Ionic Strenght Research Articles

Ion Exchange High Performance Liquid Chromatography of Leukotrienes

Abstract A novel anion exchange liquid chromatographic system has been developed for isocratic separation of leukotrienes. Hydrophobic as well as ionic forces were found to influence the separation. By optimization of solvent strenght, ionic strenght and pH, amphoteric peptidoleukotrienes could be separated simultaneously with hydroxy fatty acids such as leukotriene B4 and its ω-oxidized metabolites. To obtain a good buffering capacity of the mobile phase at optimum pH, a multicomponent buffer was developed.

Restriction enzyme accessibility and RNA polymerase localization on transcriptionally active SV40 minichromosomes isolated late in infection

The transcriptionally active SV40 minichromosomes isolated late in infection contain a nucleosome-free ORI region or gap. To analyze the chromatin structure of this subpopulation of minichromosomes extracted at different ionic strenghts in the early and late coding regions, minichromosomes were isolated in the presence of a 5, 50, or 130 m M concentration of monovalent cations and subjected to in vitro RNA elongation in either the presence or the absence of high salt and anionic detergent. The minichromosomes isolated at low ionic strength were transcriptionally more active than those isolated at physiological ionic strength. Nevertheless, in each case, the in vitro elongation complexes were present essentially on the late strand of the SV40 genome and localized preferentially in the late and 3′ early coding regions. These regions were transcribed similarly in either the presence or the absence of chromatin denaturing agents. In contrast, the in vitro elongation activity of the RNA polymerase molecules present on the late strand in the middle and 5′ end of the early coding region was inhibited in the absence of treatments to disrupt chromatin structure. In addition, as probed by restriction enzyme digestion, the ORI and late coding regions of the transcriptionally active minichromosomes were found to be more sensitive than the 5′ region of the early genes. Taken together, these results suggest that the 5′ and middle regions of the early genes of the SV40 transcriptional complexes isolated late in infection at low or physiological ionic strenght are packaged in a more compact conformation than the rest of the genome.

RNA polymerase activity in PtK1 micronuclei containing individual chromosomes: An in vitro and in situ study

Micronuclei have been induced by colchicine in rat kangaroo ( Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strenghts with or without inhibitors, and in micronucleated cells by radioautography after [ 3H]uridine pulse labelling. In vitro transcription shows that isolated micronuclei are able to take up [ 3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65–70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [ 3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characterisitic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

Concentration and recovery dependence of hyperfiltration system rejection of electrolytes by ion exchange membranes

The hyperfiltration rejection of an electrolyte by a membrane containing a significant fixed charge concentration is a strong function of the ionic strenght, making the hyperfiltration system rejection of an electrolyte dependent upon feed concentration as well as recovery. An approximate method for relating the system rejection to zero-recovery hyperfiltration parameters, applicable over a broad range of concentrations and recoveries, is described and applied to a commercial high recovery hyperfiltration system. Although concentration polarization and velocity effects are not included in this simplified model, it appears to be useful in both the design and performance analysis of high recovery hyperfiltration systems containing ion exchange membranes.

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCI, 0.2 mM ATP and 10 mM Tris-HCI buffer at pH 8.0 and 25°C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg 2+-enhanced ATPase activity of myosin at low ionic strenght. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.

Effects of Different Solvent Characteristics on the Proton-Ligand and Ni2+-, Sm3+-, AND Yb3+- Ligand Formation Constants of 5,5-Dimethyl-Cyclohexane-2-(2-Hydroxyphenyl)Hydrazono- 1,3-Dione (DCPHD) in Various Mixed Aqueous Solvents-(II)

Complex equilibria of DCPHD with proton and Ni2+, Sm3+, and Yb3+ ions has been measured in various mixed aqueous solvents, viz: isopropanol-water, acetone-water, ethanol-water, and methanol-water. Based on potentiometric equilibrium measurements of hydrogen ion concentration at 30°C, ionic strenght 0.10 M (KNO3) and in the above various mixed aqueous solvents, the values of the protonation constants of DCPHD and the stability constants of DCPHD with Ni2+, Sm3+, and Yb3+ have been evaluated. The variation of protonation and stability constants with the inverse of dielectric constant or mole fraction of the solvent was studied. Application of Fuoss expression and consideration of electrosatic and non-electrostatic effects are made to explain the values of the constants.

Assay and characterization of IgG binding to endodermal cells of the fetal rabbit yolk sac membrane

The transfer of passive immunity in the rabbit is mediated by the fetal yolk sac membrane (YSM) and is initiated by the specific binding of IgG to receptors on the microvillar surface of the endoderm from the YSM. This report describes the preparation of suspensions of endodermal absorptive cells of the YSM and their use in equilibrium binding experiments to characterize the nature of the binding reaction. Equilibrium binding is achieved in 4 h at 4°C. The system is more rapid than, affords greater reproducibility of binding data than, and utilizes only about 1 10 the amount YSM and ligand as the YSM disc assay system (Tsay and Schlamowitz, 1975) used previously. A Scatchard plot of the binding data over a wide range of IgG concentration was non-linear implying the presence of at least 2 binding elements. Apparent binding constant values for the stronger and weaker binding components in this population differed by about 50-fold. For the weaker binding system, binding decreased when temperature was increased indicating that the reaction was not entropy-driven (i.e., dominated by hydrophobic ‘forces’) and that ionic interactions might be a major factor. At low ionic strenghts the measurement of specific binding was complicated by the effects of secondary ionic interactions. At physiological ionic strength the binding of IgG was species-specific.

Dansyl Amino-Acids Behavior on a Radial Pak C18 Column. Derivatization of Grape Wine Musts, Wines, and Wine Vinegars

Abstract Investigation was carried out into the retention behaviour of dansyl amino acids with the concentration of the organic modifier, the pH and the ionic strenght of the eluting buffer. The quantitative result precision obtained using gradients of polarity and the stability of dansyl amino acids have been calculated. Finally, the dansylation conditions of grape wine musts, wines and wine vinegars are fixed.

Metal-mediated reduction of cytochrome c by thioglycolic acid

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible in comparison with rates obtained from reactions supplemented with catalytic amounts of copper or iron. Both the catalyzed and uncatalyzed reactions were independent of pH (near neutrality) but when o-phenanthroline was included in the reaction mixture, a pH dependence was induced. This pH dependence is the result of an interference of oxygen with the metal complexes. A comparison of the rate constants at zero ionic strenght, which were obtained from the application of the Debye-Hückel theory for the ionic strength dependence, demonstrated that copper complexes are superior catalysts as compared with iron complexes. Our results suggest that in the copper-mediated reaction, the catalyst is a cupric thioglycolate complex with a net charge of −2. The addition of o-phenanthroline to the reaction mixture results in a tenfold decrease in the catalytic activity and in a change in the net charge of the catalyst to −1. At pH 8 the iron-mediated reduction is catalyzed by a ferric thioglycolate complex, whereas at pH 7 a ferrothioglycolate complex provides the catalytic activity. Both complexes have a net negative charge of −2. At both pH's the catalytic activity is completely abolished by the addition of o-phenanthroline. The results demonstrate the effectiveness by which metal-sulfur complexes can facilitate one-electron transfer reactions and could there-fore serve as a model in the study of various biological oxidations.

Rotational Motion of Actin Monomer at Low and High Salt Concentration

Treatment of F‐actin pellet with 1 M MgCl2 results in a high concentration of monomer actin in solution [Biró, E. N. A. and Venyaminov, S. Yu. (1979) Acta Biochim. Biophys. Acad. Sci. Hung. 14, 31–42]. The physico‐chemical properties of this actin solution were studied by the spin‐labelling method and were compared with the normal actin solutions. The electron paramagnetic resonance (EPR) spectra produced the following results. The rotational correlation time for monomer actin at low ionic strenght, which was calculated by the extrapolation procedure, increased from 16 ns to 26 ns in the presence of 1 M MgCl2. The polymerization of actin is accompanied by a change in the polarity of the environment of the label (increasing hydrophobicity). When heavy meromyosin (HMM) was added to monomer actin at high ionic strengh, the EPR data provided evidence that HMM induced the polymerization of the MgCl2‐depolymerized actin.

Stability and structure of complexes of transition metal ions with nucleotides and related compounds

Potentiometric, spectrophotometric and kinetic techniques have been used to determine the stabilities of complexes of transition metal ions with phosphoric acid and ribose-phosphate, purine nucleosides and nucleotides [1–4]. At ionic strenght 0.1 M, dinegative phosphate groups bind to Ni 2+ and Co 2+ with stability constants close to 100 M −1 [1, 5]. The neutral purine nucleosides form only weak complexes; the binding constants depend markedly on the base involved, e.g. K = 14 for Ni 2+-inosine, and K = 2 M −1 for Ni 2+-adenosine [3]. The data are consistent with assumption that the N7 atom of the imidazole ring is the predominant binding site. Similar differences are observed also for the complex stabilities of the nucleotides: K(NiIMP) = 920 M −1, and K(Niz.sbnd;AMP)=300 [3]. The experimental overall stabilities of the nucleotide complexes can be rationalized only by assuming a chelate structure, with the metal ion being to the phosphate group and to the base. Space-filling models indicate that in the nucleotide complexes only the N7 atom an act as the binding site of the base. The kinetic data, too, can be interpreted only by a stepwise chelate formation process. Moreover, the kinetic data enable also the evaluation of the stepwise equilibria. In the case of NiAMP, about 2 3 of the complexes are present in the chelate form, 1 3 in the monodentate form. Complex formation of Ni 2+ with the dinucleoside-monophosphate ApA − is weaker (K = 2.6 M −1) than with AMPH − (K = 11) [14], despite the availability of an additional adenosine group in ApA −. This observation is attributed to the conformational properties of ApA − in solution. the ligand prefers a conformation in which the two adenines are in a stacked position. In this conformation the N7 atoms are far away from the phosphate group, and the metal ion can bind either to the phosphate or to the base, but not simultaneously to both [4]. Strong interactions were observed between Ni 2+ ions and the polynucleotide poly(A) [4]. Quantitative data were obtained by using murexide as an indicator for the concentration of free metal ions. The difference from the total metal concentration gives the amount of Ni 2+ bound to poly(A). This technique is applicable for free Ni 2+ concentrations 10 −5–10 −3 M. At the upper limit, 1 Ni 2+ is bound per 4.4 mononucleotide units (ionic strenght 0.1 M). A Scatchard plot yields a straight line, i.e. the process can formally be described as the binding of Ni 2+ to one class of independent binding sites at poly(A). Slope and intercept of the plot yield a value of 0.26 for the number of binding sites per monomer ( i.e. 4 monomers form one binding site), and a stability constants K = 8.2 × 10 3 M −1 for the binding of Ni 2+ to these binding sites. In kinetic studies of this system two reaction effects were detected. The kinetic data can be rationalized in terms of a mechanism which involves a second-order (outer-sphere) association process followed by 3 first-order steps (inner-sphere binding, probably to two phosphates and one base [6]). The high overall stability constant is mainly due to strong outer-sphere association (high charge density at poly(A)).

Novel mechanistic aspects of the reduction of iron(III) phenanthroline complexes by aquo iron(II). Temperature dependence of the substituent effect

There is strong evidence that the reduction of Fe(phen) 3+ 3 by Fe 2+ aq occurs via an inner-sphere path mediated by water molecules coordinated at the carbon atom adjacent to the ring nitrogen of the phenanthroline ligands. Water thus acts as a catalyst. The rate constant measured is related to the product Kk 2 of the equilibrium constant of the covalent hydration of Fe(phen) 3+ 3 and the rate constant of subsequent bridge formation to give (Fe(phen) 3·H 2O· Fe aq 5+. The two reaction steps are inversely influenced by the electronic effect of substituents at the phenantroline (k and k 2 increase and decrease, respectively, with acceptor substituents). These opposing effects involved can readily be invoked to account for the non-linear Hammett plot and further for the occurrence of anti-compensation of the experimental activation parameters. There is a dominant influence of the subtituents on the pre-equilibrium since the net rates are enhanced with acceptor substituents. The reaction mechanism proposed brings the following features under one roof: dependence of rate on acid and ionic strenght and substituents, the non-linear Hammett plot, the pattern in activation parameters and the anti-compensation effect.

OH Radical‐induced chemical reactions of polynucleotide complexes

AbstractPoly(A + U), the 1:1 complex of poly(riboadenylic acid), poly A, and poly(ribouridylic acid), poly U, at pH 8 and self‐complexed poly A at pH 4 exist as double helices in dilute aqueous solution. These complexes exhibit a similar behavior as native calf thymus DNA upon irradiation with 16 MeV electron pulses. Thus time resolved Rayleight light scattering measurements showed that crosslinking and double strand breakage can be clearly separated, the former proceeding faster than the latter. The extent to which the two processes occur depends on the ionic strength of the solution. At ionic strenghts exceeding 10 −1 mol/l crosslinking is the dominant process indicating that hcrit, the critical length between two single strand breaks for the accomplishment of double strand breaks, is strongly reduced. The investigation of complexes of poly A and Mg2+ ions revealed that the destruction of salt bridges is the rate determining process for the decrease of the light scattering intensity due to mainchain scission. This implies that life‐times of salt bridges can be determined.

Surface behaviour of γ-FeOOH: Point of zero charge and specific ionic interactions

The acid-base dissociation behaviour of the surface of γ-FeOOH is investigated by potentiometric acid-base titrations of the oxide suspensions in aqueous solutions. The zero point of charge (p.z.c.) of the electrical double layer of the reversible oxide solution interface is determined: in KNO 3 titrations at various ionic strenghts cross over at pH = 7.6; by coincidence with the pH of the isoelectric point, determined by microelectrophoresis, the cross over of titrations in KNO 3 is identified as the point of zero charge of the surface. Specific effects regarding monovalent anions (Cl −, ClO 4 −) are found to be absent, while SO 4 −− strongly adsorbs on the γ-FeOOH surface, thus modifying the balance of the positive and negative groups on the surface. The sequence of interaction of monovalent cations with the surface is reported: K + < Na + < Li +. Surprising and of particular interest are the strong adsorption of Na +, which has always been reported to interact only electrostatically with oxide surfaces, and the absence of specific interactions with Cl − ions.

Binding of lead cations to oligogalacturonic acids

The activity of Pb2+ ions in solutions of lead oligogalacturonates of polymerization degree DP 1 to 5 (starting concentration of the investigated solutions 3.00 mmol (COOPb0.5) l-1, the ionic strenght of the solution I = 0.01 mol l-1, electrolyte KNO3, was determined by lead specific ion electrodes. The activity aPb2+ is independent on the kind of anion in very diluted solutions of lead perchlorate and nitrate. Ions of lead become bound quite firmly even to the monomeric D-galacturonic acid (association degree of Pb2+ ions to carboxyl groups of uronic acid β = 0.43). The increase of polymerization degree is associated with a strong decrease of the lead oligogalacturonate solubilities on one hand, and increase of the bond strenght of the Pb2+ ions to these oligomers on the other. The association degree of Pb2+ ions to carboxyl groups of the pentamer (β = 0.96) almost reaches the value found for the polymeric chain of pectate molecule (D-galacturonan); here, the stoichiometric binding of Pb2+ to two carboxyl groups takes place with a great deal of probability. Lead has been found to bind highly preferentially to oligogalacturonates even in the presence of calcium ions. Results presented in this paper help to clear the mechanism of elimination of lead from the human body through pectin and its oligomeric fragments.

Structural changes in bacteriorhodopsin induced by electric impulses

Electric impulses of high field intensity (2 × 10 5 to 3 × 10 6 Vm −1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 m m NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10 −30 Fm 2 (2.2 × 10 −14 cm 3); the electric permanent dipole moment which is involved amounts to 4.7 × 10 −28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.

Substituent effect of chelated cobalt II : Saponification of m- and p-carbomethoxyphenyl (ligand) cobaloximes

Binding constants for eight axial ligands, including four primary amines, and CN, SCW, N02, and N3, to m-and p- carbomethoxyphenyl (aquo)cobaloximes have been determined in aqueous solution at 25.0 C, ionic strenght 1.0 M. These constants have been used to establish experimental conditions for the measurement of the second-order rate constants for base-catalyzed hydrolysis of these cobaloxime substituted methyl benzoates. These rate constants have been correlated via the Taft dual substituent parameter equation to provide values for the inductive and resonance substituent constants for the ehelated substituents -Co(D2H2)L. The results show that substantial resonance interaction can occur between these substituents and carbomethoxyphenyl-organic ligands when L is an unsaturated inorganic ligand capable of ir-interactions with the cobalt atom. Inductive electron donation by the substituent -Co(D2H2)L is found to increase linearly with proton basicity of L for all the inorganic ligands save on, while an unexpected inverse dependence is seen for L= RNH2.

The redox reactions of complex cobalt(II) cyanides. XI. The kinetics and mechanism of the redox addition reaction of pentacyanocobaltate(II) with 1,4-naphthoquinone in aqueous solution

the following reaction scheme was proposed on the basis of kinetic measurements on the reaction system of Co(CN) 3− 5 −1,4-naphthoquinone (NQ), whose behaviour was studied in dependence on the ionic strenght and by finding the reaction orders with respect to the reactants. ▪ ▪ ▪ ▪ products (P) K 3 It then holds for the rate of decrease in the amount of pentacyanocobaltate and for the rate of formation of products (P) in system with a large excess of Co(CN) 3− 5 that −d[Co(CN) 3− 5]/dt = 2d[P]/dt = = 2k 1k 2[Co(CN) 3− 5] 2·[NQ]/k −1 = k 2[Co(CN) 3− 5]) The derived rate relationship was confirmed experimentally and the rate constant k 1 (9.08 × 10 4 M −1 s −1) was found. The proposed mechanism is further confirmed by the dependence of the experimentally determined overall equilibrium constant K = [P eq{[C0(CN) 3− 5] 2 eq·([NQ] o −2[P]q) 2} on the ionic strength. The dependence of the logarithm of constant K on √I(1 + √I) yields a straight line with slope 9.1. The product of this reaction, which are quite unstable and thus cannot be analysed directly, have an instense absorption band at 500 nm. Differences between the mechanism of the reaction of Co(CN 3− 5] with 1,4-napthoquinone and the mechanism of the reaction with 1,4-benzoquinone are discussed.

Ionic strength effects on the reduction of CoEDTA − by Fe(CN) 4−6

The effect of ionic strength on the reduction of CoEDTA − by Fe(CN) 4− 6 is reported. The results are in direct contrast to expected behavior in that the rate constant initially increased, reaches a maximum at ionic strength 0.11 M, and then begins to decrease. The results are discussed in terms of ion-pairing between Na + and Fe(CN) 4− 6 with the proposal that the observed decrease can be attributed to the increasing presence of the unreactive ion-triplet Na 2Fe(CN) 2− 6 at higher ionic strenghts. The effect of the cyclic polyether 18-crown-6 is reported.

Characterization of adenosinetriphosphatase of squid actomyosin.

Adenosinetriphosphatase (ATPase) activity of actomyosin of the obliquely striated mantle muscle of squid was studied to confirm and supplement the previous work. ATPase activity was assayed at ionic strength of 0.15 or 0.6, Tris-maleate buffer 30 mM (pH 7.0-7.5), protein 0.1mg/ml, ATP 3.2 mM, CaCl2, MgCl2 or EDTA 3.2 mM, and 25°C; for 10 min. Effects of Ca2+ and Mg2+ were tested in the function of temperature. Both Ca2+ and Mg2+ revealed marked activation at eithe ionic strength of 0.15 or 0.6, but EDTA none. At ionic strength 0.15, Ca2+-ATPase showed a steep peak at 30°C, while Mg2+-ATPase demonstrated a gradual peak with the top at 35°C. At ionic strenght of 0.6, the macimum of the former appeared at 25°C and that of the latter at 40°C. Mg2+ ion seemed to stabilize the ATPase. When the activety was assayed at various ratios of actomyosin and ATP, the full activity levels were attained below the ratio of 0.1-0.2 mg/ml protein per 3 mM ATP. Addition of Mg2+ to Ca2+-activated ATPase, reduced the activity to the level activated with Mg2+ alone, while addition of Ca2+ to Mg2+-activated ATPase, did not alter the activity. Effects of Mg2+ appeared to overcome that of Ca2+. During 60 min incubation of enzymic reaction mixture at 25°C prior to adding ATP, the activity was reduced appreciably. This seemed to satand with the so far reported lability of the squid actomyosin which was illustrated in changes of several physiconchemical properties.