Polyvalent Anions Research Articles

Determination of trace amounts of anionic impurities in hydrochloric acid by ion chromatography

The purity of the hydrochloric acid used in semiconductor processing has a direct impact on device reliability because ionic impurities adhere to the wafer's surface during processing, and the impurities must be monitored in order to avoid both unnecessary wastes and exceeding of the permitted levels. Ion-chromatographic procedures for the determination of bromide, nitrate, phosphate, selenate and arsenate in hydrochloric acid are described. The high chloride concentration and the pH of the sample cause major problems that, for monovalent anions, can be overcome by simply diluting the sample, while matrix elimination in the presence of potassium permanganate is effective in the determination of polyvalent anions. The optimum pH range, concentration of the matrix and of the eluent, and working conditions have been investigated. The use of different eluents has been suggested for the determination of bromide and nitrate after matrix dilution, as these ions elute very near to the large chloride peak. The procedures described have been successfully used for the routine analysis of hydrochloric acid batches used for cleaning semiconductors.

Ion chromatography on a new moderate capacity anion exchange column.

The mechanisms of retention of anions on a new solvent-compatible, moderate capacity (170 microequivalent per column) anion-exchange column are described. Retention based on anion exchange is shown using both sodium hydroxide and 4-cyanophenolate eluants. Plots of the log k' vs. log C, (where k' is the capacity factor and C is the eluant concentration) are shown to be linear with slopes approximating the charge of the eluant as predicted by the standard anion-exchange equation. Sodium hydroxide is shown to be a relatively weak eluant, capable of separating monovalent anions that are difficult to separate on other columns. Sodium 4-cyanophenolate is shown to be a strong eluant, capable of eluting polyvalent anions. The retention of nonionic alkylphenones is also described. In plots of log k' vs. percentage acetonitrile in the eluant, these compounds were found not to follow the solvophobic mechanism typical of reverse-phase HPLC columns. Results are compared with other anion exchange columns. Applications include the determination of lysophosphatidyl inositol in brain microsomes and methacrylic acid in esterase-treated samples.

Organic anions exhibit distinct inhibition patterns on the low-Km and high-Km transport of S-(2,4-dinitrophenyl)glutathione through the human erythrocyte membrane

The low-Km and high-Km components of S-dinitrophenyl-glutathione (DNPSG) uptake by inside-out vesicles of human erythrocytes show different pH profiles and inhibition properties with organic anions. Both components are competitively inhibited by various polyvalent anions, including glutathione conjugates, conjugated steroid hormones and bile salts, and bilirubin ditaurate. A variety of monovalent anions, including glucuronidated and sulphated drugs and taurocholate, inhibit the high-Km system only. Taurocholate is taken up by the erythrocyte vesicles in an ATP-dependent manner. The anionic dyes fluorescein, Indocyanine Green and bromosulphophthalein inhibit the low-Km system competitively and the high-Km system non-competitively. The study shows that interactions between different types of biologically occurring conjugates can occur at the level of the transport step out of erythrocytes. The kinetic properties suggest overlapping substrate specificities for the two systems, in which the low-Km component is physiologically more important for transport of glutathione conjugates and polyvalent organic anions, whereas the high-Km component is of significance for transport of monovalent organic anions. Low- and high-Km transport of DNPSG is also observed in plasma membrane vesicles from rat, pig and bovine erythrocytes.

The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.

The Resistance of Dunaliella acidophila against Heavy Metals: The Importance of the Zeta Potential

Summary The resistance of photosynthesis and growth of the acid-resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt-resistant Dunaliella parva (grown at pH 7.6) against Al(III), La(III), Cu(II), Cd(II), Hg(II) (applied as chlorides) and W(IV) (applied as Na 2 WO 4 ), and the effects of these compounds on the zeta potential of the cells were measured in glycerol media of comparable ionic strength. Dunaliella cells with a positive zeta potential ( Dunaliella acidophila , pH 1–2) are extremely resistant to toxic di- and trivalent cations, but are sensitive to toxic anions. On the other hand, cells with a negative zeta potential ( D. acidophila and D. parva , pH 7.0) are resistant to toxic anions, but sensitive to toxic cations. The non-dissociated HgCl 2 causes similar inhibitions of photosynthesis in both Dunaliella species, independent of the pH of the medium. Di- and trivalent cations cause an increase of positive zeta potentials, but a decrease of negative zeta potentials. Toxic polyvalent anions decrease positive zeta potentials, but increase negative zeta potentials. Almost no effect of HgCh on the zeta potential was observed. The results demonstrate that the zeta potential plays an important role in cation and anion toxicity. The interpretation of effects of toxic ions on growth is more difficult than the interpretation of the effects of these ions on photosynthesis, since the chemical composition and electrical properties of the culture media are very different, leading to different activity coefficients of the toxic ions as well as competition effects between non-toxic and toxic ions. This applies especially to divalent cations, whereas trivalent cations exhibited similar differential inhibition patterns for growth to those observed for photosynthesis. The actual sensitivity of growth of D. parva cells to toxic divalent cations was masked by the high salinity of the culture medium: The growth of D. acidophila e.g., was more sensitive to Cu(II) than the growth of D. parva . Mechanisms of toxicity of polyvalent cations and anions are discussed as well as the ecophysiological benefit to an acidophilic organism of high resistance against heavy metals.

Inhibition of glutathione-conjugate secretion from isolated hepatocytes by dipolar bile acids and other organic anions

The effect of a spectrum of organic compounds on the secretion of a model organic anion, dinitrophenylglutathione (GS-DNP), by hepatocytes was tested. Previous experiments have demonstrated that the secretion of GS-DNP from isolated rat hepatocytes is predominantly mediated by a canalicular transport system for this compound. Preincubation of isolated rat hepatocytes with the bile acids cholic acid (C), taurocholic acid (TC), tauroursodeoxycholic acid (TUDC) and glyco- or tauro-lithocholic acid (GLC or TLC) had no effect on the initial efflux rate of GS-DNP. In contrast, the 3-sulphates of GLC (SGLC) and TLC (STLC) did inhibit GS-DNP efflux; half-maximal inhibition with SGLC was reached with 10 microM. The 3-O-glucuronides of both cholate and lithocholate (GlucLC) were even more potent inhibitors of transport; 10 microM-GlucLC inhibited GS-DNP transport by 89%. Other cholephilic organic anions also inhibited GS-DNP secretion, albeit at higher concentrations; at 100 microM, bilirubin ditaurate, an analogue of bilirubin diglucuronide, inhibited transport by 48%. On the other hand, a number of cholephilic cationic and neutral compounds had no effect on GS-DNP efflux. The hepatobiliary secretion of oxidized glutathione (GSSG) was also investigated. In normal isolated perfused rat liver, extensive biliary secretion of GSSG was observed upon intracellular oxidation of reduced glutathione (GSH). GSSG was also actively secreted from isolated normal hepatocytes, and this secretion could be inhibited by 95% by incubation of the cells with 100 microM-SGLC. In contrast, biliary secretion was absent in the isolated perfused liver and in isolated hepatocytes from TR- mutant rats with a hereditary conjugated hyperbilirubinaemia. These results show that the canalicular efflux of GSSG and GS conjugates can be inhibited by a wide variety of polyvalent organic anions, but not by cations, neutral compounds and unianionic bile acids. This suggests that a multispecific organic-anion transporter is responsible for transport of these polyvalent anions, which is in close agreement with the fact that the biliary transport of all these compounds is defective in the mutant TR4 rat.

Effect of ATP on the activity of bovine heart mitochondrial b-c1 complex

The effect of ATP on the reductase activity of purified bovine heart b-c 1 complex was studied. ATP stimulates the steady-state activity and the antimycin-insensitive pre-steady-state reduction of b and c 1 cytochromes, also causing changes of kinetic properties of the enzyme. There is no absolute specificity for ATP since other polyvalent anions such as EDTA and EGTA produce similar effects in the micromolar range. It is proposed that ATP stimulates the activity of the b-c 1 complex, chelating inhibitory cation(s), exerting a modulatory action on the enzyme.

Anion exchange behaviour of hydrous titanium oxide and its use for column separations

Hydrous titanium oxide (HTiO), prepared under varying conditions of precipitation, has been studied for its anion exchange behaviour. Its chemical and thermal stability has been examined. The stoichiometry of uptake for Cl− has been investigated using a36Cl radiotracer and the regeneration power of the exchanger checked. Distribution coefficients for halides and polyvalent anions have been determined and on the basis of sorption data some polyvalent anions have been separated using HTiO columns.

The pho-controlled outer membrane porin PhoE does not contain specific binding sites for phosphate or polyphosphates.

Purified PhoE-porins were reconstituted into black lipid bilayer membranes, and the selectivity and size of the reconstituted pores were determined. Addition of polyphosphates influenced the internal charge situation of the pore resulting in a shift from anion to cation selectivity. However, the pore size as judged from single channel conductances was not influenced by the addition of polyphosphates. A strong inhibition of the pore conductance only occurred when Mg2+ was also present in the aqueous phase. The inhibition of the pore function is presumably caused by the formation of a chelate between the divalent cation and the polyphosphate. Nevertheless, neither this inhibition nor the selectivity shift are specific to phosphate, because both effects can be mimicked by other polyvalent anions such as citrate. Inhibition of the PhoE pore function by polyphosphate in in vivo experiments confirmed the results of in vitro experiments that polyphosphate is only able to affect the permeability of the outer membrane toward beta-lactam antibiotics if Mg2+ is present. The outcome of the in vivo and the in vitro experiments are consistent with the assumption that the PhoE-porins do not contain a specific binding site for phosphate or polyphosphates but are anion selective because of an excess of positively charged amino acids inside or at the surface of the pore.

Influence of 8-azido-ATP and other anions on the activity of cytochrome c oxidase.

The effect of ATP and other anions on the kinetics of cytochrome c oxidation by reconstituted bovine heart cytochrome c oxidase was investigated. The following results were obtained: (1) ATP and other polyvalent anions increase the Km for cytochrome c and the Vmax (if assayed by the photometric method). The magnitude of the effect is proportional to the charge of the anion as follows from the series of increasing effectiveness: Pi less than AMP less than ADP less than PPi less than ATP less than PPPi. (2) The kinetic effects are obtained in the millimolar physiological concentration range. (3) The kinetic changes are not saturated at high concentrations. (4) A specific interaction site for ATP at the cytosolic domain of the enzyme is concluded from the increase of Km for cytochrome c after photolabelling of proteoliposomes with 8-azido-[gamma-32P]-ATP, which is protected by ATP but not by ADP. (5) No specific "binding site" for ATP could be identified by photolabelling with 8-azido-[gamma-32P]-ATP. The labelling is only partly protected by ATP or ADP.

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase.

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.

Regulation of the mitochondrial outer membrane channel, VDAC.

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channel in vitro raises the possibility that VDAC may perform a regulatory role in vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.

Substrate-specific stimulation of protein kinase C by polyvalent anions

The activity of protein kinase C (PKC) toward arginine-rich substrates was greatly stimulated by sulfate and phosphate, but not by monovalent anions. This stimulation did not require phospholipid, calcium, or diacylglycerol, and appeared to mimic the stimulation by phospholipid. Anionic proteins such as bovine serum albumin also promoted PKC activity toward certain substrates that were characterized by either high arginine or high lysine content. The mechanism of both of these stimulations appeared to be related to formation of a substrate-PKC complex which is essential to phosphorylation by PKC. Polyvalent anions bind the cationic substrate and, together with PKC, form an aggregate which allows phosphorylation. Potential physiological relevance of this stimulation is discussed.

Specific effects of ATP on the kinetics of reconstituted bovine heart cytochrome- c oxidase

Bovine heart cytochrome- c oxidase was reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured by the polarographic and photometric method under uncoupled conditions in the presence of various polyvalent anions. In order to distinguish between specific and unspecific ionic effects of ATP, the photolabelling reagent 8-azido-ATP was applied. Covalently bound ATP at the enzyme complex caused the same increase of K m for cytochrome c as free ATP, if measured by the photometric assay. The increase of K m by photolabelling with 8-azido-ATP was completely prevented by ATP, but not by ADP. The data indicate the occurrence of a specific binding site for ATP at the cytosolic side of cytochrome- c oxidase, which, after binding of ATP, changes the kinetics of cytochrome c oxidation.

Studies on the equilibrium uptake of some anions on a mixed and doped hydrous oxide

A mixed hydrous Fe(III)−Zr(IV) exhibits enhanced adsorption of anions in comparison to its constituent oxides. The uptake has been observed at pH 2.0, as a function of initial salt concentration and the product shows specific affinity for Cl−, SO42− and PO43− ions. Doping the mixed oxide with Sn(II) improves its sorption capability for halide ions, while no significant enhancement is observed in the case of polyvalent anions.

Haemolytic Anaemia Associated with Increased Cation Permeability

Volume regulation in the red cell depends on the balance between the osmotic swelling pressure contributed by the polyvalent anions 2,3-DPG and haemoglobin, and the counterbalancing osmotic pressure provided by extracellular Na+. Although changes in 2,3-DPG have been identified in some cases, increases in the electrodiffusional leak of Na+ and K+ are most likely responsible for the disorders of volume regulation leading to haemolytic anaemia. The current challenge is to identify the membrane proteins and/or lipids which play a key role in passive transport of the monovalent cations.

Synthetic imogolite: properties, synthesis and possible applications

AbstractThe unique properties of imogolite are closely related to its structure, which is a tube of 23–27 Å outer diameter and ∼10 Å inner diameter, with an AIOH outer surface and SiOH inner surface. Acid dispersions contain the long, positively-charged tubes as isolated units or small bundles, which form bulky gels in alkali, and flocculate with negatively-charged colloids, polyvalent anions, and long-chain anionic detergents. Sorption properties are associated with the 10 Å intra-tube pores and with inter-tube channels of variable dimensions. Surface acidity is less than that of layer-silicate clays. The chemical and mechanical stability, biological activity, film- and fibre-forming characteristics, and conditions of synthesis are reviewed, on the basis of both new and published findings. Areas of potential application are indicated.

Studies on the Suppression of Polarographic Maximum of Mn(II) by Some Uni- and Polyvalent Anions vis-a-vis the Kinetics of the Electrode Reaction

Studies on the Suppression of Polarographic Maximum of Mn(II) by Some Uni- and Polyvalent Anions vis-a-vis the Kinetics of the Electrode Reaction

Studies on the Suppression of Polarographic Maximum of Ga(Ill) by Some Uni- and Polyvalent Anions vis-a-vis the Kinetics of the Electrode Reaction

Studies on the Suppression of Polarographic Maximum of Ga(Ill) by Some Uni- and Polyvalent Anions vis-a-vis the Kinetics of the Electrode Reaction