Ionic Strength Phosphate Buffer Research Articles

Phosphate Ions Affect the Water Structure at Functionalized Membrane Surfaces.

Antifouling surfaces improve function, efficiency, and safety in products such as water filtration membranes, marine vehicle coatings, and medical implants by resisting protein and biofilm adhesion. Understanding the role of water structure at these materials in preventing protein adhesion and biofilm formation is critical to designing more effective coatings. Such fouling experiments are typically performed under biological conditions using isotonic aqueous buffers. Previous studies have explored the structure of pure water at a few different antifouling surfaces, but the effect of electrolytes and ionic strength (I) on the water structure at antifouling surfaces is not well studied. Here sum frequency generation (SFG) spectroscopy is used to characterize the interfacial water structure at poly(ether sulfone) (PES) and two surface-modified PES films in contact with 0.01 M phosphate buffer with high and low salt (Ionic strength, I= 0.166 and 0.025 M, respectively). Unmodified PES, commonly used as a filtration membrane, and modified PES with a hydrophobic alkane (C18) and with a poly(ethylene glycol) (PEG) were used. In the low ionic strength phosphate buffer, water was strongly ordered near the surface of the PEG-modified PES film due to exclusion of phosphate ions and the creation of a surface potential resulting from charge separation between phosphate anions and sodium cations. However, in the high ionic strength phosphate buffer, the sodium and potassium chloride (138 and 3 mM, respectively) in the phosphate buffered saline screened this charge and substantially reduced water ordering. A much smaller water ordering and subsequent reduction upon salt addition was observed for the C18-modified PES, and little water structure change was seen for the unmodified PES. The large difference in water structuring with increasing ionic strength between widely used phosphate buffer and phosphate buffered saline at the PEG interface demonstrates the importance of studying antifouling coatings in the same aqueous environment for which they are designed. These results further suggest that strong long-range water structuring is limited in high ionic strength environments, such as within cells, facilitating chemical and biological reactions and processes.

Adsorption of cations onto positively charged surface mesopores

Uwe Neue developed a theoretical treatment to account for the adsorption of ions on mesopores of packing materials the walls of which are bonded to ionic ligands but left this work unfinished. We elaborated upon this treatment and refined it, based on the equivalence that he suggested between charged surface particles and a membrane that separates two ionic solutions but is impermeable to one specification. He had written that the electro-chemical potentials in both ionic solutions are equal (Donnan equilibrium). The equilibrium between the surface and the pore concentrations is accounted for by an homogeneous electrostatically modified Langmuir (EML) isotherm model. The theoretical results are presented for four different charge surface concentrations σ0=0, 0.001, 0.002, and 0.003C/m2, using a phosphate buffer (WSpH=2.65) of ionic strength I=10mM. The average pore size, the specific surface area, and the specific pore volume of the stationary phase were Dp=140Å, Sp=182m2/g, and Vp=0.70cm3/g, respectively.The theoretical results provide the quantitative difference between the ionic strength, the pH, and the concentrations of all the ions in the pores and in the bulk eluent. The theory predicts (1) that the retention times of cations under linear conditions is lower and (2) that their band widths under overloaded conditions for a given retention factor shrinks when the surface charge density σ0 is increased. These theoretical results are in good agreement with experimental results published previously and explain them.

Determination of the binding parameters for antithrombin–heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.

Specific ion binding influences on surface potential of chromium oxide

Colloidal force measurements as a function of pH can yield the isoelectric point (IEP) of a surface immersed in an electrolyte. The condition of surface charge-potential regulation imposed by the potential-dependent binding of H + and counter-ions at the interface makes a detailed analysis of the electrostatic force non-trivial. In the current study, the specific ion binding of phosphate ions on to chromium oxide has been investigated. An atomic force microscope (AFM) has been used to measure the force of interaction between a SiO 2 sphere (∼5 mm diameter) and a chromium oxide surface in aqueous media of sodium phosphate buffer or sodium chloride over the pH range 3–11. From the force separation profiles the force at `Jump To' is plotted over the pH range studied for each ionic strength. As the IEP of SiO 2 is around pH 2 the probe interaction with the surface measures its electrostatic properties, and hence can be used to determine the IEP. The comparison of force titration plots shows the IEP of the chrome surface decreases with increasing phosphate ion concentration, from around pH 8 with no phosphate ions present, down to around pH 6 at 0.01 M ionic strength phosphate buffer. This indicates that there is specific ion binding of the phosphate to the chrome oxide surface. We have used approach of DLVO theory, together with a simple model of specific adsorption of ions at the oxide–water interface, to model the long range electrostatic repulsion force measured by the force separation plots at each pH and ionic strength. By comparing this model to the isoelectric points at several ionic strengths, we can estimate surface dissociation constants for the adsorption of protons and phosphate from the electrolyte.

Determination of dissociation constants of cytokinins by capillary zone electrophoresis

A method for the p K a determination, based on mobility data measured by capillary zone electrophoresis, was applied to cytokinins and their analogs. The combination of charged mobility standards with an uncharged electroosmosis marker, injected in the uncoated capillary simultaneously with the measured substances, allows one to minimize the number of runs, reduce their duration and, in addition, to inform on the run-to-run stability of electroosmosis and on contingent side-effects. p K a values of investigated cytokinins and their analogs ranged from 2.8 to 4.0 at 25°C in the phosphate and acetate buffers of ionic strength 0.015 M. Standard deviations of the constants, obtained by the non-linear fitting of equations for the p K a calculation, were 3–5-times lower than standard deviations from the linear fitting or from the point-to-point calculation utilizing the Hendersson–Haselbalch equation. The equation of Boltzman sigmoid offers two checks on reliability of effective mobilities that serve as the raw data in the p K a calculation.

Reversed-phase high-performance liquid chromatographic separation of32P-labeled nucleotide adducts formed by food-derived carcinogenic aminoimidazoazarenes

Carcinogenic heterocyclic amines are formed on the surface of grilled or fried meat and fish. These heterocyclic amines have been shown to form the covalent DNA adducts considered to be the first stage of the process of carcinogenesis. In this paper we describe the separation of DNA adducts formed by six food-derived mutagenic aminoimidazoazarenes.32P-Postlabeling is the most sensitive technique for analysis of DNA adducts formed by covalent bonding of chemical carcinogens to DNA. An improved method developed by combining ion-exchange thin-layer chromatography with reversed-phase high-performance liquid chromatography enabled the resolution of the32P-labeled monophosphate nucleotides modified with reactive derivatives of these food mutagens. Optimum conditions for the separation were obtained by use of a Zorbax phenyl-modified silica gel column and a gradient system consisting of acetonitrile and a phosphate buffer of high ionic strength (0.5m) at pH 2.0. Sub-femtomole quantities of labeled adducts were detected with a radioactivity flow detector coupled to the HPLC equipment. This method can be used for the characterization of DNA adducts detected in human tissues and for analysis of mixtures of DNA adducts formed in experiments on combination genotoxic effects of mixtures of food-derived heterocyclic amines.

Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase.

Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4Cl as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing > 99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a novel visible region circular dichroism spectrum which has a small distinct negative band at 366 nm and a large positive ellipticity at 454 nm with a shoulder centered at 480 nm.

Large scale procedure for fractionation of albumins and globulins from pea seeds.

The buffer extractable proteins of pea, albumins and globulins, were successively extracted in a large amount at a pilot scale. In order to preserve as much as possible the native structure of proteins, a selective solubilization step procedure was performed. Firstly, albumins were extracted with acetate buffer and secondly globulins with phosphate buffer of high ionic strength. Each extract was desalted by diafiltration without protein precipitation. From 15 kg of flour, 380 g of albumin fraction and 1000 g of globulin fraction were obtained with a protein content of 86.0% and 90.7% of dry matter respectively. The characterisation of albumin and globulin fractions by electrophoresis, ultracentrifugation and anion-exchange chromatography, showed that the cross-contamination of these two fractions was minimal.

Mature Cathepsin L Is Substantially Active in the Ionic Milieu of the Extracellular Medium

The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate–4-morpholineethane sulfonic acid (MES)–Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate–MES–Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant,kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength.Kmvalues, of ca. 5 μM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20–25 μM, specifically at pH 6.5 andI= 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L,kcat/Kmdeclining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate–MES–Tris buffer. In Hanks’ balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 werekcat, 18.9 s−1;Km, 13.5 μM; andkcat/Km, 1.4 × 106M−1s−1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks’ balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).

Electrochemical oxidation and kinetics of the decay of UV-absorbing intermediate of uric acid oxidation at pyrolytic graphite electrodes

The electrochemical oxidation of uric acid has been studied in 0.50 M NaCl and phosphate buffers of ionic strength 0.05 to 0.5 M. The kinetics of the decay of UV-absorbing intermediate generated during electrooxidation of uric acid (7,9-dihydro-1H-purine-2,6,8-(3H)-trione) has also been studied. On the basis of pseudo first order rate constants observed in different media, it has been concluded that the diimine species formed from the 2e, 2H+ oxidation of uric acid is attacked by water to give diol, which then decomposes in a series of reactions to give allantoin as the major product at pH 7.2. The possibility of attack by phosphate on diimine has been ruled out.

The binding profiles of sodium dodecyl sulfate to domain sized fragments of bovine serum albumin

The binding of sodium dodecyl sulfate (SDS) to the three fragments of bovine serum albumin, corresponding to sequences 1–306, 193–368, and 383–511 in the parent protein, was studied in a phosphate buffer of ionic strength 0.014 using gel chromatography. The cooperative binding of SDS to each fragment proceeded stepwise in a manner similar to the binding to the intact protein. The saturated quantities of the SDS binding were approximately 100, 65, and 50 mol/mol for 1–306, 193–368, and 383–511, respectively. The Scatchard equation was applied to the binding isotherms at low concentrations of SDS. The intrinsic binding constants of SDS to the fragments were 2–5 × 10 3 M −1 at 25°C. The numbers of stoichiometric binding sites were 9, 4, and 3 for 1–306, 193–368, and 383–511, respectively, indicating the appearance of new binding sites through the fragmentation.

Proteins of protoplasts from red alga Porphyra yezoensis.

To elucidate the properties of protein in red alga Prophyra yezoensis, protoplasts having lesser amount of polysaccharides were used as a starting material. Protoplasts were isolated from Por-phyra by pre-treatment with protease followed by wall digesting enzyme mixture consisting of abalone enzyme and Macerozyme R-l0. Proteins in protoplasts were then extracted with low (I=0.05) and high (I=0.5) ionic strength phosphate buffer (pH 7.5) and 0.1N sodium hydroxide, and analyzed for protein composition, SDS-polyacrylamide gel electrophoretic pattern, and amino acid composition. The average content of crude protein in protoplasts was 32.7%, the amount being 5% higher than that of the thallus. The major protein in the protoplasts was water-soluble protein and it amounts to 43.2% of total nitrogen. Salt-soluble protein was a minor fraction (3.1% of total nitrogen). Electrophoretic pattern of the protein fractions consisted of many bands, and that of the water-soluble protein fraction was closely similar to that of the corresponding fractions from the thallus. In contrast, the patterns of salt-soluble and alkali-soluble protein fractions were different between protoplasts and thallus. Amino acid composition, however, was similar in all protein fractions from both protoplasts and thallus. The proteins were rich in aspartic and glutamic acids, alanine and leucine, and poor in histidine and methionine.

A study of some polypyridylruthenium(II) complexes as DNA binders and photocleavage reagents.

The nature of the binding of several ruthenium polypyridyl complexes containing 2,2'-bipyridine (bipy), 4,4'-dimethyl-2,2'-bipyridine (DMB), 1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (DPP), 2,2',2"-terpyridine (terpy), 2,2'-biquinoline (biq), 1,4,5,8-tetraazaphenanthrene (TAP) and 1,4,5,8,9,12-hexaazatriphenylene (HAT), with calf thymus DNA, poly[d(A-T)] and poly[d(G-C)] were studied by absorption and emission spectroscopy, DNA melting techniques, and emission lifetime measurements. In low ionic strength phosphate buffer, spectroscopic changes and DNA stabilization depended on the polypyridyl ligands present, and indicated binding that varied from substantially electrostatic to intercalative. Ru(bipy)2(HAT)2+ and Ru(phen)3(2+), which bind by partial intercalation, also show a strong preference for poly[d(A-T)]. The emission quantum yields for most complexes were increased in the presence of DNA. An exception was Ru(TAP)3(2+) which has a markedly reduced emission quantum yield and lifetime in the presence of poly[d(G-C)] or CT-DNA, due to photoredox interaction with quanines. Emission decays of the complexes generally showed multiexponential behaviour. The ability of the ruthenium complexes to sensitise DNA cleavage was determined using pBR322 plasmid DNA. Ru(TAP)3(2+) is the most efficient sensitiser while uncharged complexes and complexes with very short-lived excited states do not cleave DNA.

Interaction of streptococcal lipoteichoic acid with artificial tooth pellicle

Artificial pellicles were prepared by coating hydroxyapatite beads with whole saliva. Radio-labelled lipoteichoic acid was isolated from Streptococcus sanguis NCTC 7863 by phenol extraction. Various concentrations of radiolabelled lipoteichoic acid were mixed with saliva-coated hydroxyapatite in the presence and absence of high ionic strength phosphate buffer, bovine serum albumin, gelatin, unlabelled lipoteichoic acid, Tween 20 and Triton X-100. The amount of lipoteichoic acid binding was measured by counting the residual radioactivity of the saliva-coated hydroxyapatite after thorough washing. In one experiment the binding was measured in the presence of a mucinous glycoprotein isolated from human saliva. The data were analysed by means of Scatchard and double reciprocal plots of the bound and unbound fraction of lipoteichoic acid. The lipoteichoic acid interacted hydrophobically with the saliva-coated hydroxyapatite; the interaction was complex with multiple binding sites exhibiting a range of affinities. The mean association constant was 1.1 × 10 9 M −1 and the minimum number of binding sites was 3.9 × 10 12/mm 2 of artificial pellicle. The salivary mucin competitively inhibited the interaction, which suggests that this may be one of the salivary components involved.

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 °C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.

Pressure-jump kinetics of bovine beta-casein micellization.

Two samples of highly purified bovine beta-casein supplied to us by Dr. T.A.J. Payens of the National Institute for Dairy Research, Ede, The Netherlands, were studied over a range of concentration from just below the critical micelle concentration (CMC) to 0.46%, in 0.2 ionic strength phosphate buffer (pH 7.0), at 20 and 25 degrees C. The relaxation process studied by pressure jump using a 90 degree scattered light detector was also confirmed by the temperature-jump method. In the pressure-jump experiments, the process could be separated into two general time domains, with an approximate ratio of 10-25:1, a behavior reminiscent of that found for synthetic micellar systems. The faster relaxation process was still exhibited below the CMC, however. The concentration dependence of the faster relaxation time agreed very satisfactorily with predictions from the micelle model described in the companion paper.

Biphasic photolysis of riboflavin. III. Effects of ionic strength on the photolysis.

Riboflavin undergoes a characteristic biphasic photolysis with a low-intensity light source, depending on the ionic strength of the phosphate buffer used. In higher ionic strength phosphate buffer, an initial faster phase is followed by a slower second phase, and vice versa in lower ionic strength buffer. On the basis of the circular dichroism spectra, a side chain-isoalloxazine ring interaction through hydrogen bonding is considered to be responsible for the ionic strength dependence.

A ribonuclease assay: Separation of product from undegraded aminoacyl-transfer RNA substrate

A new rapid microassay for RNase is based on the extraction of the products of the degradation of aminoacyl-tRNA into ethyl acetate. The substrate, for example, f[ 3H]Met-tRNA, is not soluble in ethyl acetate but the product f[ 3H]Met-nucleotide is extracted with high efficiency over a wide range of pH in phosphate buffer of high ionic strength. The limit digest of f[ 3H]Met-tRNA by RNase T1 in which the minimum-sized product should be f[ 3H]Met-hexanucleotide is also soluble in ethyl acetate. The assay is sensitive to 0.05 pg of RNase A, has a very low background, and is linear with enzyme concentration. The most economic substrate is unfractionated tRNA containing a small concentration of the radiolabeled aminoacyl-tRNA.

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI ( M r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2 12 12 1 with cell parameters a = 57.5 A ̊ , b = 86.2 A ̊ , c = 40.3 A ̊ . Crystals of calotropin DII ( M r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters a = 135.8 A ̊ , b = 32.0 A ̊ , c = 47.7 A ̊ , β = 103.80 ° . In both cases, there is only one molecule in the asymmetric unit.

On the Perturbation of Phospholipid Bilayers by Complement: The Use of Inside-Out Vesicles from Complement-Lysed Erythrocytes for the Demonstration of Perturbating Peptide Chains

Abstract It has been postulated by different groups that late complement components may partly insert into the phospholipid bilayer of complement lysed erythrocytes and form a trans-membrane channel. The present report examines whether complement component antigens can be detected at the inner surface of complement lysed membranes by the use of inside-out vesicles. Insideout and right-side-out vesicles from osmotically or complement lysed sensitized sheep erythrocytes (EA or EAC1-9) were obtained by an incubation in a low ionic strength phosphate buffer, pH 8.0, followed by a gentle homogenization to liberate the entrapped vesicles. Inside-out and right-side-out vesicles were separated on linear gradients of Dextran T 110 by centrifugation to equilibrium. Inside-out vesicles from EAC1-9 (IO-EAC) as well as those from osmotically lysed sensitized sheep erythrocytes (IO-EA) revealed both a very low binding of 3H-Ouabain in comparison to the respective right-side-out vesicles (RO-EAC and RO-EA). The four types of vesicles were radioiodinated by lactoperoxidase and glucose oxidase.