Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles.

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pH_i, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, ‘bulk pH values’, pH_o, were measured by a combination electrode. While pH_i = pH_o for neutral, pH_i < pH_o for anionic and pH_i > pH_o for cationic liposomes prepared in 5.0 · 10^?3 M phosphate buffers. pK_a values for the ionization of pyranine were 7.22 ± 0.04 and 6.00 ± 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-d-α-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calculated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

Quantification of surface etching by common buffers and implications on the accuracy of label-free biological assays

High throughput analyses in biochemical assays are gaining popularity in the post-genomic era. Multiple label-free detection methods are especially of interest, as they allow quantitative monitoring of biomolecular interactions. It is assumed that the sensor surface is stable to the surrounding medium while the biochemical processes are taking place. Using the Interferometric Reflectance Imaging Sensor (IRIS), we found that buffers commonly used in biochemical reactions can remove silicon dioxide, a material frequently used as the solid support in the microarray industry. Here, we report 53 pm to 731 pm etching of the surface silicon oxide over a 12-h period for several different buffers, including various concentrations of SSC, SSPE, PBS, TRIS, MES, sodium phosphate, and potassium phosphate buffers, and found that PBS and MES buffers are much more benign than the others. We observe a linear dependence of the etch depth over time, and we find the etch rate of silicon dioxide in different buffers that ranges from 2.73±0.76 pm/h in 1M NaCl to 43.54±2.95 pm/h in 6×SSC. The protective effects by chemical modifications of the surface are explored. We demonstrate unaccounted glass etching leading to erroneous results with label-free detection of DNA microarrays, and offer remedies to increase the accuracy of quantitative analysis.     

Are histones the targets for flavan-3-ols (catechins) in nuclei?

Binding of flavan-3-ols to nuclei is characteristic of Tsuga canadensis (coniferous tree). This is achieved with the DMACA reagent (p-dimethylamino-cinnamaldehyde), which stains almost exclusively monomeric and oligomeric flavan-3-ols with an intense blue colour. Deep flavanol staining also occurred when calf cells of small intestine were enriched with added catechins. In order to detect the components of nuclei that may associate with catechins, the principal components of chromatin (DNA, histones) were subjected to UV-VIS spectroscopic titration. DNA or histone sulphate containing the histones H1, H2A, H2B, H3 and H4 were dissolved in cationic and anionic buffers (Tris, phosphate) at different pH values (pH 8.0, 7.4 and 7.0) and titrated with EGCG (epigallocatechin gallate) or catechin. The results show that DNA of calf thymus and the catechins investigated form no spectroscopically detectable association equilibria. However, strong association complexes are found between histone sulphate and EGCG or catechin by means of the Mauser diagrams (A and AD diagrams). The association equilibria can be accompanied by aggregation (precipitation) of histone proteins, especially initiated by EGCG. The titration equilibria are spectroscopically more pronounced in Tris buffers at higher pH values than at lower values, whereas in phosphate buffers the opposite trend is found. Surprisingly, catechin shows nearly no interactions with histone sulphate in phosphate buffers in the pH range 7.0-8.0, which is in contrast to EGCG. Fundamentally, the targets of chromosomes for catechins seem to be the histone proteins of chromatin.     

Inhibition of shoot geotropism by neutral buffers

Submerged hypocotyl sections from Helianthus have been used to test the effect of neutral buffers on shoot geotropism. When hypocotyls have been abraded, it is found that increasing the molarity (0.25 to 20 mm) of pH 6.8 K-phosphate buffer, as well as other buffering systems, results in a strong inhibition of geotropic curvature. Buffer strength has no such effect on the curvature of nonabraded segments. One possible explanation for these data is that asymmetric shoot growth following geostimulation may require the establishment of a proton gradient across the cell walls of the shoot. When neutral buffers have access to the wall space (i.e. in abraded segments), they may prevent the establishment of such a gradient.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.     

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.     

Structural plasticity of staphylococcal nuclease probed by perturbation with pressure and pH

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys-66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys-66 is located. Above 2 kbar pressure at these pH values the protein with Lys-66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys-66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys-66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure.     

The effects of pH, buffers, and fatty acid concentration on the incorporation of radioactive arachidonate into endogenous neuronal nuclear lipids

Using neuronal nuclei (N1) isolated from cerebral cortices of 15-day-old rabbits the incorporation of [3H]arachidonate into N1 lipids was followed in vitro. Arachidonate was principally incorporated into triacylglycerol and phosphatidylinositol. When low concentrations (32 mM) of Tris-HC1 (pH 7.4) were used, rates of total arachidonate incorporation were small and phosphatidylinositol received the bulk (greater than 84%) of the arachidonate. When the concentration of Tris-HC1 (pH 7.4) or, in certain cases, the concentration of arachidonate was increased, there was a rise in total arachidonate incorporation into N1, with an increasing proportion of radioactivity entering triacylglycerol until it was the predominantly labelled lipid. Using other buffers (phosphate, imidazole, HEPES, pH 7.4), the shift from phosphatidylinositol to triacylglycerol as principal labelled lipid, with buffer concentration, was not as marked as with Tris-HC1 (pH 7.4). When the buffer concentration was maintained at 107 mM and the pH was lowered to 6.5, the three amine-containing buffers showed a sizeable decline in arachidonate incorporation into N1 lipids and a corresponding decrease in triacylglycerol labelling. The proportion of the total radioactivity in N1 phosphatidylinositol rose as the pH declined. Of the buffers used, Tris-HC1 showed the greatest changes over the pH range. Based upon pK values for the amine buffers, it is suggested that an increased proportion of the protonated amine may be inhibitory to arachidonate incorporation in N1. Studies of acyl-CoA synthetase in N1 indicated this enzyme as the site of the inhibition.     

Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions.

Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative histochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean integrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium-DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose-6-phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values.     

Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.     

Stability of 6-beta-[(hexahydro-1H-azepin-l-yl)methyleneamino]-penicillanic acid in aqueous solutions]

Stability of acqueous solutions of 6-beta-[(hexahydro-IH-azepin-I-yl)methylenamino] penicillanic acid at various values of pH and temperature was studied. It was found that inactivation of the antibiotic in both the acid and the alkaline medium proceeded according to the equation of the 1st order. At pH 1.3 and a temperature of 35 degrees the half life of the antibiotic was 7 hours. The activation energy calculated according to the Arrenius equation was 13.5 kcal/mol at pH 1.3 and 22.2 kcal/mol at pH 10.5. The antibiotic was inactivated in glycol and phosphate buffers. Its qualitative analysis was performed according to an improved iodometric method.     

Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography.

A rapid method is described for the analysis of mixtures of short-chain acyl coenzyme A thioesters by reversed-phase ion-pair chromatography on LiChrosorb RP-8 and μBondapak C₁₈ columns. The technique is applicable to separation of CoASH, acetyl-CoA propionyl-CoA, and 3-hydroxy-3-methylglutaryl-CoA, as well as dicarboxylic acids and several nucleotides commonly used as cofactors for biosynthetic reactions. The method was utilized on a preparative scale for purification of 3-hydroxy-3-ethylglutaryl-CoA from CoASH and 3-hydroxy-3-ethylglutaric acid. The counterion employed was tetrabutylammonium (phosphate), pH 5.5, in various methanol:water mixtures. Elution profiles and retention values of compounds were influenced by the concentration of counterion and mass of injected sample. Tetrabutylammonium ions could be removed from effluent by ionexchange chromatography on Amberlite IR-120 resin.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Determination of biogenic amines using heterocyclic cation and aromatic anion elution

Heterocyclic cation and aromatic anion are used in the chromatographic buffers for the analysis of monoamines and diamines on a sulfonated cation-exchange column using an amino acid analyzer. All elution buffers employed for these analyses had a pH of 5.0 to maximize the ninhydrin color reaction. These methods have been successfully used for biological samples. Using a two-column (0.8 × 12 cm) system, with a buffer flow rate of 50 ml/h, analysis can be carried out in 330 min for monoamine and diamine mixtures on 0.5 to 10 nmol of each amine.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Bromophenol blue protein assay: Improvement in buffer tolerance and adaptation for the measurement of proteolytic activity

A modification of the bromophenol blue dye binding procedure of protein estimation is described. Substritution of glycine/phosphoric acid, pH 2.6, for dilute acetic acid in the colour reagent extended the applicability of the procedure to protein solutions containing buffers of various pH values. This was, however, accompained by approximately 25% loss in the sensitivity of the procedure. The mofified reagent exhibited very marked tolerance to detergents and could be successfuly adapted for the measurement of proteolytic activity in acidic, neutral or alkaline pH ranges.     

Fractional diffusion-limited component of reactions catalyzed by acetylcholinesterase

The values of kcat/Km for the reactions of four substrates, p-nitrophenyl acetate (PNPA), propionyl-beta-methylthiocholine (PrMSCh), 3,3-dimethylbutyl thioacetate (DBTA), and acetylthiocholine (AcSCh), with acetylcholinesterase were determined as a function of increasing viscosity (eta rel) in sucrose-containing and in glycerol-containing buffers. Glycerol, or possibly some contaminant of it, was found to be a nonspecific inhibitor and sucrose a nonspecific activator of the enzyme as reflected in the dependence of kcat/Km values measured for PNPA and PrMSCh upon the concentration of these reagents. The rates of reactions of these two substrates, the first neutral and the second cationic, are chemically limited rather than diffusion limited, and they thus serve as quantitative controls or internal standards to monitor the effects of the viscosogens on the enzyme, which are not related to diffusion. The additional effect on kcat/Km over the controls observed for the rapidly reacting substrates AcSCh (cationic) and DBTA (neutral) serves as a measure of the extent to which these values of kcat/Km measure diffusion-controlled processes. The reaction rate of DBTA with the enzyme is 24% diffusion controlled as measured in glycerol-containing buffers and 16-20% as determined in sucrose-containing buffers, while that for AcSCh is 100% (in glycerol) and 24-40% (in sucrose) diffusion controlled.