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Mummification processes in Pharaonic Egypt were successful using sodium salts. Quite frequently sodium concentrations in mummified bones ranged from 300 to 4000 micromol/g. In the search for an effective inorganic conservation compound our choice fell on boric acid. The possible presence of borate in mummification salts used in Pharaonic Egypt was of special interest both historically and biochemically. In two salt samples, one from the embalming material of Tutankhamen (18th dynasty, 1336-1327 BC) and the second from Deir el-Bahari (25th dynasty, 700-600 BC) borate was found, amounting to 2.1+/-0.2 and 3.9+/-0.1 micromol/g, respectively. In five of the examined bone fragments from the Junker excavation at Giza (Old Kingdom) similar borate concentrations i.e., 1.2 micromol borate/g bone were seen. It must be emphasized that the usual borate content of contemporary autopsy is far below the detection limit. The elevated borate content in both mummification salt and ancient bone samples support the suggestion that borate-containing salt had been used. There is a striking correlation of both borate concentration and alkaline phosphatase activity. When both sodium salts and borate were essentially absent no activity at all was detectable. With increasing borate concentrations the enzyme activity rises significantly. Attributable to the distinct biochemistry of the tetrahydroxyborate anion it was of interest whether or not borate may stabilize alkaline phosphatase, an important and richly abundant bone enzyme. This enzyme was chosen, as it is known to survive more than 4000 years of mummification. In the presence of borate oligomeric species of this zinc-magnesium-glycoprotein at 400,000 Da became detectable. Attributable to this borate-dependent stabilization of the enzyme molecule a significant temperature resistant increase of the enzymic activity was measured in the presence of up to 2.5 mM borate.

Blood-tissue exchange and homeostasis within the organs depend on various interactions between endothelial and perivascular cells (Buniatian, 2001). Podocytes possess anatomical and cellular features intermediate between those of astrocytes and hepatic stellate cells (HSCs). Podocytes, like HSCs, are associated with fenestrated capillaries and, similar to astrocytes, interact with the capillaries via the basement membrane and participate in permeability-limiting ultrafiltration. The fact that podocytes come in direct contact with xenobiotics prompted us to investigate whether they express metallothionein (MT), an anticytotoxic system characteristic of astrocytes. In comparative studies, cryosections of 1- and 3-month-old rat kidney and adult rat brain, as well as podocytes and astrocytes from early and prolonged primary cultures of glomerular explants and newborn rat brain, respectively, were investigated. The cells were double-labeled with antiserum against glial fibrillary acidic protein (GFAP) and monoclonal antibody (MAb) against the lysine-containing epitope of Cd/Zn-MT-I (MAb MT) or MAb against alpha-actin. In kidney sections, MT immunoreactivity was detected in GFAP-positive glomerular cells and in interstitial fibroblasts. The pattern of staining for MT and GFAP in glomerular cells was similar to that of astrocytes in vivo. In glomerular cell cultures, MT was expressed in cobblestone-like podocytes which contained Wilms' tumor protein and lacked desmin. MT was upregulated at later culture periods, during which podocytes acquired features typical of undifferentiated astrocytes. This study hints at the existence of common regulatory mechanisms of blood-tissue interactions by neural and non-neural perivascular cells. These mechanisms appear to be used in an organ-specific manner.

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.

The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.

X-ray absorption spectroscopy has been applied to the in vivo examination of copper-resistant yeast cells. The in vivo structure of the metal-binding site of the accumulated copper has been compared to that of the purified yeast thionein. Analysis of the EXAFS spectra performed on intact yeast cells indicates that the accumulated copper is univalent and is exclusively coordinated to sulfur atoms at a distance of 219 pin with an average coordination number of 2. In contrast, the purified protein indicates a univalent copper trigonally coordinated to sulfur at a distance of 221 pm. These discrepancies are discussed in terms of copper location in the resistant yeast cells.

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.

Copper complexes with superoxide dismutase (SOD) activity show a wide range of pharmacological activities. We have investigated the effect of ([ N,N ′-bis(2-pyridylmethylene)-1,4-butanediamine]-( N,N′,N″,N″ ))-Cu(II)-chloride (CuPuPy) and a[ N,N ′-bis(2-pyridylphenyl)methylene-1,4-butanediamine]-( N,N′,N′″ a-Cu(II)-chloride (CuPuPhePy) on the multiple catalytic functions of rat brain No synthase (NOS). Both drugs inhibited the formation of l -citrulline as well as the enzymatic reduction of cytochrome c. The uncoupled oxidation of NADPH, catalyzed by neuronal NOS in the absence of l -arginine, was inhibited by CuPuPy but stimulated by CuPuPhePy, suggesting that the phenyl-substituted compound acts as a parasitic electron acceptor. Our data identify copper complexes with SOD mimicking activity as a novel class of neuronal NOS inhibitors blocking the reductase (diaphorase) activity of the enzyme.

The membrane permeability and intracellular fate of ([N,N'-bis(2 pyridyl-phenyl-methylene)-1,4-butanediamine](N,N ',N",N"')-copper(II))-diperchlorate (CuPuPhePy), a copper-diSchiff-base complex of superoxide dismutase(SOD)-mimetic activity surviving biochelation, were examined using rat hepatocytes. Lipophilicity was quantified by determining the octanol/water partition coefficients (K(p)) employing PBS as the aqueous phase. K(p)(octanol/water) was close to 1 (0.7 +/- 0.31) for Cu-PuPhePy. The complex associates with phosphatidylcholine liposomes, as deduced from ultracentrifugation and gel filtration experiments. The ability of the complex to permeate cellular membranes was proven by correlating copper release and viability of rat hepatocytes preincubated with CuPuPhePy and treated with digitonin and diethylmaleate (DEM), respectively. The toxicity and reactivity of CuPuPhePy (LD (50) approximately 10 muM for rat hepatocytes under the given conditions) were higher than those of CuSO (4)(LD(50) approximately 16 mu M) and CuZn-SOD (no toxicity in the tested range of concentration). Unlike CuSO(4) and CuZn-SOD, the toxicity and reactivity of the diSchiff-base complex were increased (LD(50) approximately 5 muM) when the concentration of intracellular glutathione was reduced to 16% of the initial content, by preincubating the cells with DEM. The toxicity of Cu-PuPhePy paralleled lipid peroxidation. This phenomenon was strongly enhanced when Cu-PuPhePy and cumene hydroperoxide (CumOOH) were simultaneously allowed to react with rat hepatocytes. This effect was intensified following preincubation with DEM. A decline in Cu(II)-EPR signals was indicative of the reduction of CuPuPhePy by GSH and liver extract, respectively. The concomitant formation of the Cu(I)-GSH complex during this reduction was monitored by the formation of luminescent Cu(I)-thiolate chromophores.

The physiologically important copper complexes of oxidized glutathione have been examined by electron spin resonance (ESR) spectroscopy in aqueous solution at neutral pH. Low temperature measurements show that the Cu(II) binding site in oxidized glutathione has the same ligand arrangement as in copper complexes of S-methylglutathione, glutamine, glutamate and glycine. The site is composed of the amino nitrogens and the carboxyl oxygens of two gamma-glutamyl residues; there is no interaction with amide nitrogens, the sulphur bond or the glycyl carboxyl groups. At high metal to ligand ratios a binuclear species exists, in which each Cu(II) binds only to one gamma-glutamyl residue. The previously reported forbidden transition detected at g = 4 is due to non-specific aggregation and not to spin coupling of intramolecular sites. Liquid solution ESR spectra show the Cu(II)-glutathione complex has a lower mobility than the corresponding Cu(II)-S-methylglutathione species. From the degree of spectral anisotropy the complex with glutathione is calculated to exist as a dimer. These results demonstrate that the physiologically relevant complex between copper and oxidized glutathione in solution is completely different from the known solid state structure determined by crystallography.

In addition to the well known catalytically accelerated O2 dismutation, Cu2Zn2 Superoxide dismutase (SOD) reversibly reduces NO to NO− with the consequence of a prolonged half-life of NO. This alternative reactivity was examined in the presence of the intact CuZn enzyme and a diSchiff base copper complex prepared from putrescine and pyridine-2-aldehyde (Cu-PuPy) which is known as a convenient active center analog of the former copper protein. The reaction of this SOD mimick with NO and NO− was monitored by electronic absorption and electron paramagnetic resonance (EPR) spectroscopy via the formation of nitrosylmyoglobin. Cu-PuPy reacted up to three times faster with NO compared with Cu2Zn2 SOD and 15 times faster in comparison with CuSO4 and copper EDTA. The oxidation rate of NO− by Cu-PuPy was up to 300% higher compared with the reactivities of CuSO4 and Cu EDTA. Cu2Zn2SOD reacted with NO− to a neglible extent only. Catalytic characteristics could be observed in the course of the oxidation of NO− in concentrations between 1 and 20 μM copper. Disturbances of the EPR properties suggested a modification of the chemical environment at the copper sites in both the copper complex and the enzyme. As a consequence, no further reactions of the nitrogen monoxides with the respective active centers were seen. In conclusion, Cu-PuPy appears to be an efficient moderator of the biochemical reactivity of nitrogen monoxides attributable to the observed increased half-life of NO.

Our knowledge to the mode of conservation of mummified structurally and functionally intact biopolymers is limited. Rib samples of a well-preserved 2300-year old ptolemeic mummy were examined whether or not functionally active Zn 2 Mg alkaline phosphatase could be detected. A protein of M rmr 170 ± 20 kDa being close to 200 kDa of the enzyme of fresh bones was successfully isolated. Both a 200 kDa protein and a distinct subunit of 60 kDa were seen in SDS-PAGE electrophoresis which was identical to those of fresh bone alkaline phosphatase. There was a significant enzymic activity of 17 mU/mg protein which could be inhibited in the presence of l -homoarginine and 1,10-phenanthroline.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

Bone samples of a ptolemeic mummy have been employed to study the mode of conservation on the intactness of Zn2Mg alkaline phosphatase in both structure and catalytic activity. A protein of Mr = 190 ± 10 kDa being identical to the 200 kDa enzyme of fresh human bones was successfully isolated. Regardless of age 200 kDa protein bands and a distinct sub­ unit at 60 kDa were seen in SDS-PAGE electrophoresis. The 200 kDa band was also monitored by activity staining. The specific activity was 120 mU/mg and 65% of the respective activity obtained in the identical preparation using fresh human tibia or rib. The enzymic activity was inhibited in the presence of 1,10-phenanthroline and ʟ-homoarginine. Radiocarbon dating supported the assignment of the mummy to the early ptolemeic period. Among the many bactericidal and fungicidal components employed for mummification were aromatic alcohols, mono-and sesquiterpenes. Pistachio resin was the major balm resin used. The microbiological sterility of the bone surface was ascertained by independent bacterial and fungal examinations

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 · 1011 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 · 106 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.

In order to exclude possible interferences in the many indirect superoxide dismutase (SOD) activity measurements using the copper-putrescine-pyridine complex (Cu-PuPy) a pulse radiolytic study on this diSchiff base copper complex has been devised and sucessfully performed. The reaction kinetics and rate constants of pulse radiolytically generated superoxide in the presence of Cu-PuPy reveal pseudo first-order characteristics. The rate constant (k 2 = 6 ± 1 × 108 m −1 s −1) is comparable to that of an Fe-SOD and is approximately a factor of 3 lower than that of bovine Cu2Zn2-SOD. The superoxide dismutating activity of Cu-PuPy shows a more pronounced temperature dependence compared with that of Cu2Zn2-SOD. Arrhenius analyses yielded activation energies of 7.8 ±0.3 and 4.6 ± 0.2 kcal mol−1 for Cu-PuPy and Cu2Zn2-SOD, respectively. The rate constant of the reaction of superoxide and Cu-PuPy is highest at pH 5.0. The possible application of Cu-PuPy for new therapeutic strategies on all types of inflammatory diseases appears to be promising.

The molecular side of copper transport in biological systems is unknown. It was attempted to examine the copper and metallothionein (MT) release into the portal blood in rats in vivo. After direct administration of Cu(II) into the jejunum the copper and MT levels were distinctively higher in the portal venous serum compared with that of the vena cava inferior. MT in gel filtrated serum samples was analyzed immunologically employing ELISA and a monoclonal antibody to rat MT-I. Affinity chromatography on Protein A-Sepharose resulted in a higher immunoreactivity in the portal compartment as deduced from an elevated MT-antibody complex. It is assumed that MT serves as a genuine transport system for cuprous copper during the mucosal-to-serosal flux of this biologically important transition metal.

The reductive activation of N,N'-bis(2-pyridylmethylene)-1-4-butanediamine (N,N′,N″,N″′)-Cu(II)-diperchlorate (CuPUPY), a di-Schiff base copper complex with antineoplastic properties, was investigated in vitro in the presence of glutathione, ascorbate, NADH or NADPH. Glutathione and ascorbate but not the pyridine dinucleotides were able to reduce the compound. The apparent second order rate constants of the reduction reaction (9.6 ± 2.0 M−1 sec−1 for ascorbate and 94.7 ±1.9 M−1 sec−1 for glutathione) indicate that glutathione is more effective by about one order of magnitude in reducing CuPUPY than ascorbate. Reduction by glutathione triggered a CuPUPY-supported redox-cycle with oxygen yielding H2O2. Whereas reduction by ascorbate was reversible, CuPUPY reduced by glutathione reacted with excess reduced glutathione (GSH) in a ligand exchange reaction yielding a GSH-Cu(I) complex which was reoxidized by O2, forming a complex between copper(II) and oxidized glutathione. These results suggest a dual role for the reduced thiol tripeptide; promoting oxidative stress induced by CuPUPY at low concentrations and inhibiting it at high concentrations. This hypothesis was verified by showing that optimum glutathione/CuPUPY ratios are needed in order to obtain maximum oxidative damage to both DNA and albumin in vitro. Evidence was obtained for the occurrence of the same reaction pathway in human KS62 erythroleukemia cells: CuPUPY was more toxic to cells in which glutathione synthesis was inhibited by buthionine sulfoximine. Moreover, ESR spectroscopy revealed alterations in the hyperfine structure of the Cu(II) spectrum, consistent with the occurrence of ligand-exchange reactions in K562 cells.

Regardless of the weak immunological response against the low- M r metallothioneins (MTs) the production of murine monoclonal antibodies (mAbs) to monomeric rat liver MT-I was successful. ELISA revealed two groups of mAbs which exhibited different specificities as examined on the native and the lysine-residue modified antigen (Ag). One “lysine-directed mAb” group, consisting of three mAbs, exhibited a specific immunoreactivity with the lysine-containing epitopes of MTs. Their role in the antigenicity of MTs was examined by modifying these residues using glutaraldehyde (GA). Titration with GA resulted in a progressive decline in Ag recognition in the immunoblot; this was completely leveled off when equimolar concentrations were reached. A similar response employing the Ga-modified protein in the ELISA was noticed. The second group of mAb cross-reacted with various MTs of different origin, indicating that the common, lysine-free NH 2 -terminus is exclusively recognized. In direct ELISA of cross-linked MTs, the observed reactivities were much more pronounced. Iodoacetamide (IA) modification of the lysines confirmed the above observations of the GA-derived Ag. Notably, the immunoreactivity was not affected when the cysteine residues were IA-carboxymethylated, nor did the subsequent loss of metals diminish the immunological response in the immunoblot.

Aqueous extracts (ENOCW) and enzymatic digests of purified Nocardia opaca cell wall fragments, virtually free of muramyl peptides, were monitored for their phagocytic response modifying reactivity on polymorphonuclear leucocytes, separated or unseparated in whole human blood. In the presence of ENOCW a 74% increased production of superoxide during the respiratory burst of TPA-activated polymorphonuclear leukocytes was observed, as compared to the unprimed control. Delipidation of this preparation resulted in a further increase in reactivity (144%). Even in the presence of whole human blood, as a model for competitive binding in biological fluids, an enhanced generation of superoxide by TPA activated blood phagocytes remained detectable. A 37–75% decreased phagocytic reactivity in samples of HIV-seropositive blood was considerably restored in the presence of ENOCW.

Active centre analogues of Cu2Zn2superoxide dismutase were devised and successfully employed. Emphasis was placed on the flexible nature of the Superoxide mimicking compounds. Di-Schiff-bases proved most appropri ate to fulfil these requirements. Both structural and functional aspects of the copper binding centre of the intact enzyme were met by these complexes. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence identical to the reaction of Cu2Zn2superoxide dismutase.

The cytotoxicity of SOD-mimics was studied in human K562 erythroleukemia cells. CuPUPY, a low molecular weight copper complex with properties typical of a Cu2Zn2 SOD active center analog was shown to display pronounced toxicity upon incubation with human K562 erythroleukemia cells, while the ligand, CuSO4 or CuEDTA did not affect vitality. Externally added catalase decreased the cytotoxic effects of CuPUPY by 50% indicating an involvement of hydrogen peroxide in toxicity. An increased oxygen uptake and glutathione oxidation by K562 cells in the presence of CuPUPY suggested that toxicity might be due to a copper-mediated redox-cycle. In fact addition of glutathione to a solution of CuPUPY resulted in glutathione oxidation, O2-consumption and H2O2-generation. CuPUPY proved to be less toxic to human lymphocytes than to K562 cells. This selectivity may be related to the low content of antioxidative enzymes in K562 cells.

Our knowledge of the conservation techniques used in the Old Kingdom of ancient Egypt is limited. Examinations of a clavicle fragment of the mummy of Idu II, secretary general of the pine wood trade office (2150 ± 50 BC), revealed saturation with a wealth of sodium and wood tar compounds, many of which were highly antiseptic as no microbial contamination was noticed. This pretreatment had ensured the preservation of bone alkaline phosphatase in an enzymically and immunologically active form. This extends the use of embalming to one thousand years earlier than previously thought.

Details of mummification techniques used in dynastic Egypt have emerged from writings in subsequent ancient texts, in which the application of oils (kedros, cedrium) derived from the cedar tree have been described by Herodotus (490–425 bc)1 and by Pliny the Elder (ad 23/24–79)2. But scholars have since argued that these products were prepared from juniper trees and not from cedar3 — an assertion that is widely accepted by Egyptologists4 but which has never been verified by chemical analysis. Here we use gas chromatography to analyse the constituents of a sample of unused entombed embalming material from 1500 bc at a site in Deir el-Bahari, Egypt, and find that its components probably originated from the cedar tree. We also identify one component, guaiacol, as having notable preservative properties.

Synopsis New evidence from analytical studies has shown that the bones of the mummy of Idu II (late Old Kingdom) were treated with products originating from pinewood. The rich abundance of pine wood tar components and sodium in the bones support the proposal that Idu II had been skeletonized at least in part during the process of embalming.4 The study also indicates that the body was treated with natron, which could possibly have been utilized in the form of a solution in which the bones were soaked. If this

Crystalline binuclear imidazole-bridged copper- and zinc-copper chromophores ([Me(pip)(im)Me(pip)](NO 3 ) 3 · x H 2 O) were synthesized and compared with the active site of Cu 2 Zn 2 —superoxide dismutase. The electron absorption profiles were of striking similarity with those of the copper chromophore of the native enzyme. This similarity was also seen in the electron paramagnetic properties (g ⊥ = 2.031 ± 0.009, g 1 = 2.230 ± 0.004 and |A 1 | = (14.8 ± 1.0) × 10 −3 cm −1 ). X-ray photoelectron spectrometry of the oxidized or the reduced superoxide dismutase models revealed the existence of 3d 9 Cu(II) (∼ 934.5 eV) and 3d 10 (Cu(I)) (∼ 931.8 eV). By way of contrast the 2p 3 2 binding energy of the copper in the Cu 2 Zn 2 —enzyme remained constant at 932 eV. In an indirect assay system for superoxide dismutation, the xanthine/xanthine oxidase-mediated reduction of nitroblue tetrazolium indicated that either enzyme model reacted at a rate of 10 8 M −1 s −1 . The rate constant of the binuclear copper—zinc complex was more than three times higher than the dicopper complex in pulse radiolysis. Rate constants determined by this method ( k 2 = 3.8 ± 2.1) × 10 8 M −1 s −1 ) were in reasonable agreement with those determined in the indirect test system.

The superoxide dismutative action of Cu-penicillamine was examined by pulse radiolysis. The second order rate constant of the reaction with superoxide was 0.4 ± 0.2 · 10 9 M −1 · s −1 , comparable to the action of Fe- and Mn-superoxide dismutases. No marked pH-dependence was seen. Neither ethylene diamine tetraacetic acid nor cyanide affected the catalytic action of Cu-penicillamine. The cyanide resistant reactivity as well as further X-ray photoelectron spectrometric measurements supported the suggestion of a Cu(I) stabilized sulphur radical being the active species involved in the catalysis of superoxide dismutation.

A naturally occurring monodisperse Cu-thionein was prepared using ammonium sulfate precipitation followed by ion exchange (DEAE 23) and gel chromatography (Sephadex G-75). The chromatographic steps were repeated at least twice, or until the Cu-thionein remained homogeneous when subjected to analytical polyacrylamide disc electrophoresis. The molecular weight of this copper protein was 9500+/-500. Up to 24.3% cysteine residues were determined, indicating the relationship to the metallothioneins. Aromatic amino acids were virtually absent, while there were about three times as many acidic amino acid residues, including aspartate and glutamate, as in metallothioneins. 10 g atoms of Cu were measured per mole of protein. The copper binding strength of thionein was extremely high. Displacement by protons (pH 1.5) and gel chromatography or dialysis employing EDTA were not effective. Dialysis against diethyldithiocarbamate produced a protein essentially free of copper. Both the ultraviolet properties and the circular dichroism measurements proved identical with those properties reported for artificially prepared Cu-thionein (see ref.[1]. The major absorption was in the far ultraviolet region with a weak shoulder at 270 nm attributable to copper charge-transfer transititions. 6 Cotton extrema were seen at 213, 283 and 302 nm (negative) and 245, 328 and 359 nm (positive). The possible role of Cu-thionein as an electron transport system was discussed.

An improved simple and efficient method for the aqueous isolation of bovine erythrocyte Cu2Zn2- superoxide dismutase was devised and successfully employed. In the last purification step the preparative separation of charge isomers of this enzyme was performed. The properties of the isomers were examined using electrophoresis, isoelectric focusing and chromatofocusing. The isoelectric points of charge isomers I and II were 5.2 and 4.9, respectively. No significant differences of chemical and spectrometrical properties, including amino acid composition, XPS-spectroscopy and EPR-measurements, were seen. The chiroptical data at 261 nm differed markedly. Aqueously isolated Cu2Zn2superoxide dismutase from the red blood cells of 1–2 year old cows had the same properties compared to the protein obtained from 12–15 year old species. For this reason, ageing of the erythrocyte was not attributed to an ageing of superoxide dismutase. Hydrogen peroxide was used as a model system for the generation of charge isomer II from charge isomer I. After treatment with hydrogen peroxide the band intensity at the position of charge isomer II increased dramatically, both during isoelectric focusing and/or gel electrophoresis. The data obtained from this species by CD-, EPR- and UV-spectroscopy are completely different from the respective parameters of the genuine charge isomer II. Therefore, it is concluded that this isomer is not generated by hydrogen peroxide inactivation of charge isomer I in vivo.

X-ray photoelectron spectra of some inorganic selenium compounds, Se-methionine, Se-cystine, Se-urea and selenodicysteine were recorded and compared with the XPS data obtained from the respective sulphur containing compounds. The oxidation state of selenium could be monitored by the observed chemical shifts of the Se(3p1/2),Se(3p3/2) and Se(3d3/2,5/2) levels. Though having a formal oxidation state near zero, the binding energy of the core electrons of Se in Se-methionine, Se-cystine and selenodicysteine was shifted by 0.4, 0.7 and 0.4 eV, respectively. This phenomenon was attributed to the rather distinct polarization of Se. The reversible oxidation of Se-cystine using H2O2 and NaBH4 could be successfully demonstrated by this XPS-technique.

The release of intact Cu(I)8-thionein from copper-resistant copper-loaded yeast cells, strain X2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physiocochemical 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.

The possible structural basis of the specific inhibitory action of cuprein on the autoxidation of catecholamine was examined. The chiroptical properties of the native enzyme were compared with both the denatured and the apoprotein and the superoxide-dismutase-active Cu(Tyr)2. Apart from the reduction of cuprein copper to Cu(I), marked and characteristic changes in the circular dichroism (CD) spectrum from 260–400 nm in the presence of adrenaline and oxygen were seen, suggesting the formation of a ternary complex. This conformational change proved dependent on the concentration of oxygen, adrenaline, and cuprein copper and was not seen when the apoprotein or the heat-denatured enzyme was used. Blocking of the vicinal hydroxyl groups of adrenaline by borate did not significantly affect the formation of the complex. This implies an essential role of the catecholamine side chain. It was assumed that the ligands in close proximity around the copper appear to be even more important for the catalytic action than the involved copper. No such specificity was measured when Cu(Tyr)2 was used. Removal of the adrenaline side chain altered the enzymic function of added cuprein copper signifiantly. During the autoxidation of 1,2-dihydroxybenzene, cuprein copper accelerated the oxidation, whereas Cu(Tyr)2 was essentially inactive.

Our knowledge on the preparation and physicochemica1 properties of metallothionein has improved in recent years [l-l 51. On the other hand, a highly polymeric sulphur and copper rich protein called neonatal hepatic mitochondrocuprein was described by Porter [ 16-221. Although this protein bears the name cuprein, it proved completely different from those intracellular cupreins containing 2 g-atoms of each of copper and zinc and which display superoxide dismutase and singlet oxygen decontaminating activities (for a review see [23] ). The neonatal type cuprein normally being present under physiological conditions in newborn species was also found in mitochondria of the livers of adults suffering from Wilson’s disease. Apart from these polymeric sulphur rich copper proteins some low molecular weight species of similar compositions were found [22,24,25] . It was assumed that the copper is in the cuprous state [24,25] and bound with the cysteine sulphur. Due to the similar amino acid composition of both the metallothionein and neonatal hepatic mitochondrocuprein Porter suggested some relationship to metallothionein [22]. In this context we were highly interested in the possibility of a direct conversion of metallothionein into Cu-thionein. The partially loaded Cu-thionein was expected to polymerize via disulphide bridges in the presence of oxygen to form the ‘neonatal type

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.

Attempts to mimick the tetranuclear Cu 4 S 8 (Cys) center of yeast copper thionein with aliphatic mercaptanes reveal the (CuSR) n polymers to be strictly favoured over species with a coordination number higher than 2 for copper. However, using thiophenolates and tetraacetonitrilecopper(I) perchlorate, crystalline tetranuclear [Cu 4 (SR) 6 ] 2− complexes were obtained as alkylammonium salts. Infrared spectra, 1 H nuclear magnetic resonance, X-ray photoelectron spectrometry and analytical data support the suggestion of a pseudo-adamantane arrangement of the Cu 4 S 6 core, copper being purely Cu(I) coordinated exclusively to thiolate sulphur. As opposed to the proposed cubane-type structure of the Cu 4 S 8 (Cys) center, i.e. four fused CuS 4 tetrahedra, cuprous thiophenolate tetranuclear complexes are demonstrated to be restricted to three-fold coordination of the metal.

Glutathione peroxidase showed an X-ray photoelectron spectroscopy signal of the Se 3d (3/2, 5/2) electrons at 54.4 eV. After the addition of the acceptor substrate H2O2, a marked shift of this signal to a value of 58.0 eV was observed. Upon subsequent treatment with the donor substrate glutathione, this chemical shift was reversed and the original signal was obtained. These data demonstrate that the enzyme-bound selenium moiety participates in the catalytic process. From the chemical shift obtained it is concluded that the enzyme shuttles between a selenol or selenol derivative in its reduced form and a seleninyl or selenonyl compound in its oxidized form.

An erythrocyte Cu2(heme b)2 protein of Mr 400000 was successfully isolated. Incubating the protein in sodium dodecyl sulfate prior to polyacrylamide gel electrophoresis caused the splitting into Mr 70000, 120000, and 200000 units. The copper was fully electron paramagnetic resonance detectable of the type II (g perpendicular = 2.0309, g parallel = 2.2122, A parallel = 175 G). The high-spin (d55/2) iron(III) showed a g value of 6.05. No magnetic interaction between copper and heme iron was detected. In a comparison of more than ten different enzymatic oxidase activities, not a single one could be assigned to the Cu2(heme b)2 protein. Azide, CO, cyanide, fluoride, and imidazole were bound to the heme iron. The binding of imidazolate suggests the accessibility of the sixth coordination site of the heme b group to fairly large ligands. Removal of the copper by ethylenediaminetetraacetic acid or cyanide resulted in an irreversible precipitation of the protein. This supports the structural contribution of the copper.

The possible time- and/or light-dependent decomposition of the purple Cu(I), Cu(II)-complex of D-penicillamine (Cu(II) 6 Cu(I) 8 (D-penicillamine) 12 Cl) 5− was examined. Superoxide dismutase activity of the freshly prepared complex was assayed using the nitroblue tetrazolium assay. The formazan colour formation was inhibited by 50% in the presence of approximately 500 μM copper. Ageing of the copper complex, especially in the light, resulted in a marked increase of EDTA-sensitive activity. Upon gel chromatography of the aged samples the original low inhibitory activity was restored. All EDTA-sensitive inhibitory activity was found in a clearly separated low M r copper-containing fraction. Aerobic irradiation with a tungsten lamp at 30 °C accelerated the decomposition of (Cu(II) 6 Cu(I) 8 (D-penicillamine) 12 Cl) 5− . ϵ Cu518 = 1800 M −1 cm −1 dropped to ϵ Cu640 = 60 M −1 cm −1 . The photochemical conversion of (Cu(II) 6− Cu(I) 8 (D-penicillamine) 12 Cl) 5− was complete within 48 h. Due to the identical electronic absorption profile of both, the decomposition product and Cu(II) D-penicillamine disulphide the latter complex was assigned to be the unknown low M r copper-compound. Circular dichroism and electron paramagnetic resonance measurements support this conclusion.

A series of copper and nickel biuret complexes has been studied by X-ray photoelectron spectroscopy (XPS). The results include one of the first reports of the XPS spectrum of a copper(III) complex. Significantly, this diamagnetic complex exhibits intense satellite structure in both the Cu 2P3/2 and Cu 2Pi/2 regions. The isoelectronic nickel(II) complex shows no comparable satellite structure.