Preliminary electrochemiluminescence studies of metal ion–bacterial diazoluminomelanin (DALM) interactions

Electrochemiluminescence (ECL) studies of the chemiluminescent (CL) polymer diazoluminomelanin (DALM) biosynthesized in nitrate reductase transfected Escherichia coli JM109 bacteria revealed noteworthy anodic ECL and even more intense cathodic ECL. Bacterial DALM (BD) ECL was also assessed in the presence of 100 ppm of 33 different metal and non-metal ions which revealed specific anodic, but not cathodic, enhancements of BD ECL with Ag⁺, Hg²⁺ and Ru³⁺. The precursors and intermediate polymers which comprise DALM, such as luminol, 3-amino-L -tyrosine (3-AT), aminomelanin (AM) and diazomelanin (DM) were screened for ECL enhancement against the same set of elemental ions. Significant anodic ECL enhancements were observed for luminol with Hg²⁺ in the presence of tripropylamine (TPA), but not for any other DALM component in combination with other elemental ions, either anodically or cathodically. Comparison of BD with luminol in the presence and absence of TPA and Hg²⁺ revealed very different ECL activity patterns and suggested different mechanisms for BD and luminol ECL. © 1998 John Wiley & Sons, Ltd.     

Further characterization of tunicate and tunichrome electrochemiluminescence

Electrophoresis, size exclusion chromatography, fluorescence, and electrochemiluminescence (ECL) data obtained from the cell-rich perivisceral fluid (‘blood’) of the tunicate, Molgula occidentalis, suggests that a yellowish protein or protein subunit of <6.5 kDa is probably responsible for the low level intrinsic ECL reported previously. Variable potential ECL scans and spectrofluorometric analyses directly indicated that chlorophylls from ingested phytoplankton were not significant contaminants of M. occidentalis blood samples. Chlorophylls were also examined indirectly from monocultures of dinoflagellate and diatom species to determine their ECL levels. While ECL was observed in these cultures, high concentrations (10⁶ organisms/mL) of dinoflagellates or diatoms were needed to produce ECL levels comparable to those observed for M. occidentalis blood. Additional work with two synthetic tunichrome isomers indicated 10-fold increases in ECL when the ‘2,3,4-tunichrome’ was reacted with Tl⁺ in 1:1 and 1:2 metal ion to ligand molar ratios. The ‘3,4,5-tunichrome’ isomer exhibited a titration curve with Hg²⁺ suggestive of the existence of at least two Hg²⁺ binding sites and a 9- to 10-fold increase in ECL output. © 1997 John Wiley & Sons, Ltd.     

Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart

The metal activator sites of NAD⁺-dependent and NADP⁺-dependent isocitrate dehydrogenases from pig heart have been probed using ¹¹³Cd- and ²⁵Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed ( −8 ppm) arising from exchange with isocitrate (−20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. ²⁵Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10 200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex. indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. ¹¹³Cd- and ²⁵Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. ²⁵Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.     

Magnetic resonance studies of the interaction of divalent metal cations with 2,3-bisphosphoglycerate

The binding of Mg²⁺ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg²⁺ and Mn²⁺ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (K_D = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures.     

Electrogenerated chemiluminescence properties of bisalicylideneethylenediamino (salen) metal complexes

The spectroscopy, electrochemistry and electrogenerated chemiluminescence (ECL) of eight bisalicylideneethylenediamino (salen) metal complexes are reported. Two of the complexes contain an unsubstituted salen ligand and either cobalt(II) or nickel(II). The others have 1,2-cyclohexanediamonio-N,N′-bis(3,5-di-t-butylsalicylidene) as the ligand, and chromium(III), aluminum(III), cobalt(II), cobalt(III) or manganese(II) as the metal center. The complexes have lowest energy absorption maxima between 350 and 430 nm. When excited at these wavelengths, the complexes emit between 417 and 594 nm in acetonitrile. Photoluminescence efficiencies (?_em) were between 0.0310 and 23.8 compared to Ru(bpy)₃²⁺ (bpy = 2,2′-bipyridine; ?_em = 1), with the aluminum complexes displaying the most intense photoluminescence. Both reversible and irreversible oxidative electrochemistry is displayed by the metal–salen complexes with oxidation potentials ranging between +0.152 and +1.661 V versus Ag/AgCl. The ECL intensity peaks at a potential corresponding to oxidation of both TPrA and the salen systems, indicating that both are involved in the ECL reaction sequence. ECL efficiencies (?_ecl) were between 0.0018 and 0.0086 when compared to Ru(bpy)₃²⁺ (?_ecl = 1) in acetonitrile (0.05 M tri-n-propylamine (TPrA) as an oxidative–reductive ECL coreactant). Also, qualitative studies using transmission filters suggest that the complexes emit ECL in approximately the same region as their photoluminescence, indicating that the same excited state is formed in both experiments.     

Torpedo californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4D motif

Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca⁺², Mg⁺², and Mn⁺² was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high‐affinity binding site for Mn⁺² and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca⁺² and Mg⁺² complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.     

Application of microcolumn ion chromatography using anion exchangers modified with dextran sulfate for the determination of alkali and alkaline-earth metal ions

Microcolumn ion chromatography using anion exchangers modified with dextran sulfate has been applied to the determination of alkali and alkaline-earth metal ions contained in guinea pig serum and bovine serum. These serums contained Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ and they were indirectly detected at 200 nm. The determination was done without any pretreatment procedure other than dilution.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

“Off-On”switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology

A novel “off-On” electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy)₃²⁺)/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the “molecular recognition” strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy)₃²⁺. Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy)₃²⁺, which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective “off-On” ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed.     

THE ESSENTIAL ROLE OF CALCIUM ION IN POLLEN GERMINATION AND POLLEN TUBE GROWTH

Brewbaker, James L., and Beyoung H. Kwack. (U. Hawaii, Honolulu.) The essential role of calcium ion in pollen germination and pollen tube growth. Amer. Jour. Bot. 50(9): 859–865. Illus. 1963.—A pollen population effect occurs whenever pollen grains are grown in vitro. Small pollen populations germinate and grow poorly if at all, under conditions which support excellent growth of large pollen populations. The pollen population effect is overcome completely by a growth factor obtained in water extracts of many plant tissues. This factor is shown to be the calcium ion, and its action confirmed in 86 species representing 39 plant families. Other ions (K⁺, Mg⁺⁺, Na⁺) serve in supporting roles to the uptake or binding of calcium. The high requirement of calcium (300–5000 ppm, as Ca (NO₃)₂·4H₂O, for optimum growth) and low calcium content of most pollen may conspire to give calcium a governing role in the growth of pollen tubes both in vitro and in situ. It is suspected that ramifications of this role extend to the self-incompatibilities of plants and to the curious types of arrested tube growth distinguishing, for example, the orchids. A culture medium which proved its merit in a wide variety of pollen growth studies included, in distilled water, 10% sucrose, 100 ppm H₃BO₃, 300 ppm Ca (NO₃)₂·4H₂O, 200 ppm MgSO₄·7H₂O and 100 ppm KNO₃.     

Biosorption of silver ions by processed Aspergillus niger biomass

Summary An alkali treated A. niger biomass was found to efficiently sequester silver ions from dilute as well as concentrated solutions (2.5–1000 ppm Ag⁺), with an ability to bind it to a level of upto 10% of dry weight. Biosorption of silver ions was not influenced by pH between 5–7. The bound Ag⁺ could be fully desorbed by dilute HNO₃ and the biosorbent regenerated by washing with Ca²⁺/Mg²⁺ solution. This biosorbent is unique in that the mechanism of metal ion sorption has been found to be exclusively by stoichiometric exchange with Ca²⁺ and Mg²⁺ of the biosorbent.     

Distance measurements between the metal-binding sites in thermolysin using terbium ion as a fluorescent probe.

A single terbium ion has been introduced into thermolysin replacing two of the four calcium ions, and the fluorescence properties of the protein-bound terbium have been studied. The fluorescence of Tb⁺³ is tremendously enhanced (～7 × 10³) upon binding and is significantly quenched when divalent cobalt is substituted for the zinc ion normally found in the enzyme. By use of the Förster equation for energy transfer the distance between the protein-bound Tb⁺³ and Co⁺² in the active site was calculated to be 13.6±0.5 A. This agrees closely with the value of 13.9 A obtained from the crystal structure and suggests that energy transfer between the two metal ions bound to the protein takes place by a dipole-dipole mechanism.     

Preliminary results on the photoluminescence and optically stimulated luminescence in Cu‐doped and Ag‐doped ZnB2X4 (B = Li,Na, K: X = Cl,Br) compounds

Photoluminescence, and optically stimulated luminescence in ZnB₂X₄ (B; Li,Na,K: X; Cl,Br) compounds doped with Cu⁺ or Ag⁺ were studied. Double humped emission bands attributable to the activators were observed in all the samples. The observed photoluminescence of Cu⁺ and Ag⁺ could be identified with 3d⁹4s¹?3d¹⁰ and 4d⁹5s¹?5d¹⁰ transitions respectively. The longer wavelength band (400–500 nm range) could be attributed to the Cu⁺ or Ag⁺ ion replacing alkali ion at the octahedral alkali site whereas short wavelength band (340–400 nm range) is attributed to a Cu or Ag ion at tetrahedral zinc site. The short wavelength band was found to be intense compared with long wavelength and gave an indication that most of the Cu or Ag ions prefered a tetrahedral Zn site compared with the octahedral alkali site. All the samples exhibit optically stimulated luminescence (OSL). The sensitivity was found to be lattice dependent. The lowest sensitivity of about 1% compared with Al₂O₃:C was observed in lithium lattices whereas highest the sensitivity of about 290% was observed in the case of Cu‐doped ZnNa₂Br₄.     

Excess Dietary Vanadium Induces the Changes of Subsets and Proliferation of Splenic T Cells in Broilers

The purpose of this 42-day study was to investigate the effects of dietary excess vanadium on immune function by determining changes of the subsets and proliferation function of splenic T cells. Four hundred twenty 1-day-old avian broilers were divided into six groups and fed on a corn–soybean basal diet as control diet or the same diet amended to contain 5, 15, 30, 45, and 60 ppm of vanadium supplied as ammonium metavanadate. When compared with those of the control group, the percentage of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ of splenic T cells were decreased in the 45 and 60 ppm groups; however, the percentage of CD3⁺ and CD3⁺CD4⁺ were increased in the 5 ppm group, and the CD₄⁺/CD₈⁺ ratios were raised in the 5 and 15 ppm groups at 14 days of age. Meanwhile, the proliferation of splenic T cells were depressed in the 45 and 60 ppm groups but raised in the 5 and 15 ppm groups. Also, the serum interleukin-2 (IL-2) and interleukin-6 (IL-6) contents were decreased in the 45 and 60 ppm groups and increased in the 5 ppm group. It was concluded that dietary vanadium in excess of 30 ppm changed the percentages of splenic T cell subsets and inhibited the proliferation of splenic T cells and reduced the serum IL-2 and IL-6 contents. The cellular immune function was finally impaired in broilers.     

DFT study of the interaction of cytidine and 2′-deoxycytidine with Li, Na, and K: effects of metal cationization on sugar puckering and stability of the N-glycosidic bond

Density functional theory (DFT) calculations were performed at the B3LYP level with a 6-311++G(d,p) basis set to systematically explore the geometrical multiplicity and binding strength for complexes formed by Li⁺, Na⁺, and K⁺ with cytidine and 2′-deoxycytidine. All computational studies indicate that the metal ion affinity (MIA) decreases from Li⁺ to Na⁺ and K⁺ for cytosine nucleosides. For example, for cytidine the affinity for the above metal ions are 79.5, 55.2, and 41.8 and for 2′-deoxycytidine, 82.8, 57.4, and 42.2 kcal/mol, respectively. It is also interesting to mention that linear correlations between calculated MIA values and the atomic numbers (Z) of the above metal ions were found. The influence of metal cationization on the coordination modes and the strength of the N-glycosidic bond in cytosine nucleosides have been studied. In all cases, the N1-C1′ bond distance changes upon introducing a positive charge in the nucleosides. It has been found that metal binding significantly changes the values of the phase angle of pseudorotation P in the sugar unit of these nucleosides. With respect to the sugar ring, metal binding changes the values of the glycosyl torsion angle and sugar ring conformation. The present calculations in the gas phase provide the first clues on the intrinsic chemistry of these systems and may be of value for studies of the influence of metal cations on the conformational behavior and function of nucleic acids.     

Solvation free energies of glutamate and its metal complexes: A computer simulation study

Most biological ion channels demonstrate a high degree of selectivity for one type of ion more than others, and in many cases, how they control attaining this is still not clear. So we have studied on some metal ion compounds of glutamate. The Glutamate and its meal ion compounds (Ca²⁺, Na⁺, K⁺ and Li⁺) were first modeled by ab initio calculations and then Monte Carlo simulation was used to calculate solvation free energies and also the complexes free energies for the related structures. The results indicated that Glutamate-Ca²⁺ have more stability in water than other metal ion. Also, it was found out that the more movement in ions; less stability of the structure would result. This trend can be seen both in gas and liquid phase.     

DFT modeling on the suitable crown ether architecture for complexation with Cs<Superscript>+</Superscript> and Sr<Superscript>2+</Superscript> metal ions

Crown ether architectures were explored for the inclusion of Cs⁺ and Sr²⁺ ions within nano-cavity of macrocyclic crown ethers using density functional theory (DFT) modeling. The modeling was undertaken to gain insight into the mechanism of the complexation of Cs⁺ and Sr²⁺ ion with this ligand experimentally. The selectivity of Cs⁺ and Sr²⁺ ions for a particular size of crown ether has been explained based on the fitting and binding interaction of the guest ions in the narrow cavity of crown ethers. Although, Di-Benzo-18-Crown-6 (DB18C6) and Di-Benzo-21-Crown-7 (DB21C7) provide suitable host architecture for Sr²⁺ and Cs⁺ ions respectively as the ion size match with the cavity of the host, but consideration of binding interaction along with the cavity matching both DB18C6 and DB21C7 prefers Sr²⁺ ion. The calculated values of binding enthalpy of Cs metal ion with the crown ethers were found to be in good agreement with the experimental results. The gas phase binding enthalpy for Sr²⁺ ion with crown ether was higher than Cs metal ion. The ion exchange reaction between Sr and Cs always favors the selection of Sr metal ion both in the gas and in micro-solvated systems. The gas phase selectivity remains unchanged in micro-solvated phase. We have demonstrated the effect of micro-solvation on the binding interaction between the metal ions (Cs⁺ and Sr²⁺) and the macrocyclic crown ethers by considering micro-solvated metal ions up to eight water molecules directly attached to the metal ion and also by considering two water molecules attached to metal-ion-crown ether complexes. A metal ion exchange reaction involving the replacement of strontium ion in metal ion-crown ether complexes with cesium ion contained within a metal ion-water cluster serves as the basis for modeling binding preferences in solution. The calculated O-H stretching frequency of H₂O molecule in micro-solvated metal ion-crown complexes is more red-shifted in comparison to hydrated metal ions. The calculated IR spectra can be compared with an experimental spectrum to determine the presence of micro-solvated metal ion–crown ether complexes in extractant phase.     

Effects of total submergence in saltwater on growth and leaf ion content of preflooded and unflooded Taxodium distichum saplings

Preconditioning by hypoxic condition would contribute higher tolerance to anoxic condition in physiological and metabolic aspects of various plant species. In expectation of improvement of salinity tolerance, effects of preconditioning with soil flooding on growth responses and photosynthetic activity in relation to Na⁺ content in leaves and roots were studied using 2-year-old Taxodium distichum saplings. A submergence experiment was performed at three salt concentrations (0, 4000, and 8000 ppm NaCl), and preconditioning was started 2 months prior to the beginning of submergence treatments. Continuous soil flooding with freshwater as a control did not inhibit growth in height and stimulated diameter increment of the saplings. Saplings submerged in saltwater and freshwater also showed no morphological changes during submergence treatments. However, leaf injury and shoot dieback were observed in drained saplings that had been submerged in saltwater. Na⁺ and K⁺ ion contents increased with increases in salt concentration in both preconditioned and unconditioned saplings, whereas ion contents in preconditioned saplings tended to be higher than those of unconditioned saplings.     

Homology modeling,docking, and molecular dynamics simulation of the receptor GALR2 and its interactions with galanin and a positive allosteric modulator

Galanin receptor type 2 (GALR2) is a class A G-protein-coupled receptor (GPCR), and it has been reported that orthosteric ligands and positive allosteric modulators (PAMs) of GALR2 could potentially be used to treat epilepsy. So far, the X-ray structure of this receptor has not been resolved, and knowledge of the 3D structure of GALR2 may prove informative in attempts to design novel ligands and to explore the mechanism for the allosteric modulation of this receptor. In this study, homology modeling was used to obtain several GALR2 models using known templates. ProSA-web Z-scores and Ramachandran plots as well as pre-screening against a test dataset of known compounds were all utilized to select the best model of GALR2. Molecular dockings of galanin (a peptide) and a nonpeptide ligand were carried out to choose the (GALR2 model)–galanin complex that showed the closest agreement with the corresponding experimental data. Finally, a 50-ns MD simulation was performed to study the interactions between the GALR2 model and the synthetic and endogenous ligands. The results from docking and MD simulation showed that, besides the reported residues, Tyr160^4.60, Ile105^3.32, Ala274^7.35, and Tyr163^ECL2 also appear to play important roles in the binding of galanin. The potential allosteric binding pockets in the GALR2 model were then investigated via MD simulation. The results indicated that the mechanism for the allosteric modulation caused by PAMs is the binding of the PAM at pocket III, which is formed by galanin, ECL2, TM2, TM3, and ECL1; this results in the disruption of the Na⁺-binding site and/or the Na⁺ ion pathway, leading to GALR2 agonism.