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PubMed1985

Comparison of histidine proton magnetic resonances of human carbonmonoxyhaemoglobin in different buffers.

We have recorded the C-2 proton resonances of the histidines of carbonmonoxyhaemoglobin A and of four abnormal human HbCOs in different buffers and at different concentrations of haemoglobin. Resonance H assigned by Perutz et al. (1985) to His HC3(146) beta, is present at both pH 7.30 and pH 6.90, but somewhat broadened when recorded in 5 to 10% HbCO A in 0.1 M-bis-Tris. The broadening disappears on tenfold dilution of the Hb with bis-Tris and the resonance then stands out sharply. Resonance H is absent at both Hb concentrations in HbCO Cowtown (His HC3(146) beta----Leu). HbCO Fort de France (His CD3(45) alpha----Arg) in 0.1 M-bis-Tris of pH 6.90 has a spectrum similar to that of HbCO A. In the same buffer a resonance marked L by Russu et al. (1982) is absent from the spectrum of Hb Abbruzzo (His H21(143) beta----Arg), whereas resonance H is present. Hb Barcelona contains an additional histidine in position FG1(94) beta; in 0.1 M-bis-Tris buffer of pH 6.90 its resonance is not resolved and resonance H is either shifted or broadened. The resonances of both histidines are resolved in phosphate buffer. At pH 6.90, spectra in 0.1 M-bis-Tris buffer are similar to those previously recorded in 0.2 M-HEPES. Addition of 0.1 M-KCl produces marked changes. Replacement of bis-Tris by 0.2 M-KCl + 0.2 M-phosphate gives rise to a different and much better resolved spectrum.

Buffers↗
PubMed1982

In vitro buffer value of brain tissue during prolonged hypercapnia.

Ventilatory adaptation to CO2 has been related to a return toward normal pH via an increase in CSF and arterial [HCO3(-)]. To examine whether the overall brain tissue can contribute HCO3(-) to surrounding fluids, we measured the in vitro CO2 buffer value (beta CO2) of control and hypercapnic rat brain homogenates and compared values with reported in vivo data. Hypercapnic rats were exposed to 7% CO2 for 3 days or 1 week. Brain homogenate was continuously tonometered for 3 h at 37 degrees C with 2%, 5% and 15% CO2 in O2. In addition, we used KOH to determine the brain buffering in the pH range 6.8-10.25. During CO2 titration, [HCO3(-)] increased gradually with time up to 90 min by about 10-15%, but the increase was blocked by a metabolic inhibitor, NaF, beta CO2, estimated per kg brain tissue from the dilute homogenate, ranged between 23.4 +/- 2.7 (SD) in controls and 26.0 +/- 1.3 meq/pH in the 7 day group, which were not significantly different. Over the same 7 days, CO2 dissociation curves were shifted upwards with similar slopes by about 6 ml/100 g tissue in association with a rise in pH of about 0.06, consistent with an accumulation of HCO3(-) without any change in buffers. No significant differences between groups were found from KOH titration curves, either in slope or position, consistent with lack of alteration in buffers as well. In vitro brain tissue beta CO2 (about 25) was less than reported in vivo values in the literature (around 40), possibly because H+ adjustments by whole body occur so rapidly in vivo. In addition, other investigators demonstrated that a major part of the increased brain cell [HCO3(-)] in prolonged hypercapnia could not be accounted for by the fixed acid production (Acta Physiol. Scand. 83: 344, 1971). By assuming the in vitro beta CO2 measures the available non-carbonic buffers, the data may be interpreted as showing that the overall brain tissue accumulates HCO3(-) from surrounding fluid during hypercapnia.

Animals↗
PubMed1990

Buffering characteristics of eel blood at the extreme conditions in the swimbladder.

CO2 binding in whole blood and true plasma of the eel was estimated by measuring CO2 content and pH in blood aliquots equilibrated with various PCO2 values over a wide range expected to occur in the swimbladder. Bicarbonate concentration, [HCO3-], was calculated using the CO2 solubility coefficient, which was measured to average 50 mumols.L-1.Torr-1 (20 degrees C). Buffer lines of non-bicarbonate buffers were obtained in plots of [HCO3-] against pH, and non-bicarbonate buffer values, beta NB, were obtained by curve fitting. In the pH range 6.6-8.2, the buffer line for oxygenated whole blood was sigmoid, while that for deoxygenated blood increased its slope monotonously with increasing pH. The beta NB for oxygenated blood displayed a maximum of about 8.6 mmol.L-1.pH-1 at pH = 7.4 and dropped down to below 1 mmol.L-1.pH-1 at higher and lower pH. Similar shapes of the buffer lines were obtained in true plasma; the [HCO3-] levels and beta NB values were, however, somewhat higher than in whole blood. These data are useful to assess the back-diffusion of CO2 and HCO3- in the rete mirabile of the fish swimbladder and to estimate the effects CO2 back-diffusion exerts on the counter-current enhancement of O2 in the rete.

Air Sacs↗
PubMed1996

The interaction of sodium dodecyl sulfate and urea with cat-fish collagen solutions in acetate buffer: hydrodynamic and thermodynamic studies.

Cat-fish collagen was extracted and characterized. Shrinkage temperature of cat-fish collagen is 54.5 degrees C. SDS-PAGE pattern indicated that the cat-fish collagen is Type I in nature. The ratio of proline and hydroxyproline is 1:2 and it suggests cat-fish collagen is vertebrate. The molecular weight of cat-fish collagen was determined by using molecular sieve chromatography and it was found to be 3 20,000 Da. The mutual interaction of cat-fish collagen with SDS and urea was studied at various temperatures. The results suggest that the aggregation of collagen is facilitated by the presence of SDS, whereas hindered by urea. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into SDS micelles, urea and the reverse phenomenon was analysed. These transfer properties are temperature-dependent. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh of collagen in buffer, SDS, and urea environments using Simha-Einstein equation and intrinsic viscosity were also calculated. The low intrinsic viscosity [eta] and high Vh value of collagen in an SDS environment compared to buffer and other environments suggested a more workable system in cosmetic and dermatological preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analysed. The calculated thermodynamic parameters varied with the concentration of collagen as well as concentration of additives. The change of thermodyanamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Denaturation enthalpy of the catfish collagen in buffer, SDS and urea environments has also been determined by differential scanning calorimetric (DSC) measurements, and the results are in good agreement with the viscosity-derived values. The assymmetry and molecular geometry of this collagen in buffer, SDS and urea environments are also computed. Overall, our hydrodynamic and thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS > buffer > urea.

Acetates↗
PubMed1980

Adverse effects of Tris. HEPES and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins.

The influence of artificial buffers, Tris, HEPES and MOPS, on PGA1, B2 and F2 alpha-induced contractile responses of isolated rat aorta and portal vein was investigated. All three buffers, in 5mM concentration, when substituted for bicarbonate and phosphate anions, differentially attenuated PG-induced contractile responses; the vein responses exhibited the greater sensitivity to artificial buffer inhibition. In the presence of the artificial buffers, the PG-induced fast phase contractile components on the arterial smooth muscle were obliterated and the slow, tonic components were markedly depressed. PG contractile concentration-effect curves were shifted, differentially, to the right, concomitant with differential reductions in maximum tensions. Reincubation of the vascular tissues in normal Krebs-Ringer bicarbonate for 30-60 min resulted in a complete restoration of full PG-induced contractions. These results support the idea that Tris, HEPES and MOPS may interfere with the binding, translocation and utilization of calcium ions in vascular smooth muscle cells. One must be cautious regarding conclusions drawn for calcium-dependent responses obtained in smooth muscles in which Tris, HEPES or MOPS was the sole buffering agent.

Alkanesulfonates↗
PubMed1989

Potentiation of the poliocidal effectiveness of free chlorine by a buffer.

Poliovirus 1 was inactivated by free chlorine at pH 9.0 three times more rapidly in boric acid (0.05 M)-NaOH buffer than in purified (carbon-filtered, deionized) water. Thus, at a given concentration of free chlorine, it took three times longer to inactivate the same fraction of the poliovirus in purified water than in the boric acid-NaOH buffer. Conversely, in a given period of time, three times more chlorine was required to inactivate a given percentage of the virus in purified water than in the boric acid-NaOH buffer. Buffers are almost always used to control pH in disinfection studies with free chlorine and with other chlorine compounds also. The use of buffers for pH control in such disinfection studies may distort the resulting data and, at least for waters that contain little salt, may cause serious overestimation of the rates at which viruses are inactivated.

Boric Acids↗
PubMed1992

The roles of intracellular buffers and bone mineral in the regulation of acid-base balance in mammals.

1. Regulation of intracellular and extracellular pH may conflict in their requirements for movement of acid or base. 2. Cells make a positive or a negative contribution to 'tissue buffering' of extracellular fluid (ECF), depending on their internal buffer value, on the tightness of their internal pH control by membrane mechanisms, and on the nature of the acid-base disturbance. 3. A role is suggested for electrogenic Na-HCO3 co-transport in some of the ion shifts that occur in acid-base disturbances. 4. The time course of 'tissue buffering' in nephrectomized mammals in hypercapnia is variable, and it is far from clear in intact, unanaesthetized mammals. 5. Buffering of ECF by Ca salts of bone mineral in acidosis can only be substantial if accompanied by Ca excretion; the release of HCO3 with Na and K is more significant. 6. The relative importance of cells and of bone mineral in the buffering of ECF is unclear.

Acid-Base Equilibrium↗
PubMed1991

Effects of intra-axonal injection of Ca2+ buffers on evoked release and on facilitation in the crayfish neuromuscular junction.

Ca2+ buffers were injected into the excitatory axon of the crayfish opener muscle. The magnitude and time course of evoked release and of facilitation were measured. EGTA (on-rate about 10(6) M-1S-1) had no effect on evoked release but reduced facilitation. BAPTA and nitr-5, buffers with similar Kd's but faster on-rates, reduced both evoked release and facilitation. However, these buffers had no effect on the time course of evoked release. These results show that fast Ca2+ buffers reduce the Ca2+ transient associated with evoked release and also the level of residual Ca2+ involved in facilitation. However, Ca2+ buffering is not the mechanism which controls the time course of release.

Animals↗
PubMed1996

Determination of proteins in the presence of imidazole buffers.

Imidazole buffers were found to interfere with the determination of soluble proteins using Lowry's classical method. The influence of the constituent elements of the buffer on the calibration line was studied statistically. By combining the data corresponding to different experimental sequences, interserial calibration curves for different concentrations of imidazole buffer (10-30 mM) were obtained. The absorbance-buffer volume dependence curves produced a good fit to second-order polynomials. The accuracy of protein determination in a medium with imidazole buffer, using appropriate calibration curves, were tested by comparison with the technique of multiple standard addition and by means of recovery studies. These experiments were performed on chick brain homogenate samples. Other important aspects of validation, such as sensitivity and accuracy, were also studied.

Animals↗
PubMed2005

Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds.

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.

Ampholyte Mixtures↗
PubMed2004

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer.

Oxidized Fe protein from Azotobacter vinelandii (Av2(0)) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10-40 degrees C, over the pH range 7-8, and in various buffers--inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein--Av2(0), Av2(0)(MgATP)2, and Av2(0)(MgADP)2--was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2(0) reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2(0)(MgATP)2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2(0)(MgADP)2 reduction was favored under more physiologically relevant conditions of 20 degrees C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10-40 degrees C and pH 7-8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 degrees C and pH 7.4, the rate of the dominant phase of Av2(0) reduction is 1.9 x 10(8) M(-1) s(-1). Under the same conditions, the rates of the two dominant phases of Av2(0)(MgATP)2 reduction are 1.2 x 10(6) and 1.5 x10 (5) M(-1) s(-1); and the rate of the dominant phase of Av2(0)(MgADP)2 reduction is 3.5 x 10(6) in M(-1) s(-1). Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Adenine Nucleotides↗
PubMed2006

Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors.

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.

Amino Acid Sequence↗
PubMed2005

Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLC-ESI-MS/MS.

Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis HLB Waters cartridges followed by optimised HPLC separation on an Atlantis dC18 column with acetic acid-hydroxylamine buffer (ionic strength 5mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30-10,000 ng/mL plasma concentration range. Correlation coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30-90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5-7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.

Acetic Acid↗
PubMed2006

Buffer salt effect on pH in the interior of an anion exchange resin.

The internal pH of Q Sepharose Fast Flow anion exchange resin in equilibrium with a bis-tris acetate buffer solution is investigated as a function of buffer salt concentration. Direct evidence of a resin phase pH shift is presented. At low buffer salt concentrations of 20 mM NaCl the resin phase pH is found to be as much as 1.1 pH units greater than that of the buffer phase, approaching to within 0.1 units of the buffer phase at salt concentrations greater than 250 mM. An ideal model with no adjustable parameters based on the Boltzmann distribution and the electroneutrality condition provides excellent agreement with experimental observations. The model assumes that small ions do not bind to the resin fixed charge sites and the agreement between the model predictions and observed resin internal pH suggests that strong electrolytes do not form ion pairs with the resin fixed charge sites.

Adsorption↗
PubMed2006

Evolution of vertebrate haemoglobins: Histidine side chains, specific buffer value and Bohr effect.

This review highlights the use of analytical tools, recently developed in the comparative method of evolutionary biology, for the study of haemoglobin (Hb) adaptation. It focuses on the functional consequences of a previously largely ignored structural feature of Hb, namely the degree and positional specificity of histidine (His) substitution in Hb chains. The importance of His side chains for hydrogen ion buffering, blood CO(2) transport capacity and the molecular mechanism of the Bohr effect in vertebrate Hbs is discussed. Using phylogenetically independent contrasts, a significant correlation between the specific buffer value of Hb and the number of predicted physiological buffer groups from Hb sequence data is shown. In a new result, the evolution of the number of physiological buffer groups in 77 vertebrate species is reconstructed on a phylogenetic tree. The analysis predicts that teleost fishes, passeriform birds and some snakes have independently evolved a much-reduced specific buffer value of Hb, possibly for enhancing the efficiency of an acid load to change oxygen affinity via the Bohr effect. This analysis demonstrates how in comparative physiology analysis of genetic databases in an evolutionary framework can identify candidate species for further experimental in vitro and whole animal studies.

Animals↗
PubMed2003

Saltatory and continuous calcium waves and the rapid buffering approximation.

Calcium waves propagate inside cells due to a regenerative mechanism known as calcium-induced calcium release. Buffer-mediated calcium diffusion in the cytosol plays a crucial role in the process. However, most models of calcium waves either treat buffers phenomenologically or assume that they are in equilibrium with calcium (the rapid buffering approximation). In this article we address the issue of whether this approximation provides a good description of wave propagation. We first compare the timescales present in the problem, and determine the situations in which the equilibrium hypothesis fails. We then present a series of numerical studies based on the simple fire-diffuse-fire model of wave propagation. We find that the differences between the full and reduced descriptions may lead to errors that are above experimental resolution even for relatively fast buffers in the case of saltatory waves. Conversely, in the case of continuous waves, the approximation may give accurate results even for relatively slow buffers.

Biophysics↗
PubMed1979

Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.

We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes.

Buffers↗
PubMed1986

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.

Arylsulfonates↗