Hans-Jürgen Hartmann

Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase

Lipid peroxidation in mitochondrial membranes results in loss of respiratory control, lack of contractibility and final lysis of mitochondria [l-3]. It may be induced by incubation of isolated mitochondria in the presence of oxygen with either chaotropic agents [4] or autoxidizable substances like ascorbate [2] or thiol compounds [3]. Besides, formation of lipid peroxides in livers of tocopherol deficient rats has been observed under in vim conditions [5 ] and may, therefore, have a pathophysiological significance. Two enzymes, GSH peroxidase (C-factor I) [l] and catalase (C-factor II) [ 11, were found to prevent peroxidation of unsaturated lipids in suspensions of mitochondria or “high amplitude swelling” which is related to lipid peroxidation [2, 3,6, 71. These observations suggest an involvement of H202 which may for instance be built according to the equation

Copper-thionein from fetal bovine liver.

It was of interest to examine whether or not a low molecular weight copper-rich metal-thionein was present in biological species which received no metal pretreatment at all. From bovine fetal liver an 8 Cu 2 Zn-thionein having a molecular weight of 11 500 was successfully isolated. 16% of the total copper present in the whole liver were recovered in this protein. During the isolation process anaerobic conditions had to be maintained to avoid uncontrolled oxidation leading to polymeric species and the loss of most of the copper. The similarity of both the present copper-thionein and the polymeric neonatal type mitochondrocuprein was shown. A comparison of different copper-thioneins containing variable amounts of copper was possible when xiCu from 280 nm to longer wavelength was determined. With respect to the ultraviolet properties there were no detectable differences between copper-thioneins prepared either in vivo or in vitro and the fetal copper-thionein. Furthermore, the positions of the Cotton effects as deduced from circular dichroism measurements were rather similar although the magnitude of the observed Cotton extrema was less pronounced and sometimes the signs were reversed. X-ray photoelectron spectrometric studies revealed a Cu(2p3/2) binding energy value of 932.9 eV. Unlike the S(2p1/2,3/2) value near 162 eV using Cu-thioneins from chicken liver or yeast the higher S(20p1/2,3/2) binding energy of 163.0 eV employing fetal Cu-thionein was attributed to partial oxidation of the protein moiety and/or a particular chemical environment. The second S(2p1/2,3/2) peak was assigned to the copper catalyzed oxidation of sulphur via OH to yield RSO-3. In the X-ray photoelectron spectrum of the apoprotein one homogeneous S(2p1/2,3/2) band at 163.7 eV was seen attributable to RSSR.

Homologous copper(1)-(thiolate)2-chromophores in yeast copper thionein

Abstract Improved isolation techniques were devised and successfully applied for the convenient isolation of Cu-thionein from Saccharomyces cerevisiae . Homogeneous copper-thioneins containing eight, two and one Cu per 10 000 g of thionein were obtained. No inorganic sulphide was determined and a constant stoichiometry of copper : cysteine-sulphur of 1 : 2 was measured regardless whatever Cu-thionein was examined. This 1 : 2 stoichiometry was supported by electron absorption spectrometry, circular dichroism and X-ray photoelectron spectrometry. A linear correlation of both e and θ values with the copper content was seen. The characteristic Cotton effects at 245, 283, 302, 328 and 359 nm disappeared irreversibly when Cu-thionein was oxidized with H 2 O 2 . Titration with p -chloromercuribenzoate affected these Cotton extrema in a similar way and the above observed stoichiometry of 1 : 2 was confirmed. The binding energy values of the copper 2p 3/2 core electrons at 932.3 eV and the absence of a satellite structure suggested the assignment of Cu(1) in the Cu-thioneins. The sulphur 2p-values at 162.7 eV were attributed to thiolate sulphur. Treatment with H 2 O 2 gave rise to the appearance of Cu(II) 2p 3/2 satellites at 940.0 and 942.4 eV as well as a shifted main signal to 933.9 eV. At the end of the oxidation all sulphur was oxidized to RSO 3 - (S 2p = 167.9 eV). The chemical and physico-chemical data allow the conclusion that homologous Cu(I)-(thiolate) 2 -chromophores are present in these microbial Cu-thioneins.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.

Cadmium-thiolate protein from the grass Agrostis gigantea

A cadmium‐binding protein rich in cysteine and acidic amino acid residues was isolated from roots of Agrostis gigantea. The molar ratio of cysteine to cadmium was 2.7:1. Electronic absorption and circular dichroism measurements were characteristic of cadmium‐thiolate coordination. The cadmium‐binding centre in the plant protein was strikingly similar to that of the well characterised vertebrate cadmiumthioneins.

A study on purified apo-erythrocuprein

Apo-erythrocuprein was prepared by passing freshly isolated bovine erythrocuprein through an EDTA-equilibrated Sephadex G-25 column at pH 3.8 for 8–10 h. The apoprotein proved to be homogeneous and metal free as examined by several physicochemical methods. The fine structure of the ultraviolet absorption spectrum was very distinct. The molar coefficient of absorption at 259 nm was only about one third (ϵ259 = 3670) of the corresponding value of the native protein (ϵ259 = 9840). The apoprotein was electrophoretically homogeneous and displayed one symmetrical boundary as determined by sedimentation velocity measurements. The s20,w value was 3.0 S. Absolutely no Zn2+ and Cu2+ were detected either with atomic absorption spectroscopy or, in the case of Cu2+, using electron spin resonance (EPR). The apoprotein was fairly stable in the pH range of 2.5–9.5, provided the exposure to the more extreme pH values did not exceed 5 min. From magnetic circular dichroism (MCD) data it was concluded that the protein contained no tryptophan. Furthermore, MCD measurements were found to be an elegant method for the detection of 2 moles of tyrosine per mole of apo-erythrocuprein. The reconstitution of erythrocuprein by incubating the apoprotein together with Cu2+ and/or Zn2+ was successful under both aerobic and anaerobic conditions. However, the quality of the aerobic reconstituted erythrocuprein was not fully satisfactory as shown by absorption spectra in the visible region, EPR and enzyme assay. From optical titrations of the apoprotein using Cu2+ and Zn2+ it was concluded that Cu2+ is able to produce both a profound increase of ultraviolet absorption and a change of the absorption profile, while Zn2+ was less active. However, Zn2+ was able to cause a shift of the visible absorption maximum by 50 nm. From enzymic studies using the reduction of cytochrome c by O2− it was concluded that 4 metal ions, two of which must be Cu2+, are required for optimal superoxide dismutase activity. No measurable differences between the native and the reconstituted protein were apparent at all if the reconstitution was performed anaerobically using a second Sephadex G-25 column which contained Cu2+ and Zn2+ in the upper section.

A pulse radiolytic study on the reaction of hydroxyl and superoxide radicals with yeast Cu(I)-thionein.

In a pulse radiolytic study employing aqueous intact yeast copper(I)-thionein at pH 7 it was shown that both superoxide and hydroxyl radicals efficiently react with this Cu(I)- and thiolate-rich protein. The reaction constant of hydroxyl radicals with Cu(I)-thionein was determined by competition kinetics and was 2.2 x 10(11) M-1 s-1 at a rate close to a diffusion-controlled limit. The reaction of Cu(I)-thionein with superoxide was also successful and proceeded at a rate of 7.5 x 10(6) M-1 s-1. According to chiroptical and luminescence emission measurements minor oxidation of the copper(I)-thiolate oligonuclear binding centres was observed, leading to the release of some Cu(II). It is important to realise the dual reactivity of this yeast Cu(I)-thiolate protein in controlling copper transport and storage as well as its distinct role in the scavenging of free radicals.

Copper dependent control of the enzymic and phagocyte induced degradation of some biopolymers, a possible link to systemic inflammation

The role of copper during inflammation is unknown. An attempt was made to examine the reactivity of copper on the oxygen free radical induced depolymerization of hyaluronic acid and synovial fluid. Thionein-copper and CuSO4 at 2 mumol/l concentrations inhibited the degradation of this biopolymer successfully. Translation of the enzymically generated excited oxygen species onto a cellular level was performed. Activated PMN cells were used to decompose hyaluronic acid in the presence of CuSO4, Cu-thionein and ceruloplasmin not exceeding physiological levels. All employed copper compounds inhibited the depolymerizing process. Furthermore, PMN cell induced bleaching of cytochrome c was also affected in the presence of both CuSO4 and thionein-copper.

Tetrahedral copper—sulphur coordination in yeast Cu-thionein: An EXAFS study

A copper containing thiolate-rich protein occurs in yeast [ 1,2]. Due to many similarities of the metalthiolate chromophore to that of the metallothioneins [3] it is presently termed Cu-thionein. During gel filtration the protein migrates with an apparent relative M, of 10 000 [ 21. On the basis of amino acid analyses the relative Mr value is 4800 [4] including 4 g-atoms of copper/molecule. The stoichiometry of copper to cysteine-sulphur was 1:2. guess for the arrangement of four copper-thiolate tetrahedra fitted the cubane type of many known Cu(1) compounds.

Preparation of pure bovine apo‐erythrocuprein by gel filtration

Studies on the bimetallic protein erythrocuprein have become of widespread interest [l-4] . Erythrocuprein can be isolated from erythrocytes by precipitation with organic solvents following gel and DEAE-chromatography. The metalloprotein contains two cupric and two zinc ions per mole and has an absorption ratio of AZ59 /A6s0 = 3 1 S. Almost no helical structure can be detected in the protein portion [ 1,2]. Isolation of a pure metal free preparation would enable the nature of this copper and zinc binding ligand to be investigated. The usual preparatjon of the a~protein was carried out with excessive dialysis against chelating agents 12-4 1. However, a labile apoprotein would not survive this process, so the native erythrocuprein was passed through a G-25 Sephadex column previously equilibrated with cyanide or EDTA buffer. A pure apoprotein is obtained free from smaller degradation products and low molecular weight Cu*” and Zn” chelates. The purest ape-erythrocuprein was obtained using EDTA as a chelating agent.