Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.     

Preparation and characterization of paramagnetic polychelates and their protein conjugates

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).     

A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.     

Copper(II) interactions with non-steroidal anti-inflammatory agents. III--3-Methoxyanthranilic acid as a potential *OH-inactivating ligand: a quantitative investigation of its copper handling role in vivo

Pharmacological activities of copper(II) complexes are a direct function of the nature of their ligands associated with the metal ion in vivo. Some of these, defined as *OH-inactivating ligands (G. Berthon, Agents Actions 39 (1993) 210-217), may act as specific "lures" for hydroxyl radicals at inflammatory sites and behave as pseudo-catalase-like agents. This property has been advanced for anthranilic acid (H. Miche, V. Brumas, G. Berthon, J. Inorg. Biochem. 68 (1997) 27-38). With a view to improve the chemical features required to render such inactive substances effective anti-inflammatory drugs through their association with copper(II), an in vitro investigation into copper(II) interactions with the anionic form of an anthranilic acid derivative, namely 3-methoxyanthranilate (Man), has been performed under experimental conditions pertaining in vivo. Copper(II)-Man complex equilibria have been determined using glass electrode potentiometry, then checked by UV-vis and mass spectrometries. Given the prime role of histidine as a copper(II) ligand in blood plasma, copper(II)-histidine-Man ternary equilibria have also been studied. Subsequent computer simulations of the distribution of copper(II) in the extracellular fluid revealed that Man can specifically mobilize Cu(II) ions under inflammatory conditions without affecting their distribution under normal physiological conditions. Thiobarbituric acid reactive substances (TBARS) tests conducted with respect to standardized copper-mediated Fenton-type reactions (P. Maestre, L. Lambs, J.P. Thouvenot, G. Berthon, Free Rad. Res. 20 (1994) 205-218) have shown that, like anthranilic acid, Man can effectively both increase the Fenton-like reactivity of copper and decrease the amount of TBARS detected in solution, i.e., act as a potential *OH-inactivating ligand.     

Determination of copper in biological materials by atomic absorption spectroscopy: a reevaluation of the extinction coefficients for azurin and stellacyanin

A new method for the determination of copper in biological materials by graphite furnace atomic absorption spectroscopy is presented. This new procedure is an extension of the classic method of standard additions, where the analyte concentration is determined in a series of identical samples to which various amounts of metal standard have been added. The concentration of metal in the original sample is determined from an extrapolation of a plot of absorbance versus added analyte. In the new method, the amount of copper is determined by the method of standard additions for different concentrations of the sample under investigation as well. From an extrapolation of the data, the concentration of copper in the absence of interfering matrix is obtained. Studies with fetal bovine serum demonstrate that the new extrapolation technique is precise. Furthermore, considerably more copper is detected than by the classic method of standard additions applied to a nitric acid treated sample. The matrix effects of phosphate, nitrate, albumin, and serum were also examined. Both phosphate and serum, at physiological pH, decrease the detectability of added copper, while nitrate and albumin were without effect. The accuracy of this method has been verified by determining the extinction coefficients of stellacyanin and azurin. The values obtained, 4.33 X 10(3) and 3.75 X 10(3) M-1 cm-1, respectively, are considerably different from those determined by the method of standard additions on nitric acid digests of these proteins, but were close to values previously reported and determined colorimetrically.     

Heme nitrosylation of deoxyhemoglobin by s-nitrosoglutathione requires copper

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.     

Titanium preferential binding sites in human serum transferrin at physiological concentrations

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.     

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level

The antioxidant and metal-chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serum-depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o-phenanthroline, bathophenanthroline disulfonic acid, and N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC-induced endothelial cell death was greater than that of copper. Zn-EDTA did not block PDTC-induced cell death, whereas Ca-EDTA and Cu-EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn-EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC-induced cerebral endothelial cell death.     

海南热带雨林土壤中胡敏酸对Pb2+、Cu2+的吸附解吸特性研究

重金属元素在土壤-溶液界面的吸附反应深刻地影响重金属行为及其生态风险。以热带次生雨林土壤中提取的胡敏酸为材料, 采用傅立叶红外光谱、扫描电镜等技术对胡敏酸进行了表征, 通过批量吸附实验研究了胡敏酸对铅、铜的吸附特性, 通过连续解吸方法研究了在不同浓度下饱和吸附的胡敏酸对重金属吸附的形成过程。结果表明, 胡敏酸的腐殖化系数较高, 阳离子交换量为52.84 mmol·g-1, 表面含有羧基和酚羟基官能团。胡敏酸对铜的吸附符合准二级动力学模型, 对铅的吸附符合准一级动力学方程; Langmuir 模型能够更好地描述单一和复合污染条件下胡敏酸对Pb2+的等温吸附行为, Freundlich 模型能够更好的描述单一和复合污染条件下胡敏酸对Cu2+的等温吸附行为。通过连续解吸实验发现, 胡敏酸对铅的吸附过程中4种结合方式的先后饱和顺序为: 物理吸附、络合、离子交换和氢键结合。胡敏酸对铜的吸附过程中4种结合方式的先后饱和顺序为: 络合、氢键结合、离子交换和物理吸附。     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

How do Gram-negative bacteria acquire copper? This study shows that the outer membrane protein OprC from Pseudomonas aeruginosa is abundant during infection and mediates highly selective acquisition of both copper redox states via an extracellular "methionine track" and an unprecedented near-irreversible binding site.     

Role of transition metal ferrocyanides (II) in chemical evolution

Due to ease of formation of cyanide under prebiotic conditions, cyanide ion might have formed stable complexes with transition metal ions on the primitive earth. In the course of chemical evolution insoluble metal cyano complexes, which settled at the bottom of primeval sea could have formed peptide and metal amino acid complexes through adsorption processes of amino acids onto these metal cyano complexes.Adsorption of amino acids such as glycine, aspartic acid, and histidine on copper ferrocyanide and zinc ferrocyanide have been studied over a wide pH range of 3.6 – 8.5. Amino acids were adsorbed on the metal ferrocyanide complexes for different time periods. The progress of the adsorption was followed spectro-photometrically using ninhydrin reagent. Histidine was found to show maximum adsorption on both the adsorbents at neutral pH. Zinc ferrocyanide exhibits good sorption behaviour for all the three amino acids used in these investigations.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Synthesis and spectroscopic and fungicidal characterization of hydroxamic acids and their metal chelates

Hydroxamic acid chelates of the type ML2, ML2', and ML2" where M = Cu(II), Ni(II) or Co(II) and L = N,2'-diphenylacetohydroxamic acid (N,2'-DPAHA), L' = 2,2'-diphenylacetohydroxamic acid (2,2'-DPAHA), and L" = 2-phenylacetohydroxamic acid (2-PAHA) have been isolated and characterized on the basis of elemental analysis and infrared and magnetic data. These metal chelates were screened for their fungicidal activity. The testing against fungi has been carried out by slide germination technique against Alternaria alternata and by inhibition zone technique against Fusarium oxysporum and Aspergillus flavus. The fungicidal activity of chelates and their parent ligand has been compared with the commercial fungicide, Dithane M-45, screened under similar conditions.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

Dual on-off and off-on switchable oligoaziridine biosensor

A water-soluble biocompatible aziridine-based biosensor with pendant anthracene units was synthesized by radicalar polymerization of N-substituted aziridines in supercritical carbon dioxide. The binding ability of the sensor towards a series of metal ions was examined by comparing the fluorescence intensities of the solutions before and after the addition of 100 equivalents of a solution of the metal ion chloride salt. A fast, simple and highly optical sensitive dual behavior, "off-on" and "on-off" response, was observed after the biosensor was exposed to the metal cations in aqueous solution. Zinc presented the highest fluorescence enhancement (turn-on) and copper presented the highest fluorescence quenching (turn-off). The response time was found to be instantaneous and the detection limit was achieved even in the presence of excess metal cation competitors. By using immunofluorescence microscopy it was also shown that oligoaziridine acts as an "on-off" probe through highly sensitive (detection limit of 1.6nM), selective and reversible binding to copper anions under physiologic conditions using living Human Fibroblast cells. The stoichiometry for the reaction of the biosensor with Cu(2+) was determined by a Job plot and indicates the formation of an oligoaziridine-Cu(2+) 1:2 adduct.     

Activation of protein-bound copper ions during early glycation: study on two proteins

This study proposes several possible pathways by which hyperglycemia could make protein-bound metal ions more redox active. These mechanisms were tested on bovine serum albumin and calf lens protein. Proteins rich in early glycation products were less capable of competing for copper ions in the presence of other ligands (e.g., glycine and calcein), suggesting that glycated proteins might have diminished stability constants of their copper chelates compared to control counterparts. When protein-copper complexes were tested for their ability to cause the oxidation of ascorbic acid, as well as the reduction of molecular oxygen to hydrogen peroxide, glycated and control proteins differed considerably in their redox abilities. Oxidative damage on proteins documented by protein carbonyl content and amino acid analysis indicates the involvement of Fenton chemistry upon metal chelation. The possible biological consequences of the observed activation of metal ions bound to early glycated proteins are discussed.