Structural characterization of a metal-based perfusion tracer: Copper(II) pyruvaldehyde bis (N4-methylthiosemicarbazone)

Copper(II) pyruvaldehyde bis(N⁴-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N₂S₂ metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.     

Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions

Prompted by the recently reported capacity of the physiologically occurring Cu(I)-[glutathione]₂ complex (Cu(I)-[GSH)]₂) to reduce oxygen, the effect of various GSH-binding metals (Co²⁺, Cd²⁺, Zn²⁺, Pb²⁺, Al³⁺, Hg²⁺ and Ni²⁺) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg²⁺ was able to substantially affect the capacity of Cu(I)-[GSH]₂ to generate superoxide. When Hg²⁺ and Cu(I)-[GSH]₂ were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg²⁺ for GSH and the observed ability of Hg²⁺ to lower the concentration of Cu(I)-[GSH]₂ (spectroscopically assessed), we suggest that Hg²⁺ displaces Cu(I) from Cu(I)-[GSH]₂, to release Cu(I) ions and form a Hg(II)-[GSH]₂ complex. The latter species would account for the Hg²⁺-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)-[GSH]₂ complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)-[GSH]₂ to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.     

Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM

The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N⁴-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 ± 12, 11 ± 3, 2.8 ± 1.3 and 51 ± 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 ± 10%; II, 18 ± 5%; III, 3.4 ± 1.5%; and IV, 38 ± 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 ± 5, 12 ± 3, 17 ± 4 and 46 ± 6% of ⁶⁷Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of ⁶⁷Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000–46,000 and 10,000–16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000–16,000 Da). These results suggest that the intracellular decomposition of tracer Cu-PTSM may result in the radiocopper entering the normal cellular pools for copper ions.     

[Cu4(H2O)4(dmapox)2(btc)]n · 10nH2O: The first two-dimensional polymeric copper(II) complex with bridging μ-trans-oxamidate and μ4-1,2,4,5-benzentetracarboxylato ligands: Synthesis, crystal structure and DNA binding studies

A two-dimensional copper(II) polymer with formula of [Cu₄(H₂O)₄(dmapox)₂(btc)]_n · 10nH₂O, where dmapox is the dianion of N,N′-bis[3-(dimethylamino)propyl]oxamide and btc is the tetra-anion of 1,2,4,5-benzenetetracarboxylic acid, was synthesized and characterized by elemental analysis, conductivity measurement, IR and electronic spectral studies. The crystal structure of the complex has been determined by X-ray single-crystal diffraction. The structure consists of crystallized water molecules and neutral two-dimensional copper(II) coordination polymeric networks constructed both by the bis-tridentate μ-trans-dmapox and tetra-monodentate μ₄-btc bridging ligands. Each btc ligand links four trans-dmapox-bridged binuclear copper(II) building blocks [Cu₂(H₂O)₂(trans-dmapox)]²⁺ and each binuclear copper(II) building block attaches to two btc ligands forming an infinite 2D layer which consists of 4+4 grids with dimensions of 13.563(5) × 15.616(5) Å. The environment around the copper(II) atom can be described as a distorted square-pyramid and the Cu?Cu separations through μ-trans-dmapox and μ₄-btc bridging ligands are 5.225 Å (Cu1-Cu1ⁱ), 5.270 Å (Cu2-Cu2ⁱⁱ), 6.115 Å (Cu1-Cu2), 9.047 Å (Cu1-Cu2ⁱⁱⁱ) and 10.968 Å (Cu1-Cu1ⁱⁱⁱ), respectively. Abundant hydrogen bonds among the crystallized, the coordinated water molecules, and the uncoordinated carboxyl oxygen atoms cross-link the two-dimensional layers into an overall three-dimensional channel-like framework. The interaction of the copper(II) polymer with calf thymus DNA (CT-DNA) has been investigated by using absorption, emission spectral and electrochemical techniques. The results indicate that the copper(II) polymer interacts with DNA strongly (K_b = 4.8 × 10⁵ M⁻¹ and K_sv = 1.1 × 10⁴) and the interaction mode between the copper(II) polymer and DNA may be the groove binding. To the best of our knowledge, this is the first report about the crystal structure and DNA-binding studies of a two-dimensional copper(II) polymer bridged both by the trans-oxamidate and btc ligands.     

Chiral 2-pyridyl alcoholate copper complexes: crystal structures of [bis(2-pyridyl alcoholate)copper(II)] and the use of [(2-pyridyl alcoholate)copper(II)]and [(2-pyridyl alcoholate)copper(I)] in asymmetric cyclopropanation of styene and allylic oxidation of cyclohexene

Chiral N,O pyridine alcohols HL¹-HL⁶ were used to form complexes with copper(II) ions. Ligands HL¹ and HL² formed complexes with copper(II) ions when Cu(OAc)₂ and HL were refluxed in methanol/ethanol mixture. Ligand HL³ formed a complex with copper(II) when deprotonated with NaH and stirred in a Cu(II) acetate THF solution. Ligands HL⁴-HL⁶ did not form complexes with copper(II) under similar conditions. Two complexes, [Cu(L¹)₂] and [Cu(L²)₂], were isolated as single crystals and characterized by X-ray crystallography. These complexes showed low catalytic activities in asymmetric reactions. However, they became active when reacted with triflic acid. Copper complexes, [Cu(L)] or [Cu(L)]⁺, formed in situ by reacting ligands HL with copper(I) or (II) ions, respectively, were also found to be active copper catalysts for asymmetric cyclopropanation of styrene with ethyl diazoacetate and allylic oxidation of cyclohexene with t-butylperoxybenzoate. Enantioselectivities up to 56% and 38% were obtained in asymmetric cyclopropanation of styrene and asymmetric allylic oxidation of cyclohexene, respectively.     

Copper(II) import and reduction are dependent on His-Met clusters in the extracellular amino terminus of human copper transporter-1

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (⁷Met-Gly-Met⁹) and Asp¹³ abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

NMR and homology modeling studies of copper(II)-halocyanin from Natronobacterium pharaonis bacteria

Halocyanin from the haloalkaliphilic archaean Natronobacterium pharaonis is a peripheral membrane type 1 blue copper protein with a single polypeptide chain of 163 amino acid residues. Halocyanin participates as putative electron carrier protein associated to an electron acceptor role for a terminal oxidase and has the lowest redox potential value reported to date for a BCP. NMR studies and homology modeling calculations were performed to evaluate the electronic properties of Cu(II)-halocyanin from Natronobacterium pharaonis. The copper coordination site properties of Cu(II)-halocyanin are discussed. The ¹H NMR spectra, isotropic chemical shifts and relaxation times for halocyanin are compared with those of other BCPs such as azurin, amicyanin, plastocyanin and stellacyanin. The wild-type Cu(II)-halocyanin presents almost the same ¹H NMR spectra in comparison with Cu(II)-plastocyanin as expected from a similar coordination symmetry. However, minor differences were found. In order to get some insight on these differences, a computational model for Cu(II)-halocyanin from N. pharaonis was built. Model is based on sequential homology of halocyanin with two different families of proteins: plastocyanins and pseudoazurins. Homology modeling was performed using two different structural templates and copper ion was added for further refinement of the coordination site. Proposed structure was in good agreement with NMR experimental information and is the first three-dimensional model reported to date of an halocyanin. Small differences were found in the copper coordination site with respect to other BCP with known structure. This work is also an interesting example of expertise-driven homology modeling across different protein families.     

Calcium-mediated enhancement of copper tolerance in Elodea canadensis

The alleviative effects of exogenous calcium on copper phytotoxicity were investigated in Elodea canadensis plants. There was a significant accumulation of Cu in the plants after their exposure to 0.01 mM Cu accompanied by many symptoms of toxicity. Increased uptake of Cu severely reduced content of photosynthetic pigments, soluble proteins, and free proline. The total antioxidant capacity (T-AOC), reduced glutathione (GSH), and non-protein thiol (NP-SH) were severely suppressed in Cu-stressed plants resulting in a rapid increase in content of superoxide anion (O₂ ^·?), hydrogen peroxide, lipid peroxidation, and cell death. Simultaneous application of Ca markedly increased the content of photosynthetic pigments, soluble proteins, free proline, T-AOC, GSH, and NP-SH, and reduced oxidative damage as indicated by lowered content of MDA, O₂ ^·?, and H₂O₂; and decreased cell death. Furthermore, application of Ca reduced Cu uptake and effectively reversed the Cu-induced nutrient imbalance.     

Iron and Copper Act Synergistically To Delay Anaerobic Growth of Bacteria

Transition metals are known to cause toxic effects through their interaction with oxygen, but toxicity under anoxic conditions is poorly understood. Here we investigated the effects of iron (Fe) and copper (Cu) on the anaerobic growth and gene expression of the purple phototrophic bacterium Rhodopseudomonas palustris TIE-1. We found that Fe(II) and Cu(II) act synergistically to delay anaerobic growth at environmentally relevant metal concentrations. Cu(I) and Cu(II) had similar effects both alone and in the presence of ascorbate, a Cu(II) reductant, indicating that reduction of Cu(II) to Cu(I) by Fe(II) is not sufficient to explain the growth inhibition. Addition of Cu(II) increased the toxicity of Co(II) and Ni(II); in contrast, Ni(II) toxicity was diminished in the presence of Fe(II). The synergistic anaerobic toxicity of Fe(II) and Cu(II) was also observed for Escherichia coli MG1655, Shewanella oneidensis MR-1, and Rhodobacter capsulatus SB1003. Gene expression analyses for R. palustris identified three regulatory genes that respond to Cu(II) and not to Fe(II): homologs of cueR and cusR, two known proteobacterial copper homeostasis regulators, and csoR, a copper regulator recently identified in Mycobacterium tuberculosis. Two P-type ATPase efflux pumps, along with an F_oF₁ ATP synthase, were also upregulated by Cu(II) but not by Fe(II). An Escherichia coli mutant deficient in copA, cus, and cueO showed a smaller synergistic effect, indicating that iron might interfere with one or more of the copper homeostasis systems. Our results suggest that interactive effects of transition metals on microbial physiology may be widespread under anoxic conditions, although the molecular mechanisms remain to be more fully elucidated.     

Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H₂N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine ε-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K_D,Zn = 1.6 × 10⁻⁹ M and K_D,Cu = 5.0 × 10⁻¹² M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K_D values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.     

Using lignimerin (a recovered organic material from Kraft cellulose mill wastewater) as sorbent for Cu and Zn retention from aqueous solutions

Adsorption of copper and zinc in lignimerin (an organic material mainly composed by lignin, carbohydrate fragments and some extractives) and its acid derivative (H-lignimerin), recovered from Kraft cellulose mill wastewater was examined. A Box–Behnken experiment design, used to optimize lignimerin recovery process, revealed that the type of solvent used for precipitation is a determining factor in the amount of substance obtained. Conversely, batch adsorption studies at pH 4.0 revealed that the maximum adsorption capacities, modeled by the Langmuir equation, were 666.7 and 370.4 mmol kg⁻¹ for Cu(II) and Zn(II), respectively in lignimerin and 232.6 and 312.5 mmol kg⁻¹ for Cu(II) and Zn(II), respectively in H-lignimerin. The adsorption of Cu(II) and Zn(II) through deprotonated hydroxyl and carboxylic groups was the dominant mechanism that may explain the adsorption in both materials. The adsorption capacities indicated that lignimerin, with a molecular mass between 50 and 70 kDa, has a potential use as an organic sorbent for removing copper and zinc from liquid resources.     

Copper metabolism in analbuminaemic rats fed a high-copper diet

Copper metabolism in male Nagase analbuminaemic (NA) rats was compared with that in male Sprague Dawley (SD) rats fed purified diets containing either 5 or 100 mg Cu/kg diet. Dietary copper loading increased hepatic and kidney copper concentrations in both strains to the same extent, but baseline values were higher in the NA rats. There was no strain difference in true and apparent copper absorption nor in faecal endogenous and urinary copper excretion. NA rats had higher levels of radioactivity in kidneys at 2 hr after intraperitoneal administration of ⁶⁴Cu. As based on the distribution of added ⁶⁴Cu, about 70% of plasma copper appeared to be in the non-protein compartment in the NA rats, whereas in SD rats, it was only about 1%. It is concluded that the NA rats are able to maintain a relatively normal metabolism of copper, even after dietary copper challenge. In the NA rats, zinc concentrations in kidneys, liver and urinary zinc excretion were elevated when compared with SD rats. The high-copper diet did not affect tissue zinc concentrations and apparent zinc absorption in both strains of rats.     

An omp gene enhances cell tolerance of Cu(II) in Sinorhizobium meliloti CCNWSX0020

The main aim of this work was to study molecular characterization of a DNA fragment conferring resistance to Cu(II) in Sinorhizobium meliloti CCNWSX0020. The strain CCNWSX0020, resistant to 1.4 mmol l^?1 Cu(II) in tryptone-yeast extract medium was isolated from Medicago lupulina growing in mine tailings of Fengxian County, China. The availability of the complete genome sequence of S. meliloti CCNWSX0020 provides an opportunity for investigating genes that play significant roles in Cu(II) resistance. A copper resistance gene, with a length of 1,445 bp, encoding 481 amino acids, designated omp, was identified by cDNA-amplified fragment length polymorphism from S. meliloti CCNWSX0020. The expression of omp gene strongly increased in the presence of Cu(II). The omp-defective mutants display sensitivities to Cu(II) compared with their wild types. The Cu(II)-sensitive phenotype of the mutant was complemented by a 1.5-kb DNA fragment containing omp gene. BLAST analysis revealed that this gene encoded a hypothetical outer membrane protein with 75 % similarity to outer membrane efflux protein in Rhizobium leguminosarum bv. viciae 3841. These studies suggested that the omp product was involved in the Cu(II) tolerance of S. meliloti CCNWSX0020.     

Metal-directed synthesis of a chiral acyclic pentaamine and pendant-arm macrocyclic hexaamine derived from an amino acid

l-3-Phenylpropane-1,2-diamine (dapp) was prepared by a three-step synthesis based on l-phenylalanine and characterized, including determination of stability constants with M²⁺ ions (Ni, Cu, Zn, Cd). The reaction of L-3-phenylpropane-1,2-diamine as the [Cu(dapp)₂]²⁺ complex ion with formaldehyde and nitroethane in basic solution yields the acyclic (5-methyl-5-nitro-1,9-diphenyl-3,7-diazanonane-1,9-diamine)copper(II) complex ion, [Cu(1)]²⁺, as the major product. In addition, small amounts of the macrocyclic complex ion (2,10-diphenyl-6,13-dimethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecane)copper(II), [Cu(2)]²⁺, form. Reduction of the [Cu(1)]²⁺ ion with zinc in aqueous acid yields the acyclic polyamine 5-methyl-1,9-diphenyl-3,7-diazanonane-1,5,9-triamine (3), an analogue of the previously reported pentaamine 5-methyl-3,7-diazanonane-1,5,9-triamine. Using the bis(l-3-phenylpropane-1,2-diamine)palladium(II) as precursor and an excess of other reagents, the macrocyclization reaction to produce [Pd(2)]²⁺ proved more successful. Reduction and recomplexation to copper(II) allowed isolation of the 2,9-dibenzyl-6,13-diammonio-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane)copper(II) ion, [Cu(4H₂²⁺)]⁴⁺. The acyclic complex [Cu(1)]²⁺ promotes the hydrolytic cleavage of plasmid DNA modestly; a mechanism to support this observation is presented.     

Effects of interaction between65Zn,cadmium, and copper in rats

Distribution and retention of zinc in the presence of cadmium and copper was studied in rats exposed repeatedly to these metals. The experiment was performed on white rats of the Wistar strain. The animals were divided into four groups/five rats each: 1)⁶⁵ZnCl₂; 2)⁶⁵ZnCl₂+CdCl₂; 3)⁶⁵ZnCl₂+CuCl₂; and 4) control group. Rats were administered sc every other day for two weeks:⁶⁵ZnCl₂−5 mg Zn/kg; CdCl₂−0,3 Cd/kg; and CuCl₂−2 mg Cu/kg. The zinc content was measured in rat tissues by γ-counting. Effect of Cd and Cu on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein were also examined. Whole body retention of zinc under the influence of cadmium was lower than that observed in animals treated with zinc alone. However, copper increased twofold the whole body retention of zinc. Cadmium elevated the accumulation of zinc only in the kidneys nuclear fraction and liver soluble fraction. In the kidneys and liver, copper elevated the accumulation of zinc, in the nuclear, mitochondrial, and soluble fractions. The level of metallothionein-like proteins (MT) in the kidneys after a combined supply of zinc and copper was significantly increased with respect to the group of animals treated with zinc alone. These results indicated complex interactions between cadmium, copper, and zinc that can affect the metabolism of each of the metals.     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

A potential copper radiopharmaceutical for imaging the heart and brain: Copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone)

An investigation of the biodistribution of lipophilic copper-64 (half-life = 12.7 h) compounds has been initiated in order to screen potential tracers that could be used to measure regional cerebral and/or myocardial blood flow when labeled with generator-produced ⁶²Cu. The ⁶⁴Cu complex of pyruvaldehyde bis(N⁴-methylthiosemicarbazone), [⁶⁴Cu]Cu-PTSM, was prepared and found to be lipophlic (octanol/saline partition coefficient, log p = 1.97 ± 0.03). Biodistribution studies following i.v. injection of [⁶⁴Cu]Cu-PTSM into rats show tracer uptake by the brain and heart. At 1, 5, and 15 min post-injection 3.3, 3.0 and 2.7% of the injected dose was found in the brain. Corresponding brain to blood ratios (per gram) were 3.2, 3.6 and 4.0 respectively. Heart to blood ratios of 7.6, 7.6 and 7.3 were observed at these same time points.     

Use of Monochlorobimane to Determine the In Vivo Effect of Cadmium,Copper and Lead on Glutathione Status in Mercenaria mercenaria Brown Cells

This study was undertaken to demonstrate the synthesis of glutathione (GSH) in Mercenaria mercenaria brown cells to test the hypothesis that failure to achieve 100% mortality with metal treatment is the result of high concentrations of GSH heterogeneously distributed in the brown cell population and to determine the effect of Cd²⁺, Cu²⁺, and Pb²⁺ on the GSH status in brown cells. The monochlorobimane (MCB) assay appeared to be selective for GSH in brown cells and a close relationship between the levels of GSH measured by MCB and a standard enzymatic method was found. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. The technique was used to establish that GSH was synthesized in brown cells and was heterogeneously distributed in the brown cell population. Metal concentrations as high as 40 mM cadmium, 6.0 μM copper, or 20 mM lead did not deplete but caused decreases in brown cell GSH concentration that differed significantly (P < 0.05) from controls. The decrease caused by cadmium, copper, and lead was not in a dose dependent manner, whereas, the decrease caused by N-ethylmaleimide (NEM) was. It appears that the partial kill phenomenon associated with metal toxicity may be due to the heterogeneous distribution of GSH in the brown cell population.     

Copper complexes of some potentially binucleating tetradentate phthalazine-hydrazone ligands. Spontaneous reduction to form copper(I) derivatives

The tetradentate phthalazine-hydrazone ligands PHT and DMPH, formed by the reaction of 1,4-dihydrazinophthalazine (DHPH) and p-tolualdehyde and 2,5-dimethylbenzaldehyde respectively form predominantly copper(I) derivatives when reacted with copper(II) salts in solvents containing small amounts of water. The derivatives Cu(I)(PHT)X (X=NO₃⁻, ClO₄⁻) were produced by reaction of copper(II) salts with PHT in methanol, while in aqueous acetonitrile ligand hydrolysis occurred with no complex formation. In aqueous acetonitrile the hydrolysis occurs at one azomethine centre, generating initially a hydrazino derivative and p-tolualdehyde, followed by copper(II) reduction and nitrogen evolution and the formation of p-toluic acid and a cyanobenzene derivative (A) resulting from phthalazine ring cleavage. The copper(I) complexes of both PHT and DMPH can also be synthesized directly by reaction of copper(I) salts with the ligands in acetonitrile and copper(II) complexes of PHT can be synthesized with electronegative and coordinating anionic groups, e.g. Cl, Br.