Mol Of Cu Research Articles

Metalloselenonein, the selenium analogue of metallothionein: synthesis and characterization of its complex with copper ions.

We used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein. Metalloselenonein binds 3 mol of Cu(I) per mol. This adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the copper-metalloselenonein complex shows a positive band around 245 nm attributable to asymmetry in metal coordination.

Cu(I) analysis of blue copper proteins.

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.

200 KD neurofilament protein binds AL, CU and ZN

200 Kd human and bovine neurofilament proteins were isolated from spinal cord and purified to homogeneity. The purified proteins were shown to be metal-binding proteins which bind at least one mole of Al, one mole of Cu and four moles of Zn. Neither dephosphorylation of the protein nor equilibrium dialysis against 25 mM EDTA, pH 7.0, removed these metals. This is the first report of a human protein which stoichiometrically binds Al.

Order-disorder studies on copper substituted lithium aluminate and lithium gallate spinels

Copper substituted lithium aluminate becomes disordered when 0.2 mole of Cu 2+ replaces 0.1 mole of Li 1+ and Al 3+, respectively. The 1:3 ordering persists only up to 0.35 mole of Li oct, and the order-disorder transition temperature increases with increasing Li oct content. The gallium containing Li-spinels require more Li oct due to clustering of ions which tends to disrupt the 1:3 ordering.

Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans.

Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly to homogeneity by an anaerobic procedure that results in a product with high specific activity. The enzyme is a dimer of about Mr 144,000 composed of two subunits of apparently equal Mr and contains 4 mol of Cu per mol of subunit. The isoelectric point is 4.3; specific activity at 25 degrees C, pH 7.1, is 122 mumol X min-1 X mg of protein-1; and Km is about 7 microM N2O under the same conditions. The N2O- and O2-oxidized forms of the enzyme had principal absorption bands at 550 and 820 nm; the dithionite-reduced form, at 650 nm. The extinction coefficient at 550 nm for the oxidized enzyme is about 5300 (M subunit)-1 X cm-1. Ferricyanide-oxidized enzyme and enzyme exposed to O2 for a couple of days at 4 degrees C exhibited additional bands at 480, 620, and 780 nm and had very low specific activities. Cu-EPR signals were observed with oxidized and reduced forms of the enzyme with g perpendicular values at 2.042 and 2.055, respectively. The O2-oxidized enzyme had g parallel and A parallel values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g parallel region. The enzyme may therefore contain at least one Cu atom approximating the "Type 1" class. Spin counts against Cu-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the O2-oxidized enzyme and 7-15% in the enzyme as prepared and in the reduced enzyme. Much of the Cu thus appears to be EPR silent. Nitrous oxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluoride among other anions were found to accelerate this process. In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfectomarina and Rhodopseudomonas sphaeroides, particularly the former. Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions.

Selective extraction of trace metals associated with hydrous aluminum oxides

In soil/sediment analysis, subdivision of trace metal content into different categories is usually based on selective extraction schemes. To assess the disposition of metal ions bound to aluminum hydrous oxides in such schemes, suspensions of Al(OH)3gel, gibbsite, or alumina were loaded with up to 5 μ mol of Cu, Pb, Cd, or Zn ions prior to being extracted for 24 h with one of fifteen different chemical solutions. The percentage of sorbed ion retrieved varied along the reagent sequence: NaCl, CaCl2 < MgCl2, NH4NO3 < CH3COONH4, Na citrate, Na4P2O7 < EDTA, DTPA < CH3COOH, H2C2O4, HCl, HNO3. In each system, the recovery value varied with the initial surface loading (a function of sorption pH) and reflected changes in metal species form, e.g., bonded M2+, (MOH+), M(OH)2. With low loading levels up to 40% was displaced by salt solutions; with 1 to 2 μ mol sorbed, as little as 10% was displaced by acids or complex formers but this increased to ~90% with higher loadings. The relationship between sorption pH, amount sorbed, and extraction value was complex, and since in selective extraction schemes classification is based on recovery values, changes in initial retention parameters (e.g. system pH) lead to varying fractions of the different metal ions being classified as "ion exchangeable", "chemisorbed", and "incorporated in the lattice".

Fe-S centers in lactyl-CoA dehydratase.

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of the two copper ions in bovine serum amine oxidase.

With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS)

Metal substitution of Neurospora copper metallothionein.

The binding of diamagnetic Zn(II), Cd(II), and Hg(II) and paramagnetic Co(II) and Ni(II) ions to the apo form of Neurospora metallothionein (MT) was investigated by various spectroscopic techniques. In contrast to native copper MT, which was shown to bind 6 mol of Cu(I)/mol of protein (Lerch, 1980), all substituted forms reveal an overall metal to protein stoichiometry of 3. The charge-transfer (CT) transitions of the complexes containing diamagnetic metal ions as well as the d-d transitions of those with paramagnetic metal ions are indicative of a distorted Td coordination. Electron paramagnetic resonance and absorption measurements of the Co(II) derivative are in agreement with the presence of a metal-thiolate cluster in this protein. Metal titration studies of the apoprotein reveal characteristic spectral features for the derivatives containing two metal equivalents as compared to those with a full complement of three metal ions. The former features are indicative of an exclusive Td type of metal-sulfur coordination whereas the latter suggest that the third metal ion is coordinated in a different fashion. This finding is in agreement with the presence of only seven cysteine residues in Neurospora MT as opposed to nine cysteine residues in the three-metal cluster of the mammalian MT's [Winge, D.R., & Miklossy, K.-A. (1982) J. Biol. Chem. 257, 3471].

Refrigeration into the microkelvin range

We describe a double stage nuclear demagnetization refrigerator (4.2 moles of PrNi 5 in 6 T and 10 moles of Cu in 8 T) which has cooled about 2 kg Cu to a measured electronic temperature of 50 μK. Temperatures are determined by NMR measurements of nuclear magnetization and from spin-lattice relaxation times of Pt and Cu.

The Formation and Structure of Copper(II) Complexes with Cyclodextrins in an Alkaline Solution

Abstract Copper(II) forms a 2: 1 complex with cyclodextrin in an alkaline solution. The complexes with α- and β-cyclodextrins were isolated from solutions by the addition of ethanol. They are labile, and, upon heating, the Cu(II) is reduced to Cu(0). The role of the hydroxide ion was examined by means of potentiometrie and conductometric titrations; it was found that the reaction of 1 mol of α-cyclodextrin with 2 mol of Cu(OH)2consumes 2 mol of the hydroxide ion, whereas 3 mol of the hydroxide ion are consumed for the reaction of β-cyclodextrin. Polarimetric measurements showed that the optical rotation of cyclodextrin changes from dextrorotatory to levorotatory upon the formation of the Cu(II)-cyclodextrin complex. It was suggested, with a molecular model, that the two pairs of C2 and C3′ secondary hydroxyl groups of contiguous glucose units are cross-linked by the Cu(OH−)2Cu ion bridge in the α-cyclodextrin complex and by Cu(OH−)(O2−)Cu ion bridge in the β-cyclodextrin complex. Furthermore, the circular ring of cyclodextrin may be distorted to the ellipsoidal form by the complex formation, causing a pronounced change in the optical rotation.

Formation, circular dichroism and x-ray photoelectron spectroscopy of hepatic Zn-thionein.

The formation of the powerful Zn binding protein called Zn-thionein was examined using male albino rats and [14C]cysteine, as cystein is known to be the most abundant constituent of this metal protein. 65% of the hepatic [14C]cysteine was incorporated into the protein portion of freshly prepared Zn-thionein. The protein was isolated by a combination of ethanol/chloroform treatment and various chromatographic steps, including ion exchange and gel filtration. 4.7 mol of Zn, 0.02 mol of Cd and less than 0.001 mol of either Cu or Hg were found per 12 000 g of portein. It was presumed that considerable amounts of Zn were lost during these isolation procedures, with the consequence of disulphide gridge formation. Indeed, the presence of R-S-S-R was deduced from circular dichroism and X-ray photoelectron spectroscopy. Due to the clearly detectable disulphide chromophore in the circular dichroism spectrum, it was possible to assign the shoulder at S 2p1/2,3/2 = 162.7 eV of the X-ray photoelectron spectrum of native Zn-thionein to R-S-S-R and not to strongly polarized sulphur. Upon reducing R-S-S-R-containing native Zn-thionein with dithiothreitol, all oxidised thiolate moieties of the thionein molecule could be restored. The addition of ZnCl2 with the subsequent desalting of extraneously bound Zn2 yielded a homogeneous Zn-thionein with 9.6 mol Zn2 per mol protein. A stoichiometry of ZnRS 1:3 was seen, which confirmed earlier reports of the existence of the mixed Cd,Zn-thionein. The conversion of mixed Cd,Zn-thionein into homogeneous Zn-, Cd-, Hg- and Cu-thionein by the gel filtration technique proved successful. From chiroptical measurements, the extraordinary contribution of the metal chromophores to the circular dichroism was seen. Due to the differences in the geometry of complexes formed by the respective metal ions, dramatic changes in the protein portion were expected. Polyacrylamide disc electrophoresis of purified native untreated Zn-thionein resulted in the appearance of two or more bands. This phenomenon was attributed to the different migration rates of cystine-thionein and thiolate-rich Zn-thionein, and was consistent with the spectral properties of the above Zn-protein species. By contrast, only one single band was monitored when a homogeneous metal-thionein was electrophoresed.

The binding of Cu 2+ to actin without loss of polymerizability: The involvement of the rapidly reacting SH group

G-or F-actin binds 1 mole of Cu 2+ per mole of actin without denaturation and without replacement of the essential Ca 2+ or Mg 2+. The association constant is ~10 16. Denaturation of actin, e.g., by EDTA treatment, abolishes the high-affinity Cu 2+ binding. The absorption spectrum of Cu-actin shows a new intense absorption band centered at 347 nm (ϵ = 3000 M −1 cm −1) and a weak band centered at 550 nm (ϵ = 130 M −1 cm −1). The tryptophyl fluorescence of actin is strongly (70%) quenched by the binding of Cu 2+. Circular dichroic spectra show multiple bands associated with each absorption band indicating an asymmetrical environment for the bound Cu 2+ and suggest that histidine is one of the ligands. Electron-spin resonance studies show that Cu 2+ maintains its valence when bound and also suggest that there are approximately three nitrogen ligands. Chemical modification studies of the sulfhydryl groups provide evidence for the involvement of one SH group in the binding of Cu 2+ to actin.

Circular dichroism of iron, copper, and zinc complexes of transferrin

The absorption banda of copper-transferrin are optically active with well-defined positive dichroic bands in the visible range having ellipticities of [ θ] 620 = 3,620 deg cm 2 dmole −1 and [ θ] 426 = 6,700 deg cm 2 dmole −1 in terms of bound copper. In iron-transferrin there are also two bands; a negative ellipticity band with [ θ] 455 = −16,000 deg cm 2 dmole −1 and a positive ellipticity band with [ θ] 360 = 6,000 deg cm 2 dmole −1 in terms of bound iron. Additional copper binding to iron-transferrin produces an absorption band at 675 nm with an associated negative ellipticity band. Zinc binding to apotransferrin, 2 moles per mole, affects 1 mole of tyrosine per mole of Zn 2+ bound, and the generated tyrosinate spectrum is optically active. There is optical activity in apotransferrin in the 280 nm region attributable to tryptophan, and optical activities in the 270 and 230 nm regions suggest possible disulfide contributions. Neither the tryptophan optical activity nor the optical activities in the region of 270 nm and 230 nm are affected by metal binding. Apotransferrin has an estimated α-helical content of 17–18%, and there is no observable change in this value when 2 moles of Cu 2+ or Zn 2+ are bound per mole of protein.

Current yield during electrolysis of metal salt solutions with cations in different valency states

The number of moles of Cu transferred per faraday from a Cu anode to a Cu cathode during the electrolysis of an acid CuSO 4 solution containing a small concentration of Cu + ions under steady-state conditions is estimated with the help of data for the electrode reactions involved and conditions of convective transport of Cu + ions.