pH was changed to 7.7 (buffered) or 7.2 (nonbuffered), and this was tested alone and after pretreatment with Na(+)-H(+) exchange blockade (cariporide) to define their protective effects. METHODS: Twenty-four Yorkshire-Duroc pigs (27-34.5 kg) underwent 30 minutes of normothermic global ischemia, followed by 30 minutes of aortic clamping during protection with buffered (n = 12) or nonbuffered (n = 12) glutamate-aspartate-enriched blood cardioplegic solution. Twelve hearts (6 buffered and 6 nonbuffered) were pretreated with intravenous cariporide (5 mg/kg) 15 minutes before ischemia. RESULTS: Severe and comparable left ventricle dysfunction followed buffered or nonbuffered cardioplegia: Preload recruitable stroke work recovered to 56% +/- 21% and 45% +/- 20% of baseline levels; creatine kinase MB, conjugated dienes, and myeloperoxidase activity markedly increased; moderate myocardial edema occurred; and endothelin-1 increased 2-fold more than baseline values. Cariporide pretreatment caused a similar return of preload recruitable stroke work to 86% +/- 9% and 90% +/- 6% after buffered or nonbuffered cardioplegia (P <.05 vs nonpretreated groups), allowed only minor creatine kinase MB and conjugated diene changes, and reduced endothelin-1 release 3-fold compared with hearts without sodium-hydrogen exchange blockage. CONCLUSIONS: The severe ischemia-reperfusion injury of 30 minutes of normothermic ischemia is not altered by an acidic or alkalotic pH cardioplegic solution. Correction of damage is achieved by adding Na(+)-H(+) exchange blocker therapy before treatment with buffered and nonbuffered solutions; thus, sodium-hydrogen exchange inhibition plays a more vital role in recovery than pH management.

Acidosis↗
PubMed2000

Determination of baseline human nasal pH and the effect of intranasally administered buffers.

The nose is becoming a common route of drug administration, however, little is known about the pH of the human nasal cavity. Local pH may have a direct effect on the rate and extent of absorption of ionizable compounds and hence this study was performed to investigate normal pH values and whether pH could be manipulated by various buffers. Twelve healthy volunteers participated in a study to measure pH in the anterior and posterior sites of the nasal cavity. Miniature pH electrodes were placed 3 cm apart in the nasal cavity and a baseline was recorded for 30 min once the pH had stabilized. One hundred microlitres of isotonic solution was sprayed into the nostril and the pH was measured for 4 h post-dose. The following five formulations were tested: formulation A--sodium chloride (0.9%) at pH 7.2; formulation B--sodium chloride (0.9%) at pH 5.8; formulation C--Sorensens phosphate buffer (0.06 M) at pH 5. 8; formulation D--Sorensens phosphate buffer (0.13 M) at pH 5.8 and formulation E--formulation as (c) but adjusted to pH 5.0. Each formulation also contained saccharin sodium (0.5%) as a taste marker for nasal clearance. The time at which each subject detected the taste of saccharin was noted. The 30-minute baseline recording prior to administration of the nasal spray formulation demonstrates that there was both considerable intersubject and intrasubject variation in nasal pH. The average pH in the anterior of the nose was 6.40 (+0. 11, -0.15 S.D.) when calculated from H(+) values. The pH in the posterior of the nasal cavity was 6.27 (+0.13, -0.18 S.D.). The overall range in pH was 5.17-8.13 for anterior pH and 5.20-8.00 for posterior pH. Formulation A caused the pH in the anterior part of the nasal cavity to reach a maximum of 7.06 in 11.25 min from the baseline of pH 6.14 (P<0.05). The mean baseline pH was 6.5 for the posterior part of the nose which did not change over the recording period. Formulation B caused the anterior pH to increase from pH 6. 60 to 7.25 within the first minute. This fell back to a mean pH of 7.07 over the first hour which was still significantly above the baseline. It remained at this value for the remainder of the recording period. The initial average posterior pH was 6.32 and again this did not significantly change over the recording period. Formulation C produced a sustained increase in anterior nasal pH from a baseline pH of 6.57-7.12. A small transient decrease was observed in the pH in the posterior of the nose but baseline pH of 6. 6 was re-established within 15 min post dose. Formulation D significantly reduced anterior nasal pH from 6.30 to 5.87 by 30 min reaching a pH of 5.95 by 90 min where it remained for the remainder of the recording period. The posterior baseline pH was 6.3 and introduction of the pH 5.8 buffer caused a slow increase over 90 min to pH 6.6. Formulation E increased anterior pH from 6.1 to 6.7 for the remainder of the recording period. It had an insignificant effect on posterior nasal pH. The mean (+/-S.D.) time to taste saccharin for formulations A to E was 13.42+/-10.21, 14.67+/-8.37, 11.67+/-8.08, 10.08+/-7.6, 9.80+/-6.73 min, respectively. There was no significant difference between the clearance times for the different formulations. In conclusion, average baseline human nasal pH is approximately 6.3. Nasal anterior pH can be decreased when buffers of 0.13 M and above are used. Mildly acidic solutions produce an increase in pH presumably due to reflux bicarbonate secretion. Posterior nasal pH was not altered by administration of any buffer except the 0.13 M buffer at pH 5.8. This produced a rise in posterior pH.

Administration, Intranasal↗
PubMed1998

Serotonin-stimulated calcium responses in human platelets: assay buffer dependency.

In Fura-2 labelled human platelets, the observed calcium response to serotonin stimulation is increased if the assay is conducted in a Krebs buffer compared with the HEPES buffer system usually used. The serotonin response in the Krebs buffer system was concentration dependent with an EC50 value of approximately 0.3 microM and was blocked by nanomolar concentrations of the serotonin2-receptor antagonist methiothepin. In a series of samples collected from 11 persons, a reasonable correlation was seen between the response to 1 microM serotonin and the serotonin2A-receptor density measured in corresponding membrane preparations. The calcium response to serotonin was reduced by treatment with hydrogen peroxide. It is concluded that the use of the Krebs buffer gives a more sensitive response of the Fura-2 loaded human platelets to serotonin stimulation than does the use of HEPES buffer and that the increased sensitivity is achieved without affecting the pharmacological properties of the response. For the response to thrombin, the response intensity is affected by the pH of the buffer used but is not sensitive to the buffer composition.

Blood Platelets↗
PubMed1998

Hypothermic preservation of isolated rat lungs in modified bicarbonate buffer, EuroCollins solution or St Thomas' Hospital cardioplegic solution.

OBJECTIVES: Inadequate preservation solutions limit lung storage times and, consequently, transplant programs. To address this problem we established an isolated, ventilated and perfused rat lung preparation. Here we report the effects of hypothermic storage in EuroCollins solution, St Thomas' Hospital cardioplegic solution and a modified bicarbonate buffer solution. METHODS: Lungs from male Wistar rats (230-330 g) were perfused via the pulmonary artery with modified bicarbonate buffer (37 degrees C, 15 ml/min, constant flow) and ventilated by positive pressure (tidal volume:1.6-1.8 ml, 80 breaths/min). Vascular resistance (pulmonary artery pressure:perfusate flow ratio) and airways compliance (tidal volume:tracheal pressure ratio) were measured. After a control perfusion period (20 min), lungs were flushed with, then immersed in, bicarbonate buffer (4 degrees C) for varying periods (0-24 h). After storage, lung function was assessed during 20 min reperfusion. Having established a suitable period for study, storage in EuroCollins, St Thomas' Hospital cardioplegic solution or bicarbonate buffer were compared. RESULTS: Pulmonary compliance (ml/cmH2O) was significantly (P < 0.05) reduced in lungs stored for 6 h in modified bicarbonate buffer (0.026 +/- 0.008), EuroCollins solution (0.013 +/- 0.002) or St Thomas' Hospital solution (0.025 +/- 0.005) compared to unstored lungs (0.068 +/- 0.007). Vascular resistance, (1.32 +/- 0.13 cmH2O/ml per min) in unstored lungs, was similar in lungs stored in St Thomas' Hospital solution but increased significantly in lungs stored in modified bicarbonate buffer (3.22 +/- 0.78 cmH2O/ml per min) or EuroCollins solution (4.66 +/- 0.57 cmH2O/ml per min). CONCLUSIONS: Hypothermic storage of rat lungs for 6 h in modified bicarbonate buffer or St Thomas' Hospital solution causes less increase in vascular resistance on reperfusion than EuroCollins solution.

Animals↗
PubMed1997

Use of nonvolatile buffers in liquid chromatography/mass spectrometry: advantages of capillary-scale particle beam interfacing.

The use of nonvolatile buffers like phosphate and citrate is usually incompatible with mass spectrometric detection or may heavily interfere with ion generation. In the case of conventional HPLC flow rates, the continuous buffer deposition produces fouling of the mass spectrometer ion source and may worsen the performance of any LC/MS interface as well. Our research group demonstrated that reducing the mobile phase flow rate in a particle beam interface improves the nebulization and enhances the overall performance. We have also assumed that such a modification may better handle potentially instrument harmful solvents or nonvolatile HPLC buffers. Since the presence of salts does not interfere with the electron ionization process, the particle beam interface can be considered, in principle, particularly suitable for those chromatographic separations that benefit from nonvolatile buffers. The microscale flow rate interface, developed in our laboratory, generates an aerosol from 1 microL/min of mobile phase flow rate. Under these conditions, the absolute amount of a nonvolatile buffer actually introduced into the system is approximately 1/1000 of that carried by a conventional HPLC column, slowing its deposition. This assumption was verified with a real-world application requiring the use of a phosphate buffer at a maximum concentration of 10 mM. It involved the determination of dexamethasone, an anti-inflammatory drug, in human blood after its administration to a patient. Several crucial mass spectral and chromatographic parameters were monitored during a 35-day period of intense use. Neither appreciable signal modification nor evident buffer deposition was observed during the test period, with only a normal and limited run-by-run and day-by-day variation of the instrument response.

Anti-Inflammatory Agents↗
PubMed1992

Relationship between intracellular proton buffering capacity and intracellular pH.

In a recent publication the widely held view that the intracellular proton buffering power [defined as the amount of acid or base that has to be added to the cytosol to change the intracellular pH (pHi) by one pH unit] increases as the intracellular pH decreases, has been challenged, with the opposite relationship being proposed. In that publication, buffering was defined not in terms of pH change, but in terms of the change in proton concentration. The reason for this re-definition was the fear that the conventional analysis, using as it does a logarithmic function (pHi), could bias the outcome in favor of an increasing buffering power with decreasing pHi. The new system uses a "buffering co-efficient," defined as the number of protons necessary to be added to the cytosol to change the intracellular proton concentration by 1 mM. We report the use of both of these methods to analyze the relationship of pHi and buffering power, using human peripheral leucocytes loaded with the pH-sensitive fluorophore BCECF examined over a very wide range of pHi values (pHi 6.0 to 7.5). The most common method for pHi perturbation for the measurement of buffering is used, the rapid diffusion of ammonia across the cell membrane. In this study, analysis for both a bicarbonate-containing "open" system and for a Hepes-buffered "closed" system was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed2001

Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells.

Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P(3) to activate the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca(2+) buffering. Dialysis with Ins(1,4,5)P(3) in weak Ca(2+) buffer did not activate any macroscopic I(CRAC) even after precautions had been taken to minimize the extent of Ca(2+) entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P(3) for >150 s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) pump blocker thapsigargin activated I(CRAC), and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P(3). This indicates that the Ins(1,4,5)P(3) receptors had not inactivated much over this timecourse. When external Ca(2+) was replaced by Ba(2+), Ins(1,4,5)P(3) still failed to generate any detectable I(CRAC) even though Ba(2+) permeates CRAC channels and is not taken up into the intracellular Ca(2+) stores. In strong Ca(2+) buffer, I(CRAC) could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P(3), stimulation of these receptors with carbachol did not activate I(CRAC) even after inhibition of PKC. The inability of Ins(1,4,5)P(3) to activate macroscopic I(CRAC) in weak Ca(2+) buffer was not altered by inhibition of Ca(2+)-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P(3) to activate I(CRAC) under conditions of weak intracellular Ca(2+) buffering is not due to strong inactivation of the Ins(1,4,5)P(3) receptors. Instead, a futile Ca(2+) cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca(2+) to ensure that the threshold for activation of macroscopic I(CRAC) has not been exceeded.

Animals↗
PubMed2002

Adenophostin A and ribophostin, but not inositol 1,4,5-trisphosphate or manno-adenophostin, activate the Ca2+ release-activated Ca2+ current, I(CRAC), in weak intracellular Ca2+ buffer.

Under physiological conditions of weak intracellular Ca(2+) buffering (0.1 mM EGTA), the second messenger Ins(1,4,5)P(3) often fails to activate any detectable store-operated Ca(2+) current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P(3) for Ins(1,4,5)P(3) receptors] consistently activates the current under similar conditions. Here, whole-cell patch clamp experiments have been performed to examine how adenophostin A can activate the store-operated Ca(2+) current (I(CRAC)) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca(2+) buffer, saturating concentrations of adenophostin A activated I(CRAC) maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P(3). In a weak Ca(2+) buffer, adenophostin A consistently activated I(CRAC), but the current was submaximal. High concentrations of Ins(1,4,5)P(3) or the non-metabolizable analogue Ins(2,4,5)P(3) were largely ineffective under these conditions. The size of I(CRAC) to adenophostin A in weak Ca(2+) buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase ('SERCA') pumps with thapsi-gargin or enhancing mitochondrial Ca(2+) uptake, although blocking the mitochondrial Ca(2+) uniporter with Ruthenium Red did not suppress the activation of the current. Changing the levels of free ATP in the recording pipette did not enhance the size of I(CRAC) evoked by adenophostin A. We also examined two structurally distinct analogues of adenophostin A (manno-adenophostin and ribophostin), for which the affinities for the Ins(1,4,5)P(3) receptor are similar to that of Ins(1,4,5)P(3) in equilibrium binding experiments. Although these analogues were able to activate I(CRAC) to its maximal extent in strong buffer, ribophostin, but not manno-adenophostin, consistently activated the current in weak buffer. We conclude that adenophostin A and ribophostin are able to activate I(CRAC) in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P(3) and manno-adenophostin and is not related to equilibrium affinities.

Adenosine↗
PubMed2005

Effects of buffer properties on cyclodextrin glucanotransferase reactions and cyclodextrin production from raw sago (Cycas revoluta) starch.

Results from the present study have shown that the ionic species of buffers, pH values and reaction temperature can affect the enzyme unit activities and product specificity of Toruzyme (Novo Nordisk A/S Bagsvaerd, Denmark) CGTase (cyclodextrin glucanotransferase). Applying a similar reaction environment (acetate buffer, pH 6.0; temperature, 60 degrees C), the CGTase was found to be capable of producing pre dominantly beta-cyclodextrin from either raw or gelatinized sago (Cycas revoluta) starch. Changing the buffer from acetate to phosphate reduced the yield of beta-cyclodextrin from 2.48 to 1.42 mg/ml and also affected the product specificity, where production of both alpha- and beta-cyclodextrins were more pronounced. The decrease in the production of cyclodextrins in phosphate buffer was significant at both pH 6.0 and 7.0. However, changing the buffer to Tris/HCl (pH 7.0) showed a significant increase in beta-cyclodextrin production. Increasing the ionic strength of sodium acetate and Tris/HCl buffers at pH 6.0 and 7.0 to equivalent ionic strength of phosphate buffers showed no significant effects on cyclodextrin production. Higher yield of cyclodextrins at pH 7.0 when Tris/HCl was used might be due to the binding of chloride ions at the calcium-binding sites of the CGTase, resulting in the shift of the optimum pH close to physiological environment, leading to an increase in the activities and specificity.

Buffers↗