Effect of metal ions on the fluorescence of leucine aminopeptidase and its dansyl-peptide substrates

The effect of Cu(II), Ni(II), Zn(II), Mg(II), and Mn(II) on the fluorescence of porcine kidney cytosol leucine aminopeptidase and three of its dansyl(Dns) peptide substrates, Leu-Gly-NHNH-Dns, Leu-Gly-NH(CH2)2NH-Dns, and Leu-Gly-NH(CH2)6NH-Dns, has been investigated. These five metal ions were chosen for study because each binds to the regulatory metal binding site of leucine aminopeptidase. Since the binding is relatively weak, kinetic studies of the different metalloderivatives of the enzyme are normally carried out in the presence of large molar excesses of these metal ions that can potentially affect both the enzyme and substrate. The fluorescence of all of the dansyl-peptides, as well as several other dansyl species, is quenched by Ni(II) and Cu(II), but not by Mg(II), Mn(II), or Zn(II). The absorption spectra of these dansyl substrates are also perturbed by Ni(II) and Cu(II). The rate at which maximal quenching for some dansyl species is attained after mixing with Ni(II) and Cu(II) is slow and the quenching is reversed on addition of EDTA. These results indicate that the quenching is the result of complex formation between the fluorophores and these metal ions. The association constants for the metal complexes have been determined from Stern-Volmer plots. In addition to complex formation, Ni(II) and Cu(II) cause the degradation of Leu-Gly-NHNH-Dns through a two step mechanism involving loss of dansic acid. Ni(II) and Cu(II) also partially quench the fluorescence of leucine aminopeptidase through contact with its surface accessible Trp residues. These observations indicate that care must be taken in stopped flow fluorescence studies of reactions between this enzyme and its dansyl substrates to avoid adverse effects brought about by Ni(II) and Cu(II).     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Antifungal potential of binary and mixed-ligand complexes of N,2'-diphenyl acetohydroxamic acid.

Chelating potential of N,2'-DPAHA with 3d metal ions such as Cu(II), Ni(II), Zn(II), and Cd(II) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligand complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(II), Zn(II), and Cd(II) is presumably due to pi-bonding effects. In the stabilization of the Cu(II) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to pi-bonding effects. Fungicidal activity of N,2'-DPAHA and its binary complexes with Cu(II), Ni(II), and Co(II) was examined against Fusarium oxysporum using the inhibition zone technique. Binary complexes of Zn(II) and Cd(II) with N,2'-DPAHA and mixed-ligand complexes M(II)-Gly or Phen-N,2'-DPAHA, where M(II) = Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) were screened against Alternaria alternata by slide germination technique. All mixed-ligand complexes exhibited fungicidal activity but did not improve significantly compared to binary complexes. Synergistic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:1) of the secondary ligand (N,2'-DPAHA), and the fungicidal potential of the mixed-ligand complex involving N,2'-DPAHA as secondary ligand was not increased.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

A switch‐off fluorescence probe towards Pb(II) and cu(II) ions based on a calix[4]pyrrole bearing amino‐quinoline group

A new fluorescence receptor calix[4]pyrrole‐N‐(quinoline‐8‐yl) acetamide (CAMQ) containing a pyrrolic ring connected via the meso‐position was synthesized, purified and characterized by elemental analysis, NMR and mass spectroscopy. This compound was examined for its fluorescence properties towards different metal ions e.g. Ag(I), Hg(II), Co(II), Ca(II), Ni(II), Zn(II), Cr(II), Ba(II), Fe(II), Cu(II), Pb(II)and Mg(II) ions by spectrophotometry and spectrofluorometry. It was concluded that the compound (CAMQ) possessed significantly enhanced selectivity for Pb(II) and Cu(II) ions in dimethyl sulfoxide (DMSO) even at very low concentrations (1 μM). It exhibit ‘turn‐on’ fluorescence when exposed to Pb(II) and Cu(II) and did so in preference to other metal ions. The binding constants, stoichiometry and quantum yields have been determined. The quenching mechanism was assessed using the Stern–Volmer equation and was also discussed.     

Manganese-containing superoxide dismutase from Escherichia coli: reversible resolution and metal replacements.

Exposure of the manganese-containing Superoxide dismutase of Escherichia coli to pH 3.2, in the presence of 0.7 m guanidinium chloride, causes a rapid loss of manganese and of activity. The apoenzyme so produced can be reconstituted by addition of MnCl₂ followed by neutralization. In contrast, manganese cannot be restored to the apoenzyme by adding MnCl₂after neutralization. The reconstituted enzyme is indistinguishable from the native enzyme in terms of its catalytic activity or electrophoretic behavior on polyacrylamide gels. Co(II), Ni(II), Zn(II), Fe(II), or Cu(II) could compete with Mn(II) during reconstitution of the apoenzyme. In the cases of Co(II), Ni(II), and Zn(II), it was shown that, in preventing reconstitution by Mn(II), they were themselves bound to the enzyme in stoichiometric amounts, in place of Mn(II). The binding of Fe(II) was also explored and was distinct in that the enzyme could bind more than stoichiometric amounts of this metal. None of the derivatives, in which Mn(II) had been replaced by another metal, were catalytically active. Nevertheless, these derivatives could be again resolved by exposure to acid guanidinium chloride and could then be converted back into the active holoenzyme by neutralization after addition of MnCl₂. It appears that the active site of this enzyme can accommodate and can tightly bind several metals other than manganese, but exhibits activity only with manganese. It also appears that movement of metal out of or into this site is only feasible at low pH and in the presence of a chaotropic agent. A substantial amount of the cobalt-substituted enzyme was prepared and its optical properties were recorded.     

Bivalent Metal Chelates with Pentadentate Azo-Schiff Base Derived from Nicotinic Hydrazide: Preparation,Structural Elucidation,and Pharmacological Activity

Azo-Schiff base ligand (N′-((E)-2-hydroxy-5-((E)-(2-hydroxyphenyl)diazenyl)benzylidene)nicotinohydrazide) and its Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) chelates were prepared and elucidated. The geometrical structures of the prepared chelates were characterized by several spectroanalytical techniques and thermogravimetric analysis. The obtained data revealed that the chelates have (1M:1L), (1M:2L), (1M:3L), and (1M:4L) molar ratios. The infrared spectra displayed that the H₂L ligand behaves in a pentacoordinate fashion in chelates of Mn(II), Ni(II), and Cu(II) ions. However, in Zn(II) and Pd(II) chelates, the ligand is coordinated as a tetradentate species (NONO) through nitrogen atoms of azomethine and azo groups as well as oxygen atoms of phenolic hydroxy, and carbonyl groups. Besides, it was concluded that the oxygen atoms of carbonyl and hydroxy groups along with the azomethine nitrogen atom of the ligand are bounded with Co(II) ion in metal chelate ( 2 ). According to the measured molar conductance values, the chelates of Cu(II), Zn(II), and Pd(II) are weak electrolytes, but Mn(II), Co(II), and Ni(II) chelates are ionic. The azo-Schiff base ligand and its prepared metal chelates were tested for their antioxidant and antibacterial properties. The Ni(II) chelate was found to be considered an effective antioxidant agent. In addition, the available antibacterial data suggest that the Ni(II) and Co(II) chelates may be employed as inhibitor agents against Proteus vulgaris, Escherichia coli, and Bacillus subtilis bacteria. Furthermore, the data showed that, in comparison to the ligand and other metal chelates, copper(II) chelate (4) exhibited higher action against Bacillus subtilis bacteria.     

Regulation of the phosphorylation of calpain II and its inhibitor

Phosphorylation of calpain II (or its inhibitor) by the catalytic subunit of cyclic AMP-dependent protein kinase (A-PK), cyclic GMP-dependent protein kinase (G-PK), and protein kinase C (PK-C) was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Among these protein kinases, the catalytic subunit of A-PK exhibited the strongest phosphorylations of both calpain II and its inhibitor. Arachidonic acid and staurosporine effectively inhibited phosphorylation regardless the type of kinase tested. Despite its lack of effect on the phosphorylation of calpain II by the catalytic subunit of A-PK, sphingosine moderately enhanced the phosphorylation of calpain II by G-PK. Other agents, including phosphatidylethanolamine, phosphatidylinositol and 1, 2-dioleoyl-sn-glycerol, had no significant effect.     

Adsorptive Removal and Recovery of U(VI), Cu(II), Zn(II), and Co(II) from Water and Industry Effluents

Tamarind fruit shell (TFS) was converted to a cation exchanger (PGTFS-SP-COOH) having a carboxylate functional group at the chain end by grafting poly(hydroxyethylmethacrylate) onto TFS (a lignocellulosic residue) using potassium peroxydisulfate-sodium thiosulfate redox initiator, and in the presence of N, N ′-methylenebisacrylamide as a cross-linking agent, followed by functionalization. The chemical modification was investigated using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and potentiometric titrations. The feasibility of PGTFS-SP-COOH for the removal of heavy metals such as U(VI), Cu(II), Zn(II), and Co(II) ions from aqueous solutions was investigated by batch process. The optimum pH range for the removal of meal ions was found to be 6.0. For all the metal ions, equilibrium was attained within 2 h. The kinetic and isotherm data, obtained at optimum pH value 6.0, could be fitted with pseudo-second-order equation and Sips isotherm model, respectively. The Sips maximum adsorption capacity for U(VI), Cu(II), Zn(II), and Co(II) ions at 30°C was found to be 100.79, 65.69, 65.97, and 58. 81 mg/g, respectively. Increase of ionic strength decreased the metal ion adsorption. Different wastewater samples were treated with PGTFS-SP-COOH to demonstrate its efficiency in removing metal ions from wastewater. The adsorbed metal ions on PGTFS-SP-COOH can be recovered by treating with 1.0 M NaCl + 0.5 M HCl for U(VI) ions and 0.2 M HCl for Cu(II), Co(II), and Zn(II) ions. Four adsorption/desorption cycles were performed without significant decrease in removal capacity. The results showed that PGTFS-SP-COOH developed in this study exhibited considerable adsorption potential for the removal of U(VI), Cu(II), Zn(II), and Co(II) ions from water and wastewaters.     

Homo and hetero dioxygen complexes of Co(II), Cu(II), Ni(II) and Pb(II) with the ligand 3,7,11,19,23,27-hexaaza-33,34-dihydroxy-15,31-dimethyltricyclotetratriaconta-1(32),13,15,17(34),29(33),30-hexaene

In this paper the oxygenation of HDTHCo homo and heterodinuclear complexes with Cu(II), Ni(II) and Pb(II) in aqueous solution by control of the stoichiometry of metal ions and HDTH as well as p[H] of solution was investigated (HDTH is a dinucleating 28-membered hexaazadiphenol macrocyclic ligand, 3,7,11,19,23,27-hexaaza-33,34-dihydroxy-15,31-dimethyl-tricyclo-tetratriaconta-1(32),13,15,17(34),29(33),30-hexaene). The pH potentiometric method was utilized successfully to determine oxygenation constants and to determine the distribution of species present in the solution as a function of p[H]. Spectrophotometry was used to investigate the oxygenation process of the homo and heterodinuclear complexes. The X-ray crystal structure of homodinuclear complexes of Ni(II) is also reported. These studies suggested autooxidation takes place during the oxygenation of homo and heterodinuclear Co(II) complexes of the macrocyclic ligand. The neighboring effect increases in the order Ni(II)<Cu(II)<Pb(II)<Co(II). Pb(II) stimulates the neighboring Co(II) to accept dioxygen in its sixth vacant position. Ni(II) is not helpful to Co(II) in its oxygenation.     

Synthesis, characterization and antitumor studies of Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes of N-salicyloyl-N'-o-hydroxythiobenzhydrazide

A new ligand N-salicyloyl-N'-o-hydroxythiobenzhydrazide (H2Sotbh) forms complexes [Mn(HSotbh)2], [Fe(Sotbh-H)(H2O)2], [M(Sotbh)] [M=Co(II), Cu(II) and Zn(II)] and [Ni(Sotbh)(H(2)O)2], which were characterized by various physico-chemical techniques. M?ssbauer spectrum of [Fe(Sotbh-H)(H2O)2] reveals the quantum admixture of 5/2 and 3/2 spin-states. Mn(II), Cu(II) and Ni(II) complexes were observed to inhibit the growth of tumor in vitro, whereas, Fe(III), Co(II), Zn(II) complexes did not. In vivo administration of Mn(II), Cu(II) and Ni(II) resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with Mn(II), Cu(II) and Ni(II) complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H2Sotbh and its metal complexes in tumor regression and tumor growth associated immunosuppression.     

Increased expression and function of vascular alpha1D-adrenoceptors may mediate the prohypertensive effects of angiotensin II

The peptide Angiotensin II (Ang II), part of the renin-angiotensin system (RAS), participates in the control of systemic arterial pressure. Ang II participates in increasing smooth muscle tone, and its positive effects on smooth muscle cell DNA synthesis are inhibited by treatment with prazosin, an alpha(1)-adrenoceptor agonist. Ang II also induces the expression of alpha(1)-adrenoceptor, especially the alpha(1D) subtype. Other findings suggest that the molecular signals activated by Ang II and by alpha(1D)-adrenoceptor might interweave, thus leading to the augmentation of smooth muscle tone and hypertension.     

Synthesis and characterisation of copper(II), zinc(II) and cobalt(II) complexes of salicylglycine, a metabolite of aspirin

Copper(II), nickel(II) and cobalt(II) complexes of the aspirin metabolite salicylglycine (H₂L), of stoichiometry M(HL)₂·solvate, have been prepared and characterised. In these complexes salicylglycinate is coordinated to the metal via its carboxylato group and possibly also its amide oxygen in the copper(II) complex. Under basic conditions copper(II) forms the complex Cu(LH₋₁)·2H₂O·MeOH, in which the ligand is coordinated to the metal via its carboxylate and phenolate oxygen atoms and the deprotonated peptide nitrogen atom.     

A new tripodal poly-imine indole-containing ligand: Synthesis, complexation, spectroscopic and theoretical studies

A novel flexible tripodal ligand derived from 3-methylindole, (“InTREN” L), and its mononuclear Zn(II), Cu(II), Ni(II), Hg(II) and Pd(II) complexes are described. All compounds gave analytically pure solid samples. Characterisation of the compounds was accomplished by ¹H NMR, IR and absorption spectroscopies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and elemental analysis and their geometry optimized using density functional theory (DFT).Time-dependent-density functional theory (TD-DFT) calculations have been used to assign the lowest energy absorption bands of the free ligand and the Zn(II) complex. The system is a very good candidate for in situ recognition/coordination effects by MALDI-TOF-MS spectrometry and absorption spectroscopy. The presence of three indole groups in InTREN opens up the possibility to synthesize new three-dimensional self-assembly supramolecular structures.     

Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

ESR study of the non-enzymic scission of xyloglucan by an ascorbate-H2O2-copper system: the involvement of the hydroxyl radical and the degradation of ascorbate

Xyloglucan is degraded by a mixture of copper(II), hydrogen peroxide and ascorbate. In the presence of ascorbate and/or hydrogen peroxide, copper(II) species were rapidly reduced to copper(I), which react with hydrogen peroxide. Spin-trapping experiments showed that hydroxyl radicals formed and attacked xyloglucan causing its degradation. The formation of a carbon-centred ascorbyl (C-ascorbyl) radical and its degradation with the formation of oxalate, was also caused by hydroxyl radicals. As a consequence, the features of the bis(oxalate) copper(II) complex clearly appeared in the frozen solution ESR spectra. The formation of carbon-centred radicals on the xyloglucan is the trigger for a series of possible molecular rearrangements which led to its oxidative scission.     

Specific nickel(II)-transfer process between the native sequence peptide representing the nickel(II)-transport site of human serum albumin and L-histidine.

The kinetics and mechanism for Ni(II)-transfer of the native sequence tripeptide, L-aspartyl-L-alanyl-L-histidine-N-methylamide (AAHNMA), representing the Ni(II)-transport site of human serum albumin (HSA) and L-histidine (L-His) was studied in forward and reverse reactions in the pH range 6.5 to 9.0 at I = 0.2 and 25 degrees C. For the Ni(II)-transfer from Ni(II)-(L-His)2 to native sequence peptide, the rate-determining step is the formation of a mixed-ligand complex of NiH-1AB by deprotonation of peptide nitrogen from NiAB where A and B denote the anionic forms of AAHNMA and L-His, respectively. For the Ni(II)-transfer from Ni(II)-peptide to L-His, the rate-determining step is a bond breaking between Ni(II) and peptide nitrogen to form NiH-1A by protonation to a peptide nitrogen of NiH-2A. The equilibrium constants for the metal-transfer reaction of MH-2A + 2HB in equilibrium MB2 + A (A = Ni(II), Cu(II] were 10(3.29) and 10(0.78) for Ni(II) and Cu(II), respectively. NiB2 is 324 times as stable as CuB2. Furthermore, the ratio of Ni(II)/Cu(II) in the rate constants for the reaction of MB2 with A was found to be 2.8 x 10(-4). Thus, despite the similarities of Cu(II) and Ni(II) in the metal-binding sites of HSA and in reaction mechanism, Ni(II)-(L-His)2 complex is so stable thermodynamically and kinetically, compared to the Cu(II)-(L-His)2 complex, that Ni(II) is hardly transferred from Ni(II)-(L-His)2 to native sequence peptide. These findings may support specificities in the Ni(II)-transfer, its organ distribution, and its excretion through urine in vivo.     

Synthetic and pharmacological studies on some transition metal chelates involving N-pyrimidino benzamide-2-carboxylic acid as ligand

N-pyrimidino benzamide-2-carboxylic acid (NPBCA) and its Cu(II), Ni(II), Co(II), Zn(II), and Mn(II) chelates have been synthesized and characterized by using elemental analyses, molar conductance, molecular weight determination, magnetic moment, infrared, and electronic spectra. Antifungal activity of the synthesized compounds has been screened on common fungi, viz., Aspergillus niger, Aspergillus nidulense, and Candida albicans at 28 degrees C and antibacterial activity has been observed on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria at 37 degrees C. Anti-inflammatory and ulcerogenic potential of the synthesized compounds have been discussed.