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This chapter focuses on thiyl radicals associated with transition metal ions some of which are able to stabilize these radicals in biological systems. Of special interest was the reactivity of iron, cobalt, and manganese. Chromium and vanadium toxicity has been found to be related with the generation of thiyl radical intermediates. The chapter examines copper-thiolate reactions in the authors' laboratory. A common feature of ionic sulfur-centered radicals is their intense electronic absorption in the visible wavelength region. Due to this phenomenon pulse radiolysis was extensively used. In general, the measurement of thiyl radicals is monitored by means of electron paramagnetic resonance (EPR) spin trapping. 5,5-Dimethyl-1-pyrroline-N-oxide, the tetramethyl derivative tetramethylpyrroline oxide, and N-t-butyl-α-phenylnitrone are frequently used as spin traps. Direct identification of the various sulfur-centered radicals by EPR is difficult. In the case of the thiyl species the direct detection is possible under special conditions.

Zusammenfassung An Ratten wurde untersucht, wie sich die Absorption des Kupfers bei Zulage verschiedener mono-, oligo- und polymerer Aminosaure-Cu-Komplexe verhalt. Die relativen Cu-Absorptionsraten wurden in erster Linie uber die Cu-Speicherung der Leber bestimmt. Die Cu-Gehalte in Niere und Gesamtkorper, die ebenfalls jeweils ermittelt wurden, scheinen hierfur weniger geeignet. Es ergaben sich folgende Ergebnisse: 1 Bei Zulage einiger mono-, oligo- und polymerer Aminosaure-Cu-Komplexe lag die relative Absorptionsrate des Kupfers hoher als bei Verabfolgung der gleichen Kupfermenge in Form von Cu-Sulfat (Cu-Aquokomplex). 2 Die Cu-Absorptionsrate nahm mit steigendem Polymerisationsgrad (Molekulgrose) der jeweiligen Cu-Aminosaurekomplexe ab. 3 Bei Verabfolgung von Cu-Leucinkomplexen war die Cu-Absorption wesentlich hoher als bei den Cu-Alaninkomplexen. 4 Die Cu-L-Aminosaurekomplexe warden besser absorbiert als die entsprechenden Komplexe mit den D-Stereoisomeren. Insgesamt zeigt sich, das die jeweiligen Cu-Aminosaurekomplexe als gesamte Komplexe absorbiert werden durften und das das Ausmas der Cu-Absorption nicht nur von der Stabilitat und Grose des Komplexes beeinflust wird, sondern auch davon, wieweit die Absorption der Liganden per se reguliert wird.

Zusammenfassung An 12 Schweinen wurden jeweils im Gewicht von etwa 15 und 30 kg Retention und Ausscheidung von Fluor untersucht. In beiden Versuchsreihen wurde etwa die Halfte des im Futter enthaltenen Fluors angesetzt. Die Absolutwerte der Retention und Excretion anderten sich mit dem Korpergewicht, die Relativwerte jedoch nicht.

Zusammenfassung Fur Untersuchungen uber den Einflus verschiedener Nahrungsbestandteile auf die Kupfer-Absorption wurde eine in vitro-Versuchsmethode ausgearbeitet. Dabei wird die Dialysegeschwindigkeit von Kupfer-Ionen mittels der Umlaufdialyse gemessen. Grundlagen und Methodik der fur physiologische Verhaltnisse modifizierten Dialyse werden beschrieben.

Zusammenfassung An drei Gruppen zu je 4 Schweinen wurden im Gewicht von 15 und 30 kg Stoff-wechselversuchsreihen durchgefuhrt, um Retention und Ausscheidung von Fe, Zn, Mn, Co und Cu bei unterschiedlichen CuSO4 · 5H2O-Zulagen (0; 0,5 und 1,0 g/kg Futter) zu untersuchen. 1. Durch die CuSO4-Zulage wurde die Co-Retention bei 15 kg Lebendgewicht ver-doppelt. 2. Bei 30 kg Lebendgewicht wurde der Co-Ansatz durch beide CuSO4-Spiegel etwa verdreifacht. Auserdem wurde ebenfalls mehr Fe, Zn und Mn retiniert. 3. Beide CuSO4-Konzentrationen erhohten den Gehalt an Cu und Co im Harn. In-vitro-Versuche zeigten, das die erhohte Retention an Spurenelementen durch eine verstarkte Absorption aus dem Magendarmtrakt verursacht wurde.

Zusammenfassung An drei Gruppen zu je vier Schweinen wurden im Gewicht von jeweils etwa 15 und 30 kg Stoffwechselversuchsreihen durchgefuhrt, um Retention und Aussdieidung von Cu und Mo bei unterschiedlichen Zulagen von CuSO4 · 5H2O (0, 0,5 und 1,0 g/kg Futter) zu untersuchen. Beide Cu-Zulagen bewirkten einen hoheren Cu-Ansatz, jedoch verursachte nur die hohere Zulage eine verstarkte Mo-Retention. In beiden Versuchsreihen war die renale Aussdieidung beider Elemente erhoht, wahrend sich die rectale Mo-Excretion verringerte.

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.

Unused ancient Egyptian embalming material unearthed at Deir el-Bahari (c. 1500 BC) shed new light on the potential preparation methods for various embalming materials in Pharaonic Egypt. Analyses revealed the presence ofphenols, guaiacols, naphthalenes and sesquiterpenoids. These components were attributed to a wood tar oil produced by a dry distillation or smouldering process from the true cedar tree. The preservation effects were elucidated at the molecular level. Bone alkaline phosphatase, an enzyme that is tightly bound in deeper regions of the bone mineral, served as a marker to reveal the efficacy of the investigated compounds. A very similar embalming material was extracted from a Ptolemaic torso (340 ′ 170 BC), showing powerful bactericidal and fungicidal activity on the mummified tissue and bones. Here, we show that both the unused and the used embalming materials are in striking accordance with Pliny's description of liquid 'cedrium', with the abundantly present guaiacol proving the most effective conserving compound.

We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-Å resolution. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag 7 -MT and similar, but not identical, to that of Cu 7 -MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.

The development of new, gentler methods/or the conservation of Old Master paintings, with an emphasis on the removal or thinning of deteriorated varnish, is of high priority. In the course of the deterioration process, oxidative attacks lead to progressive formation of carboxylic acid groups, while many ester bondings are expected to be weakened. A stable RbOH polyethylene glycol complex was a promising tool to react with these moieties and to successfully break down the polymeric varnish layers The remnants were easily wiped off mechanically. Control studies employing 8 6 RbOH and 1 4 C polyacrylate showed the complete absence of residual cleansing components on the paint surface.

In Pharaonic Egypt from the Old Kingdom up to the Ptolemaic Period the deceased were pre-treated in the course of the mummification process using a wealth of embalming components including resins and many different wood tars. GC/MS studies allowed the elucidation of a great number of clearly separated compounds found in the ancient embalming material. Phenols, guaiacols, naphthalenes, monoterpenes, sesquiterpenoids, oxidised diterpene resin acids and triterpenoids were noticed. These results and particularly the detection of an unused embalming material shed some new light on the possible way as to how the different embalming materials might have been prepared and applied. It was striking to see the accordance of the present data with the well-known treatises by Herodotus (490-425 B. C.) and by Pliny the Elder (23/24-79 A. C.). The impact of the historical observations on modern science and in return the dramatic promotion of ancient texts stimulated by the present study is intriguing. An enzyme, alkaline phosphatase, bound inside mummified bones was a useful tool to reveal the efficacy of the embalming materials. Initial data showed that alkaline phosphatase isolated from embalmed bones from the Old Kingdom and the Ptolemaic Period was significantly more abundant and displayed a higher activity compared to the properties of the corresponding enzyme from non-treated mummified bones. Additionally, in a model study porcine ribs were pre-treated with four selected embalming compounds - guaiacol, limonene, α-pinene and p-cymene - and subsequently air-dried. Among the four selected compounds guaiacol was the most reactive species in the course of the preservation process. The specific activity of bone alkaline phosphatase rose 12-fold compared to that of the control. The enzyme itself remained unharmed as the observed relative molecular mass was surprisingly identical with the contemporary enzyme. It was again striking that the guaiacol derivatives were richly abundant in the unused embalming material mentioned above.

It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast 13C-and 15N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the 13C- and 15N- labeled Cu7-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.

Recent advances in mass spectrometry make it possible to utilise isotope variations of transition metals to address some important issues in solar system and biological sciences. Realisation of the potential offered by these new isotope systems however requires an adequate understanding of the factors controlling their isotope fractionation. Here we show the results of a broadly based study on copper and iron isotope fractionation during various inorganic and biological processes. These results demonstrate that: (1) naturally occurring inorganic processes can fractionate Fe isotope to a detectable level even at temperature V1000‡C, which challenges the previous view that Fe isotope variations in natural system are unique biosignatures; (2) multiple-step equilibrium processes at low temperatures may cause large mass fractionation of transition metal isotopes even when the fractionation per single step is small; (3) oxidation^reduction is an importation controlling factor of isotope fractionation of transition metal elements with multiple valences, which opens a wide range of applications of these new isotope systems, ranging from metal-silicate fractionation in the solar system to uptake pathways of these elements in biological systems; (4) organisms incorporate lighter isotopes of transition metals preferentially, and transition metal isotope fractionation occurs stepwise along their pathways within biological systems during their uptake. > 2002 Elsevier Science B.V. All rights reserved.

The structure of the complex [CuII(PuPy)](ClO4)2 (PuPy = L = 1,8-bis(2-pyridyl)-2,7-diazaoctadiene-1,7) and the structure of the corresponding copper(I) complex were determined. In CuIIL(ClO4)2, a model compound with CuZnSOD activity, the unit CuIIL2+ has a tetrahedrally distorted square-planar N4 coordination geometry. The copper(I) complex with L was found to be dimeric, (CuIL)2(ClO4)2.DMF (DMF = N,N-dimethylformamide). The binuclear unit (CuIL)2(2+) has a helical structure with two ligands L bridging the two copper atoms to provide tetrahedral N4 coordination of each copper(I). In solutions of (CuIL)2(ClO4)2.DMF, solvent-dependent dissociation occurs according to D reversible 2M (D = (CuIL)2(2+); M = CuILSx+; S = solvent). Stopped-flow spectrophotometry was used to determine the rate constants for the dissociation of the dimer D (kM) and dimerization of the monomer M (kD) for S = acetonitrile and DMF. Equilibrium constants Kdim = kM/kD were determined spectrophotometrically. In aqueous solution, the oxidation of the dimer (CuIL)2(2+) by CoIII(NH3)5Cl2+ and cis- and trans-CoIII(en)2Cl2+ follows a second-order rate law, rate = kox[(CuIL)2(2+)][Co(III)]. Data for rate constant kox and for the activation parameters delta H++ and delta S++ are presented. In DMF, the oxidation of (CuIL)2(2+) by CoIII(NH3)5Cl2+ occurs via the monomer CuIL(DMF)x+ and the dissociation of (CuIL)2(2+) becomes rate-controlling. The reduction of CuIIL2+ by RuII(edta)H2O2- was found to be too fast to be resolved by stopped-flow spectrophotometry. The kinetic results are discussed mechanistically in terms of the redox switch aspects of the system.

The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz. The protein part constitutes 53 amino acids. A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered. A family of 30 structures was generated. Root-mean-square deviation (rmsd) values from the average structure of 0.32 ± 0.13 A and 0.61 ± 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2–40. The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined. The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior. If the same metal–sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available. The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.

The reaction of yeast Cu-MT with nitric oxide (NO) was examined. A release of copper from the Cu(I)-thiolate clusters of the protein by this remarkably important reagent was observed in vitro. The characteristic spectroscopic signals of the Cu(I)-thiolate chromophores levelled off in the presence of a two-fold molar excess of NO expressed per equivalent of thionein-copper as monitored by UV-electronic absorption, circular dichroism and luminescence emission. At the same time all of the copper became EPR detectable. The oxidized metal ions could easily be removed from the protein moiety by gelfiltration. The reversibility of the copper releasing process is of special interest. The specific fluorescence and dichroic properties of the previously demetallized protein could be recovered up to 85% under reductive conditions. Moreover, no difference in the electrophoretic behaviour was seen compared to the untreated Cu-MT. Thus, NO may act as a potent metabolic source for the transient copper release from Cu-MT. In the course of an oxidative burst this highly Fenton active copper is able to improve the efficacy of biological defence mechanisms.

Zusammenfassung In Versuchen an Ratten wurde gepruft, inwieweit die Cu-Absorption durch Zulage von Glucon-, Citronen-, Salicyl- oder Oxalsaure zur Diat beeinftust wird. Wahrend die Cu-Absorptionsrate bei Zulage von 25 mg dieser Sauren je kg Futter nicht ver-andert wurde, war sie bei Zulage von 250 mg Citronen- oder Oxalsaure vermindert.

Aquo-[N,N′-bis(2-(6-methyl-pyridyl) methylene)-1,4-butanediamine] (N,N′ ,N″,N‴)-copper(II) perchlorate, [Cu (II)-(Pu-6-MePy)-(H2O)] (ClO4)2, a copper(II)-di-Schiff base was synthesized and reduced to the Cu(I) form. The successful preparation was controlled by IR and in the case of the Cu(II) complex additionally by EPR spectroscopy. [Cu(II)-(Pu-6-MePy)(H2O)] ClO4)2 has an electronic absorption maximum at 717 nm of e717= 108 M-1 cm−1. The EPR parameters are g⊥ = 2.059 and g⊥ = 2.235 which are very close to those of the respective values of Cu(II) in intact Cu2Zn2 superoxide dismutase (SOD). The electronic absorption maximum of the Cu(I) complex is at 474 nm (e717= 12 123 M−1 cm−1) and, as expected, no EPR is seen. The crystal structure of [Cu(II)-(Pu-6-MePy) (H2O)](ClO4)2 shows a monomer while the reduced Cu(I) form reveals a dimer. Both complexes comprise the same SOD mimetic activity of 0.3% compared to that of the cytosolic enzyme. As the reduction of the Cu(II) complex proceeds within 10–40 min it is unlikely that the Cu(I) dimer is formed during the reduction oxidation cycle of superoxide dismutation which occurs at a timescale of milliseconds. The Cu(I) dimer is a promising superoxide dismutase mimicking compound in an aerobic reducing environment as, for example, in cytosol.

Controversy exists over whether embalming was already being performed in the Old Kingdom in the course of mummifying the deceased.

Bone samples of different archaeological sites and age have been employed to examine both the possible catalytic and immunological activity of Zn 2 Mg alkaline phosphatase. In six samples a protein of Mr 180±20 kD being closeto 200 kD of the contemporary enzyme was successfully isolated. The specific enzymic activity ranged from 5 to 100 mU/mg protein. In the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzymic activity was diminished. Likewise heating to 100°C and replacement of Zn(II) by Cd(II) resulted in a dramatic loss of activity. Distinct immunological activity against the human enzyme was demonstrated in the Superdex 200 gel filtrate of one Anglo-Saxon femur bone (Bidwell, UK, ad 429–664) extract.

Chemical nucleases are compounds that are able to bind to the surface of nucleic acids and are involved in the generation of free radicals which cleave nucleic acids. The aim of this study was to investigate the interaction of copper(II) tetrabenzo [b,f,j,n] [1,3,9,13] tetraazacyclohexadecine (Cu(TAAB)2+) with DNA and determine the DNase-like activity of this complex. Dichroic properties, electronic absorption and electrophoretical techniques were used to study the reaction of the complex with DNA. Cu(TAAB)2+ affected the dichroic properties and u.v. absorption of calf thymus DNA under anaerobic conditions. The anaerobic melting point of DNA was not affected in the presence of Cu(TAAB)2+. The hyperchromicity of DNA at 260 nm in the presence of Cu(TAAB)2+ with both ascorbic acid and oxygen was determined and compared with the known chemical nuclease Cu(phenantroline)2. Plasmid DNA in the presence of ascorbic acid and oxygen was effectively cleaved by Cu(TAAB)2+ in a concentration dependent manner. It is concluded that the Cu(TAAB)2+ complex is a potential chemical nuclease.

It was attempted to monitor the immunological response of monoclonal antibodies directed to human alkaline phosphatase in ancient Egyptian bones from the ptolemeic period. The intactness of the respective epitopes of the bone enzyme was successfully demonstrated in an ELISA. Fortunately, the mummified bone was not contaminated by fungi and bacteria due to the fungicidal and bactericidal reactivity of the ancient pretreatment employing resins of pistachio for mummification. The enzyme was enriched using gel chromatography, anion exchange and affinity chromatography to yield 310 ± 7 mU/mg. The enzymically active fractions of the wheat-germ lectin affinity chromatography were subjected to ELISA. The best binding affinity was detected using the monoclonal antibody BAP A while the reactions of all the other four antibodies BAP B, BAP G, BAP 4A5 and BAP 5D4 were substantially diminished.

A pulse radiolytic study using the cyclic tetrameric Schiff base N-coordinated copper complex Cu(TAAB)2+ has been performed. The reaction of the Cu(TAAB)2+ complex with superoxide revealed pseudo first-order characteristics with the rate constant of k2 = (2.9 ± 0.5) × 108 mol−1 s−1 dm3. The complex survive presence of competing serum albumin in physiological concentrations. The complex stability constant K = 1.15 × 1018 (log K = 18.06) is two orders of magnitude higher than that of Cu(II)-serum albumin (log K = 16.2). Transient changes of the stability during the oxidation/reduction process and in the presence of 600 /μmol l−1 albumin did not affect significantly either the electronic absorption of the complex or its catalytic activity.

[ N , N ′-bis(2-pyridyl-phenyl)methylene-1,4-butanediamine]-( N , N ′, N ″, N ‴)-copper( II ), Cu(II)PuPhePy, a copper(II) di-Schiff base in both its chloride and perchlorate form, has been prepared and crystallized with a yield of 50 ± 10%. IR spectra confirmed the successful preparation. The electronic absorption ϵ 690 was 136 M −1 cm −1 . The EPR parameters g ⊥ = 2.048 and g ∥ = 2.217 were in close agreement with the respective values of intact Cu 2 Zn 2 SOD. Comparison of the crystal structures of this complex and the copper binding centre of Cu 2 Zn 2 SOD revealed a virtually identical coordination chemistry. The pulse-radiolytically-determined superoxide dismutation proceeded even in the presence of a fourfold molar excess of EDTA at a rate of 0.48 × 10 9 M −1 s −1 . In the indirect nitroblue tetrazolium assay 0.27 μM of Cu(II)PuPhePy(ClO 4 ) 2 were required to cause 50% inhibition. The thermodynamic stability constant log K = 18.33 ± 0.77 was calculated from CD data. The stability being two orders of magnitude higher than that of serum albumin, a competitive biological Cu(II) chelator and its ‘model’ EDTA was deduced. Of all the many superoxide dismutase mimics examined, the present Cu complex is in closest agreement in both structure and catalytic function with the active centre of intact Cu 2 Zn 2 SOD.

Arthritis develops in DBA/1xB10A(4R) mice and Wistar rats upon intraplantar injection of potassium peroxochromate (K3CrO8), and is here quantified by whole blood chemiluminescence (CL) and 99mpertechnetate-imaging (99mTcO4-), and related to overt disease symptoms (the arthritis index). During the aqueous decay of K3CrO8 to chromate (VI), the chromium(V)-bound oxygen is released as superoxide, hydroxyl radicals, singlet oxygen and hydrogen peroxide, the same reactants, which are produced by activated phagocytes during inflammation. Reactive oxygen species (ROS) trigger the breakdown of the sulfhydryl-dependent antioxidant defence system and induce the nuclear factor kappa B-dependent expression of pro-inflammatory cytokines, which prime phagocytic NADPH oxidases to the enhanced production of ROS. During both the acute inflammatory response and the onset of the secondary response in non-injected paws, the phorbolester-stimulated ROS production of phagocytes was significantly enhanced (p0.001) and correlated well to the arthritis index (r = 0.797) and the uptake of 99mTcO4- into inflamed joints. Chromate(VI), formed during the decay of K3CrO8, contributes to the progression of arthritis by inhibition of glutathione reductase, thereby increasing intracellular H2O2 concentrations. In addition, Cr(VI) reduced to Cr(V) by ascorbate, catalyzes hydroxyl radical production in the presence of hydrogen peroxide. A stable loop forms, in which ROS, continuously produced by Cr(VI)/Cr(V) redox-cycling, drive the primary response into chronic self-perpetuating inflammation. We see the main application of K3CrO8-induced arthritis and its assessment by both 99mTcO4- imaging and chemiluminescent immunosensoring of phagocytic activity in unseparated blood as for the rapid screening of novel anti-rheumatic drugs and treatments.

Iron-substituted yeast metallothionein, Fe(II)-yeast-MT, has been studied by Mossbauer spectroscopy. The iron in the protein is in the high-spin ferrous state. As maximum metal content, 4 Fe(II)/molecule has been determined, with the 4 metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between the Fe(II) ions via bridging thiolates. In case the iron titration is less than 4 Fe(II)/apoprotein, the ions are magnetically noninteracting, with each individual Fe(II) behaving similar to Fe(II) in reduced rubredoxin.

In an EPR study employing yeast copper(I) thionein, GSH and Cu-GSH it was shown that thiyl radicals could be successfully generated from the thiolate sulfur via oxidation by photochemically formed superoxide at 77 K. The g-value was 2.036. Essentially no EPR detectable copper(II) was monitored under the experimental conditions, indicating that the oxidation reduction process is restricted to the thiolate sulfur. The Cu(I)-thiolate chromophores remained fully intact as deduced from chiroptical and luminescence measurements. Thus, copper thionein is supposed to be actively involved in the scavenging of oxygen free radicals by a reversible thiolate oxidation reduction cycle. The coordinated Cu(I) seems to serve as a prominent candidate to stabilize the transiently formed thiyl radical.

Iron(II)-substituted yeast metallothionein has been studied with Mossbauer spectroscopy. The iron in the protein is in the high-spin ferrous state. A maximum metal content of four iron(II)/molecule has been determined, with the four metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between Fe2+ via bridging thiolates. In the case where the iron titration gives a value of less than four iron(II)/apoprotein, the metal ions are magnetically non-interacting, with each individual iron(II) behaving like iron(II) in reduced rubredoxin.

The stoichiometry and molecular architecture of Cu-thionein is unknown. Thus, the oligonuclear copper-binding sites of chemically synthesized α and β fragments employing continuous flow solid phase peptide synthesis technique have been studied and compared with those of the apo-MT, intact Cd 5 Zn 2 -MT and its substituted form Cd 7 -MT.

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.

This title is not available to purchase from Royal Society of Chemistry. Please visit www.bioinorganic-chemistry.org/mils for title information. These sulfur-rich chelators, being important in metal ion homeostasis, find increasing attention. MILS-5, written by 30 internationally recognized experts, focuses on this hot topic. The reader is supported by about 20 tables, more than 80 illustrations and nearly 2000 references. This book is an essential resource for scientists working in a wide range of disciplines from environmental toxicology and inorganic biochemistry all the way through to physiology and medicine.

Small cysteine-rich proteins sharing most if not all of the general features used to define the metallothionein (MT) superfamily are found in yeast and fungi. Unlike MTs from mammalian sources, most of the known yeast and fungal MTs are Cu(I) rather than Zn(II) or Cd(II) binding proteins. The sequences of fungal MTs reported so far are quite diverse, in such a way that fungal MTs are assigned to six different families. Family 8 contains the MTs with the highest similarity to the N-terminal domains of mammalian MTs. The best characterized member of this family is isolated from the ascomycete Neurospora crassa. It represents a copper-induced polypeptide of only about 25 amino acid residues and harbors a single cluster made up of six Cu(I) that are bound to its seven cysteine residues. The MTs assigned to families 9 and 10 are MT-1 and MT-2 found in the human pathogenic yeast Candida glabrata. The regulation of these proteins employing a copper sensitive transcription factor shares the same principle as were described for the MTs found in Saccharomyces cerevisiae, Cu-MT and Crs5, that are assigned to families 12 and 13. S. cerevisiae Cu-MT is the only MT, of which the structure including its Cu(I)8-thiolate core has been revealed. It should be emphasized that this is the largest known Cu cluster in biological systems. Besides the presentation of these well studied aspects, the open questions of Cd(II) and Zn(II) binding in yeasts and fungi are addressed and future directions of the MT research are discussed.

The aqueous decay of K3CrO8 was used to compare the reactivity of Cu2Zn2 superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate or biuret type Cu(II) chelates these di-Schiff-base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence by 50%. Furthermore, a lucigenin amplified chemiluminescence assay based on isolated polymorph nuclear leucocytes in the absence and presence of whole, unseparated blood was developed and successfully employed. CuPu(Im)2 and CuPu(Py)2 equivalent to 0.5 and 0.8 SOD units, only, were required to inhibit the photon emission by 50% in the absence of bovine serum albumin. Even in the presence of 600 microM albumin mimicking the competitive copper chelation in biological fluids Cu-Pu(Py)2 and CuPu(Im)2 remained active, whereas the carboxylate- and biuret type chelates Cu(Sal)2 and Cu(Ser)2 reacted like CuSO4. The same reactivity of these low M, SOD mimics was seen in human blood.

A peptide corresponding to the α-domain of rat liver metallothionein-2 was chemically syn­thesized employing the solid phase peptide synthesis technique. Its luminescence properties that depend on the coordinated Cu(I) have been studied using luminescence spectrometric titration in the presence of Cu(I). Unlike the intact metallothionein which has been converted into the Cu species, the emission and excitation spectra of the Cu-α-fragment showed a red shift by 20 nm and 65 nm, respectively, suggesting a more compact and stable luminophore in the a-domain. Saturation of Cu(I) coordination was reached in the presence of 6.5 mol eq Cu(I) when the α-fragment was used and 12 mol eq Cu(I) were specifically bound by the intact metallothionein. The emission bands were homogeneous and no decline of the cluster struc­ture was observed when excessive Cu(I) was added after saturation. A rearrangement of the Cu-cluster in metallothionein during its formation seems to be plausible.

The structural intactness of many biopolymers in mummified tissues is well documented. By way of contrast no functional evidence of an isolated protein is known. The well defined air dried mummy of a deceased 16±2 year male teenager 1200 B. C. prompted us to search for the possible presence and function of Cu2Zn2superoxide dismutase or remnants thereof. Using two different assay systems unequivocal proof of a specific Cu2Zn,superoxide dismutase activity in brain extract was demonstrated. Fast protein liquid chromatography of the extract on Superose 12 revealed an approx. 5 kD Cu-containing polypeptide to which activity was assigned. It is assumed that during the ageing process considerable portions of the 31.3 kD homodimer were cleaved leaving this active core up to the present date. This would be the first case of the presence of an enzymically active component surviving the past 3000 years of mummification.

In unseparated human blood the reactivity of yeast copper (I)-thionein on TPA-activated polymorphonuclear leukocytes was evaluated and compared with low Mr copper chelates exerting Cu2Zn2 superoxide dismutase mimetic activity. Cu, 18 microM, in the form of Cu-thionein was sufficient to inhibit the superoxide production of activated human blood phagocytes by 50%. Furthermore, the scavenging of hydroxyl radicals and singlet oxygen by Cu(I)-thionein was determined, using the 2-deoxyribose fragmentation assay induced by decaying K3CrO8 and the NADPH oxidation caused by UVA illuminated psoralen, respectively. The inhibitory reactivity of Cu-thionein in both assays was compared with that of serum proteins including albumin, ceruloplasmin, transferrin, and ferritin. The galactosamine/endotoxin-induced hepatitis in male NMRI mice was used to evaluate the antiinflammatory reactivity of Cu-thionein in vivo. The serum copper, superoxide dismutase, and sorbitol dehydrogenase concentrations, as well as the activity of polymorphonuclear leukocytes in unseparated blood seemed most appropriate to quantify the protective capacity of Cu-thionein in the course of an oxidative stress-dependent liver injury. The intraperitoneal application of 32.5 mumols/kg thionein-Cu limited this damage to 45%.

In unseparated human blood the Cu2Zn2 superoxide dismutase mimetic reactivity of several differently coordinated low Mr copper chelates on TPA-activated polymorphonuclear leukocytes was evaluated and compared to their apo-chelates, CuSO4, and the native enzyme. Similar to intact superoxide dismutase, 350-400 nM Cu flexibly complexed in a di-Schiff base mode in CuPu(Py)2 and CuPu(Im)2, respectively, was sufficient to inhibit the oxidative burst-dependent superoxide production of human blood phagocytes by 50%. Acetate- or biuret-type copper chelates behaved like CuSO4. The catalytic superoxide dismuting reactivity of the di-Schiff base active center analogs of SOD was confirmed using isolated porcine PMNs. Even in the presence of 600 microM albumin as a model for competitive copper chelation in biological fluids CuPu(Py)2 and CuPu(Im)2 remained active. The stability during the Cu(I)/Cu(II) redox cycling was demonstrated in the presence of activated PMNs and albumin, taking advantage of the electron paramagnetic properties of CuPu(Py)2 and CuPu(Im)2.

The elucidation of the molecular side of copper transport in biological systems is a promising task. In this context the transfer of ingested copper into the portal blood plasma was examined. Intralumenal addition of 200 microM copper caused the release of Cu-thionein into the venous effluent. This Cu-thionein became detectable after prior perfusion of the porcine small bowel using a modified isotonic phosphate-buffered saline (Pi/NaCl) medium. The protein was characterized by gel chromatography, luminescence, electronic absorption and immunological identification. ELISA and immunoblotting employing a murine monoclonal antibody to rat liver metallothionein-I proved to be most convenient. Using buffer-loaded sacs of porcine jejunum into which Cu2+, Zn2+ and Cd2+ were added, the release of metallothionein into the serosal fluid was successfully seen by ELISA. The observed excretion of metallothionein into the portal compartment may be a genuine metal transport system for many biochemically active metals.

CuPu(Py)2 and CuPu(Im)2, two novel dischiffbase coordinated low Mr active centre analogues of Cu2Zn2 superoxide dismutase, were shown to effectively catalyze the production of hydroxyl radicals in the presence and absence of TPA-activated polymorphonuclear leukocytes. These stable copper chelates exhibited a pronounced anticarcinogenic reactivity in male Sprague Dawley rats implanted with Walker 256 carcinosarcoma cells. When four doses of 5 mumol/kg CuPu(Py)2 and CuPu(Im)2, respectively, were administered intratumorally, reduction in tumor size, delay of metastasis and a significant increase in survival of the hosts were observed, resulting in 75% of total remissions. 60% of the animals recovered totally from the carcinosarcoma, when CuPu(Py)2 was applicated intravenously.

Sulphur containing amino acid residues are found among the structural and functional constituents of many proteins. The ease of oxidising sulphur in these biologically important polymers is well known. At present, there is no simple and fast technique available to monitor the respective oxidation states using chemical reactions. X-ray photoelectron spectroscopy has proven to be both convenient and efficient to follow these electron transport reactions. In the present survey a choice of some sulphur and selenium bearing proteins and metalloproteins will be examined upon excessive irradiation, treatment with hydrogen peroxide and cleavage of metal sulphur bonding. It will also be shown that the use of this technique in general will allow a quick decision on the rate of deterioration of a protein. The limitations of this spectrochemical approach will be debated.

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.

In addition to the established control of acid secretion of the class of proton pump inhibitors (PPI) reactivity from the pyridyl methyl sulphinyl benzimidazole type a second independent anti-inflammatory reactivity was observed in vitro. This inhibitory reactivity was clearly noticed using three different assays where the aggressive hydroxyl radicals were successfully trapped in a concentration dependent manner. There is unequivocal evidence that the proton pump inhibitors having the sulphoxide group are able to scavenge hydroxyl radicals which are generated during a Fenton reaction. By way of contrast, the corresponding thioethers were substantially less active. No detectable effect was seen in the superoxide radical scavenging system. In conclusion, pantoprazole as well as the other proton pump inhibitors have a pronounced inhibitory reactivity towards hydroxyl radicals.

Superoxide dismutases are ubiquitously distributed in all biological systems including prokaryotic, eukaryotic and plant cells. These proteins contain either copper and zinc or manganese or iron in the active centre, respectively. During the catalytic activity, the superoxide anion is converted into dioxygen and hydrogen peroxide. Cu2Zn2 superoxide dismutase is one of the most intensively studied metalloproteins. The primary structure of many of these proteins has been elucidated. A convincing structure-function correlation was deduced from biophysical and chemical data. The influence of the protein backbone on the reactivity of the metal was thoroughly studied. Of special interest was the catalytic centre of Cu2Zn2 superoxide dismutase. The molecular architecture of the phylogenetically older iron and manganese enzymes is far less understood. At present, the clinical and biochemical aspects of reactive oxygen species enjoy a marked interest, as they are thought to be responsible for the oxygen derived cell-damage. This may reflect the overwhelming number of different assay systems described for the evaluation of superoxide dismutase activity. The most suitable and convenient assays are briefly discussed. The model chemistry for superoxide dismutases of low molecular mass complexes of transition metals is reviewed. Emphasis is placed on the biological significance of superoxide dismutase active copper ligands, frequently used as antiinflammatory drugs. The interaction of reactive oxygen species with superoxide dismutases and the biochemical and clinical relevance of this enzyme are critically summarized.

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocy- anin from Carcinus aestuarii contains three major and two minor structural sub- units. Here, we reveal the primary structure of the γ-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spec- troscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the bind- ing metal ion. To study the quenching effect of the active site copper ions in hemocy- anins, the copper complex Cu II (PuPhPy) 2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains con- nected to three O-glycosylated and one N-glycosylated sites were found.

Dried varnish is rich in many ester moieties, which may be broken down into small, soluble compounds by esterase activity or alkaline hydrolysis. Two methods for varnish removal have been developed, including the treatment of either lipase or RbOH / PEG-400 crown ether which allow aged oil varnishes or paint coverings to be removed or thinned. These techniques are designed to proceed in a controlled manner without damaging lower paint or base layers. Unfortunately, lipase did not react with the aged ester groups of dried linseed oil varnish. Surprisingly, the varnish came off in the presence of Tris buffer alone which, in addition, formed reactive metal complexes. A better choice was the use of high M(r) alkali ion polyethylene glycol-400 (PEG-400) crown ether type chelates. PEG-400 complexes alkali ions including rubidium and other alkaliions impeding the diffusion of their basic counter ions into lower varnish or paint layers. Possible migration of alkali metal ions into the paint layer during alkaline varnish removal was determined by labelling the cleansing solutions with (86)Rb. Fortunately, varnish is degraded on the surface only. Lower paint or varnish layers are not attacked even if chemically similar to the varnish or over painting to be removed as virtually no (86)Rb was detected on the paint surface.

It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast 13C-and 15N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the 13C- and 15N- labeled Cu7-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.