Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Cu(II) transfer into apo-Cu2Zn2-superoxide dismutase from Cu-thionein oxidized by activated leukocytes

The leukocyte-induced oxidative cleavage of yeast Cu(I)-thionein was examined. Oxidation was followed by the progressive decline of the specific Cotton bands attributed to the Cu(I)-thiolate chromophores between 400 and 270 nm. Despite many potent and competitive copper binding sites certainly present in leukocytes, the reconstitution of apo-Cu₂Zn₂-superoxide dismutase was expected due to its higher thermodynamic stability. Both enzymic activity measurements and characteristic Cu(II) EPR properties of Cu₂Zn₂-superoxide dismutase supported a successful reconstitution. The most favoured pathway for releasing Cu(II) from Cu-thionein was suggested to be an enzyme- controlled oxidation.     

Copper-thionein in leucocytes

Summary Upon incubation of peripheral leucocytes with copper sulphate a dramatic cellular copper uptake reaching levels of 25–50-fold compared to that of the natural copper content was measured. The orange-red fluorescence of the copper-treated white blood cells was assigned to the formation of Cu(I)-thiolate clusters in Cu(I)-thionein. A protein of 6–8 kDa was isolated from homogenized bovine leucocytes and characterized by its electronic absorption and amino acid composition to be identical to the above Cu(I)-thionein. More than 70% of the intracellular copper was attributed to this protein in its monomeric and polymeric form. Cu-thionein formation was more pronounced in monocytes than in granulocytes. As most intriguing phenomenon, the release of this Cu-thionein from leucocytes, was also noticed. The occurrence of Cu-thionein in leucocytes and the excretion of the intact Cu(I)-thiolate protein is of considerable interest with respect to the observed elevated copper levels in white blood cells and plasma during tumor malignancies and inflammatory processes.     

Differently bound copper(I) in yeast Cu8-thionein

The reactivity of yeast Cu-thionein in the presence of the Cu(I)-chelators, bathocuproinesulphonate and cuproine, was examined to distinguish between possible differently coordinated Cu(I). Electronic absorption measurements revealed that two out of eight coppers of the protein reacted within seconds with the chelator. At the same time, the shape and magnitude of the characteristic Cotton bands attributable to the Cu(I)-thiolate chromophores remained constant. Due to the successful removal of circular dichroic silent copper, all specific theta Cu values rose by 53% of the original value. Thus, it is strongly suggested that two or more distinct types of Cu(I) ought to be present in Cu8-thionein. In the light of the many different Cu/cysteine ratios of Cu-thioneins from vertebrate and microbial origin, possible interconversion reactions of the Cu(I)-thiolate centres seem to be likely.     

Cobalt(II) and zinc(II) binding to a ferredoxin maquette

We have examined the Co(II) and Zn(II) affinity of the prototype ferredoxin maquette ligand, NH(2)-KLCEGG.CIACGAC.GGW-CONH2 (IAA), which was originally designed to bind a [4Fe-4S] cluster. UV-Vis spectroscopy demonstrates tight 1:1 complex formation between Co(II) and IAA. The intensity of the S-->Co(II) charge transfer bands at 304 and 340 nm and the ligand field bands between 630 and 728 nm indicate Co(II) coordination by the four cysteine thiolates of IAA in a pseudo-tetrahedral geometry. A dissociation constant value of 5.3 microM was determined for the Co(II)-IAA complex at pH 6.5. Zn(II) readily displaces Co(II) from IAA as evinced by loss of the Co(II) spectral features. The dissociation constant for Zn(II), 20 pM at pH 6.5, was determined be competition experiments with Co(II)-IAA. These results demonstrate that the ferredoxin maquette ligand is an excellent ligand for Zn(II).     

Copper(I) transfer into apo-stellacyanin using copper(I)-thiourea as a copper-thionein model.

The direct incorporation of Cu(I) from [Cu(I)(thiourea)3]Cl, a structural analogue of Cu-thionein, into apo-stellacyanin, was successful both aerobically and anaerobically. A characteristic c.d. band of Cu(I)-stellacyanin at 270 nm (0 = -12.5 X 10(3) degrees X cm2 X dmol-1) was seen. On oxidation with hexacyanoferrate(III) or by air, the correct Cu(II) binding into the active centre of this 'Type 1' Cu-protein was deduced from chiroptical measurements which were supported by e.p.r. data. Thus Cu-thiourea turned out to be an excellent Cu(I)-donor in aqueous systems for the complete reconstitution of mononuclear Blue copper proteins.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

The role of Cu(I)-thiolate clusters during the proteolysis of Cu-thionein

Rat liver Cu,Zn-[35S]thionein and yeast Cu-thionein were subjected to proteolysis in vitro using equilibrium dialysis. The partially copper-loaded vertebrate thionein (2-7 Cu/mol) was affected by different proteases including thermolysin, proteinase K, protease from Streptomyces griseus and lysosomal enzymes. Unlike the 2Cu-thionein the respective 7Cu-thiolate-centred metallothionein was hardly proteolytically digested. In contrast to fully copper-loaded native yeast Cu-thionein both the H2O2-oxidized and the metal-free protein were effectively cleaved in the presence of proteinase K. It is important to realize that the native Cu(I)-thiolate chromophore survives the proteolytic attack. When the copper-sulphur bonding is broken and the same amount of copper is unspecifically bound to the thionein portion, proteolysis proceeds identically with respect to the rate observed in the presence of the apoprotein. The unsuccessful proteolysis of native Cu-thionein is not attributable to a simple copper-dependent inhibition of the proteinases. It is suggested that prior to proteolysis the copper-sulphur clusters must be destroyed.     

Copper-release from yeast Cu(I)-thionein by hypothiocyanite (OSCN−)

In the course of an oxidative burst oxygen free radicals and hypothiocyanite (OSCN^–), a transiently abundant derivative of thiocyanate (SCN^–), are formed in the presence of activated polymorphonuclear leukocytes (PMNs). At the same time Cu(I)-thionein is present and the question arose whether or not thiocyanate and its oxidized form may transiently release highly Fenton active copper to improve the efficacy of the above mentioned oxidative burst. Thus, the reaction of yeast Cu-thionein with OSCN^– was examined. Indeed, a release of copper from the Cu(I)-thiolate clusters of the protein was observed ex vivo. Both the chiroptic and luminescence emission signals of Cu-thionein essentially levelled off in the presence of a 15-fold molar excess of OSCN^– expressed per equivalent of thionein-copper. The effective copper-releasing activity of this reagent was confirmed by equilibrium dialysis. The demetallized protein could be reconstituted under reductive conditions. SCN^– did not affect the copper-thiolate bonding. It rather acts as a potent metabolic source for the transient copper release from Cu-thionein in the presence of activated PMNs.     

Reaction of a model siderophore with Ni(II), Co(II) and Zn(II) in aqueous solution: kinetics and spectroscopy

A spectroscopic study was performed showing that the [Fe(III)(L(2-))(2)](1-) (L(2-)=dopacatecholate) complex reacts with Ni(II), Co(II) and Zn(II) in an aqueous solution containing S(2)O(3)(2-) resulting in the soluble [M(L(1-))(3)](1-) (L(1-)=dopasemiquinone; M=Ni(II), Co(II) or Zn(II) complex species. The Raman and IR spectra of the [CTA][M(L(1-))(3)] complexes, CTA=hexadecyltrimethylammonium cation, in the solid state were obtained. The kinetic constants for the metal substitution reactions were determined at four different temperatures, providing values for DeltaH(not equal), DeltaS(not equal) and DeltaG(not equal). The reactions were slow (k=10(-11) Ms(-1)) and endothermic. The system investigated can be considered as a simplified model to explain some aspects of siderophore chemistry.     

Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and 'blue' copper apoprotein

It was of interest to examine whether yeast Cu-thionein could be used to transfer the thiolate bound copper directly into the copper binding site of 'blue' apoproteins which contain free thiol groups. In particular apo-stellacyanin was used in the present study and it was found to be able to accept Cu(I) from yeast Cu-thionein, without any detectable unspecific Cu(II) intermediate, both aerobically and anaerobically.     

Synthesis,spectroscopic, and structural characterization of the first aqueous cobalt(II)-citrate complex: toward a potentially bioavailable form of cobalt in biologically relevant fluids

Citric acid represents a class of carboxylic acids present in biological fluids and playing key roles in biochemical processes in bacteria and humans. Its ability to promote diverse coordination chemistries in aqueous media, in the presence of metal ions known to act as trace elements in human metabolism, earmarks its involvement in a number of physiological functions. Cobalt is known to be a central element of metabolically important biomolecules, such as B12, and therefore its biospeciation in biological fluids constitutes a theme worthy of chemical and biological perusal. In an effort to unravel the aqueous chemistry of cobalt in the presence of a physiologically relevant ligand, citrate, the first aqueous, soluble, mononuclear complex has been synthesized and isolated from reaction mixtures containing Co(II) and citrate in a 1:2 molar ratio at pH approximately 8. The crystalline compound (NH4)4[Co(C6H5O7)2] (1) has been characterized spectroscopically (UV/vis, EPR) and crystallographically. Its X-ray structure consists of a distorted octahedral anion with two citrate ligands fulfilling the coordination requirements of the Co(II) ion. The magnetic susceptibility measurements of 1 in the range from 6 to 295 K are consistent with a high-spin complex containing Co(II) with a ground state S=3/2. Corroborating this result is the EPR spectrum of 1, which shows a signal consistent with the presence of a Co(II) system. The spectroscopic and structural properties of the complex signify its potential biological relevance and participation in speciation patterns arising under conditions consistent with those employed for its synthesis and isolation.     

Reaction of DNA-bound Co(II)bleomycin with dioxygen.

The aerobic oxidation of Co(II)bleomycin bound to calf thymus DNA has been investigated in relation to the mechanism of reaction in solution in the absence of DNA. Kinetics of dioxygenation of the Co(II) complex were followed by spectrophotometric and electron spin resonance spectroscopy as well as dioxygen analysis. The reaction is slower than when carried out in solution; its rate is inversely related to the ratio of DNA base pairs to Co(II)bleomycin. The subsequent oxidation reaction, observed spectrophotometrically and by dioxygen analysis, is second order in cobalt complex. The calculated second order rate constant is also inversely related to the base pair to metal complex ratio. Once this ratio exceeds three, the reaction rate slows significantly with each additional increment of DNA added to the starting reaction mixture. Taking advantage of the high stability of O(2)-Co(II)bleomycin bound to greater than a 3-fold excess of DNA base pairs, it could be demonstrated that the rate constant for oxidation of two O(2)-Co(II)bleomycin molecules is much slower than that for O(2)-Co(II)bleomycin plus Co(II)bleomycin. With the same technique it was observed that the metal centers of O(2)-Co(II)bleomycin and Fe(II)bleomycin also undergo oxidation. The binding to DNA of both solution products of the oxidation of Co(II)bleomycin by O2 was examined by 1H NMR spectroscopy. Peroxy-Co(III)bleomycin, Form I, binds with higher affinity than Co(III)bleomycin, Form II. At lower ionic strength, the size of the DNA binding site for each form is about 2 base pairs/molecule of drug.     

Spectral studies of cobalt (II)- and Nickel (II)-metallothionein

The zinc and cadmium of native rabbit metallothionein-1 were replaced stoichiometrically with either cobalt (II) or nickel (II). The electronic, magnetic circular dichroic (MCD), and electron spin resonance spectra of Co (II)-metallothionein reflect distorted tetrahedral coordination of the cobalt atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple cobalt-tetrathiolate complexes, implying that their coordination chemistry is analogous. Ni (II) complex ions and Ni (II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The circular dichroic bands associated with ligand-metal charge-transfer transitions in the non-d-d region of Co (II)- and Ni (II)-metallothionein afford additional evidence for the similarity in tetrahedral microsymmetry of the two metal derivatives. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters.     

Synthesis, characterization and antimicrobial activities of some mixed ligand complexes of Co(II) with thiosemicarbazones and N-protected amino acids

The reaction of cobalt(II) chloride with a new class of thiosemicarbazones viz; cis-3,7-dimethyl-2,6-octadienthiosemicarbazone(CDOTSC; L(1)H) and 3,7-dimethyl-6-octenethiosemicarbazone (DOTSC; L(2)H) and N-phthaloyl derivative of DL-glycine(A(1)H), L-alanine(A(2)H) or L-valine(A(3)H) in 1:1:1 molar ratio in dry refluxing ethanol have been studied. All the isolated complexes have the general composition [Co(L)(A)]. Tentative structures are proposed for these complexes based upon elemental analysis, electrical conductances, magnetic moment, molecular weight determination and spectral (IR, electronic) studies.The ligands and Co(II) complexes have been tested for their antibacterial and antifungal activities against three bacterial strains S. aureus, B. subtilis, E. coli and two fungal strains F. moniliformae and M. phaseolina. Attempts have been made to establish a correlation between the antibacterial and antifungal activity and the structures of products.     

Analogous copper(I) coordination in metallothionein from yeast and the separate domains of the mammalian protein

The three-dimensional structures of both vertebrate Cu12-metallothionein (class 1) and yeast Cu8-thionein (class 2) are still unknown. The different copper:protein stoichiometry compared with that of the (ZnCd)7-metallothioneins was expected to alter the metal-thiolate cluster structure considerably. In order to avoid possible domain interactions in the hepatic rat metallothionein, separate chemically synthesized alpha- and beta-domains were used rather than the apoprotein. Apo yeast thionein, and the alpha- and beta-domains of rat liver metallothionein-2 were reconstituted by Cu(I) titration. Reconstitution steps were monitored using spectroscopic methods including luminescence emission and circular dichroism. Upon UV irradiation a linear increase in intensity of the orange-red luminescence was observed near 600 nm up to 6 Cu eq using either compound regardless of the different cysteine sulfer content (yeast thionein 12S, alpha-domain 11S, beta-domain 9S). The characteristic dichroic properties of the yeast copper-protein between 240 and 400 nm were in good agreement with those of the respective class 1 metallothionein domains. All observed Cotton bands were of similar shape and appeared in the same wavelength regions. However, the molar ellipticities were less pronounced in the alpha- and beta-fragments employed. There appears to be a striking similarity between the oligonuclear Cu(I) binding centers in all metallothionein species.     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Preparation of Radioactive Labelled (57Co) Sulfitocobalamin

Abstract

Farquharson and Adams (Br. J. Nutr. 36, 127-135 (1976)) have identified sulfitocobalamin (S0₃?Cbl) as one of the naturally occurring cobalamins (Cbls) in foods. We have devised a method of making radioactive labelled S0₃?Cbl for invivo and in vitro studies of this form of Cbl. ⁵⁷Co labelled cyanocobalamin (⁵⁷Co CN-Cbl) was acid photolyzed to ⁵⁷Co hydroxocobalamin (⁵⁷Co OH-Cbl) followed by ligand substitution with S0₃ ^?2 ion from aqueous sodium (meta) bisulfite in the dark. The resulting ⁵⁷Co SO₃?Cbl was purified by organic extraction and cation ex-change chromatography. The final preparation was >99% Co⁵⁷ S0₃?Cbl with an overall yield of >70%, stable for up to four weeks at 20°C in the dark, and capable of binding to the human Cbl binding proteins Transcobalamin II (TC II), Intrinsic factor (IF) and Salivary R. This method allows a simple 1 day preparation of high specific activity labelled ⁵⁷Co S0₃?Cbl for biological studies.