Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability.

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum.

The photosynthetic oxygen evolution capacity of Agmenelium quadruplication suspended in four hydrogen ion buffers (pH 7.4, 0.05 M) and its synthetic marine growth medium was measured with an oxygen electrode. High rates of oxygen evolution were obtained in the growth medium and N-tris(hydroxymethyl)-methylglycine (Tricine) buffer. Compared to oxygen evolution in the growth medium, rates in phosphate buffer and N-tris(hydroxymethyl)-2-aminoethanesulphonic acid (TES) buffer were sometimes reduced by up to 30% and rates in tris (hydroxymethyl) amino-methane (Tris) were consistently reduced by 50%. An incubation-rinsing procedure caused inhibition of oxygen evolution in TES, phosphate, and Tris by 50 to 100%. Oxygen evolution could be restored to cells rinsed in TES or phosphate by resuspension in growth medium or in buffer plus magnesium and calcium ions. Bezoquinone-supported oxygen evolution was not affected by rinsing with any buffer tested except Tris. Ferricyanide was photoreduced at a low rate by cells rinsed in Tes but at a high rate in TES plus magnesium and calcium ions. We interpreted our results to mean that, in Agmenellum quadruplicatum, inhibition of photosynthetic oxygen evolution by Tris occurs at the level of photosystem 2 while the effects of TES and phosphate are on electron-transport occurring after the rate-limiting reaction.     

Studies of glutamate dehydrogenase: analysis of functional areas and functional groups.

1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.     

Determination of indomethacin in plasma by micellar electrokinetic chromatography with UV detection for premature infants with patent ducts arteriosus

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3-10.0 microg/mL; the detection limit (signal-to-noise ratio=3; injection 0.5 psi 5s) was 0.1 microg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.     

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.     

The secretory activity of algal cells in buffered media

Summary The effect of the activity of algal cells on the buffering characteristics of suspending fluid was studied onChlorella 7-11-05 cells suspended in Tris buffer or in sodium citrate-citric acid buffer. It was observed that changes in the buffering capacity of the suspending fluid depended on the concentration of the original buffer, pH, and the nature of the buffer.In Tris buffer, which is not consumed byChlorella cells, the buffering capacity, measured in terms of the van Slyke's buffer index (), increased with time. In sodium citrate-citric acid buffer, which is used byChlorella cells in the course of their metabolism, not only the buffer index () changed, but also the pH at which the suspending fluid had maximum buffering capacity shifted to a new value. In both Tris and citrate buffers the newly formed buffer systems had a maximum buffering capacity at pH 6.4 corresponding to the pK₁ of carbonic acid.This work was supported by funds from the National Aeronautics and Space Administration.Scientific Article A 1237, Contribution No. 3742 of the University of Maryland Agricultural Experiment Station.     

Influence of iron on outer membrane components ofDesulfovibrio vulgaris

The outer membranes (OM) ofDesulfovibrio vulgaris were isolated from cells grown in either iron-rich or iron-free medium by the Sarkosyl method. OMs from iron-free cells had a greatly simplified band pattern on PAGE, but retained the lipopolysaccharide and three major polypeptides (OMPs 1, 2, and 3) seen in OMs from iron-rich cells. These OMPs may be involved in iron uptake by the bacteria. Incubation of iron-free cells with ferrous sulfate prior to OM purification resulted in the increased retention of some bands normally seen only in iron-rich OM, indicating that iron may protect these OM proteins from Sarkosyl treatment. Iron is thus an important element in the stabilization of theD. vulgaris OM     

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

The effect of pH on the production of pertussis toxin by Bordetella pertussis.

The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields.     

New buffers to improve the quantitative real-time polymerase chain reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

New Buffers to Improve the Quantitative Real-Time Polymerase Chain Reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

Optimal isolation conditions for rainbow trout (Salmo gairdneri) hepatic ribosomes

1. Isolation conditions for rainbow trout (Salmo gairdneri) liver ribosomes were optimized. 2. Optimal initial buffer (Buffer I) concentrations were 250 mM sucrose, 50 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 5 mM MgSO4.7H2O. 3. Optimal concentrations for post-105 supernatant buffer (Buffer III) were 25 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 8 mM MgSO4.7H2O.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

Optimized dipicolinate activation for viable counting of Bacillus stearothermophilus spores

Spores of Bacillus stearothermophilus were exposed to calcium and sodium salts of dipicolinic acid (DPA) in phosphate and Tris acid maleate buffers over the range pH 4.5–10.0. The exposed spores were enumerated using a standard plate counting technique from which the kinetics of colony formation were determined and maximum colony counts were obtained for each condition examined. Exposure of the spores to calcium-DPA (50-40 mmol/l) in Tris acid maleate buffer pH 9.0 maintained at 10°C was found to produce an optimal response. Following this method the total viable population of a spore suspension was enumerated. This was demonstrated statistically using the Wilcoxon rank-sum test for significance. Calcium-DPA was found to produce activation in spores but further germinants and nutrients were required for colony formation. The Ca-DPA treatment was found to be effective in enumerating both naturally dormant spores and heat injured spores.     

Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: study of long-range stability

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The stability of this hexagonal lattice structure in long-term incubation at 4 C was investigated. The hexagonal lattice structure was stable for at least 220 days when the LPS was suspended in distilled water, but it had been disintegrated into a rough mesh-like structure when the LPS was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.5, at 4 C for 60 days. Half of the Mg bound to the LPS was released when the LPS was suspended in Tris buffer for 60 days, whereas Mg was not released when it was suspended in distilled water even for 220 days. By contrast, it was stable for at least 220 days in Tris buffer containing 5 mM MgCl2. The LPS suspended in Tris buffer for 60 days, at which time the structure had been disintegrated, could be restored to the original hexagonal lattice structure within 24 hr by addition of 5 mM MgCl2. From these results it is concluded that the hexagonal lattice structure of the LPS retains long-range stability if Mg bound to the LPS is not released from the LPS.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

Possible Relationship Between Yeast Deoxyribonucleases and Deoxyribonucleic Acid Yield

The deoxyribonucleic acid (DNA) yield and deoxyribonuclease (DNase) activity of several yeasts were correlated. Debaryomyces castellii and Debaryomyces franciscae were found to contain active DNases which carry out DNA hydrolysis, whereas the amounts of DNA as determined by extraction with Sarkosyl buffer (pH 7.8) were found to be small. On the other hand, Candida parapsilosis, Saccharomyces carmosousae, and Lodderomyces elongisporus produced no detectable DNases active at pH 7.8, and their DNA yield was correspondingly high. L. elongisporus was found to possess DNase only at pH 4.0.