Intact mummified bone alkaline phosphatase.

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 Zn2Mg alkaline phosphatase could be detected. A protein of M(r) 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.

Copper deficiency and erythrocuprein (2Cu, 2Zn-superoxide dismutase).

The activities of 2Cu,2Zn-superoxide dismutase, ferroxidase (ceruloplasmin), catalase and glutathione peroxidase were measured in the blood of rats during copper depletion. Two control groups of animals were used; one received the regular diet containing all essential components including copper and the other group was maintained on a diet, containing 1% the amount of copper in normal diet, copper being supplied as Cu(Leu)2 in the drinking water. Both groups showed no detectable differences, either in the copper content of blood or in the measured four enzymic activities. Excessive copper (injected intraperitoneally) caused only an insignificant rise in the enzymic activities (0-10%) compared to either control. After starting copper depletion ferroxidase activity decreases to 15% on the 15th day, while the 2Cu,2Zn-superoxide dismutase activity decreases to 40% on the 45th day. Ferroxidase activity shows rapid but transient changes immediately after perturbation in plasma copper levels. By contrast, the 2Cu,2Zn-superoxide dismutase activity more closely parallels the overall copper deficiency. Dietary repletion with copper raises the 2Cu,2Zn-superoxide dismutase activity to 94% and the ferroxidase activity to 80% of the control values within 36 h. Apart from the copper-dependent anemia catalase activity was decreased. However, 15 days after the start of the copper depletion catalase activity rises again and reaches the control value on the 40th day and a 30% stimulation was even seen on the 58th day. Upon copper repletion catalase activity reaches 166% of the control within 14 days. No copper-dependent differences of glutathione peroxidase activity were seen regardless whatever copper level was present in the rats.

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil

Copper(I) and copper(II) in complexes of biochemical significance studied by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopic measurements of copper complexes of biochemical significance were carried out to permit the conclusion whether or not copper is present in the Cu(I) or Cu(II) state. Only one single homogeneous signal in the X-ray photoelectron spectra of the Cu(I) 2p1/2 and 2p3/2 levels was seen regardless whatever Cu(I) complex was used. By contrast one more or less split satellite in addition to the main 2p copper signal appeared when Cu(II) complexes were studied. The extent of satellite splitting was dependent on the nature of the ligands coordinated with Cu(II). Thus, a strong splitting was observed in the spectra of Cu-(trifluoroacetylacetonate)2 and Cu-(biuret)2Cl2 were Cu(II) is exclusively bound to oxygen having a formal double bond. No such splitting was seen in Cu(II) chelates where the metal was bound to single bonded oxygen and/or nitrogen. It excited great interest to see that in the antiferromagnetically coupled Cu(II) complexes Cu2-(succinate)2-4H2O, Cu-(HCCO)2, CuO and in the completely diamagnetic Cu2-(u,3-diphenyltriazene)4 complex Cu(II) could be detected. The reaction of Cu(I) and Cu(II) with the thiol sulphur of either cysteine, penicillamine or alpha-mercaptopropionylglycine yielded Cu(I) complexes. During the X-ray exposure of the different samples photoreduction of Cu(II) was not observed. For example, the satellite structure of the copper 2p levels using Cu(II)-cystine remained unchanged during the measurement.

Involvement of superoxide in the catalytic cycle of diamine oxidase.

The reaction of pig kidney diamine oxidase (amine:oxygen oxidoreductase (deaminating) (pyridoxal-containing), EC 1.4.3.6) could be significantly inhibited by superoxide dismutase active copper chelates but not by native 2Cu,2Zn-superoxide dimutase (cuprein). The ligands alone as well as Cd2+, a heavy metal of similar toxicity to Cu2+, showed no inhibition whatsoever. This indicates that .O-2 participates in the catalytic cycle and is produced at a site scarcely accessible to such a large molecule as cuprein. A mechanism for the second, aerobic step of the diamine oxidase reaction is suggested.

Differently bound copper(I) in yeast Cu8-thionein.

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

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.

Functional aspects of the superoxide dismutative action of Cu-penicillamine.

The superoxide dismutative action of Cu-penicillamine was examined by pulse radiolysis. The second order rate constand of the reaction wpith superoxide was 0.4 +/- o.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.

Transient thiyl radicals in yeast copper(I) thionein.

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.

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.

Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein.

Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.

Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems.

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1 : 2 complex, namely Cu2(indomethacin)4 x L2, in the unit cell. Surprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu2(acetate)4 was seen. Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. the superoxide dismutating activity was successfully demonstrated in Me2SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 x 10(9) M-1 x s-1 in strictly aqueous systems dropped only slightly to 1.1 x 10(9) M-1 x s-1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO2-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 x 10(-10) mol x ml-1 of Cu2(indomethacin)4. The inhibitory action dropped to 25% when Cu2Zn2superoxide dismutase was employed. The formation of copper x indomethacin in rat serum after administration of indomethacin was shown in vitro and vivo.

Substrate-induced redox change of selenium in glutathione peroxidase studied by x-ray photoelectron spectroscopy.

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.

Copper-thionein in melanoma.

The phenomenon of an elevated copper concentration in melanoma tumors was examined. It was demonstrated that 50-60% of total tissue copper is associated with metallothionein. The amino acid composition, electronic absorption and fluorescence were identical to that of the many known vertebrate Cu-thioneins. The immunological identification of melanoma tissue metallothionein was successful. The elevated Cu-thionein concentration in melanoma tumor tissue is not yet understood. It appears to be a common concept that in most tumors transient changes of the copper status parallel the metallothionein levels.

Erythrocuprein (Cu2Zn2 superoxide dismutase) is the major copper protein of the red blood cell.

The copper balance in the red blood cell deserved special attention as the exact amount of copper being bound in erythrocuprein (Cu2Zn2 superoxide dismutase) is poorly understood. An improved aqueous isolation of erythrocuprein revealed that essentially all erythrocyte copper is found in this protein. This fact is supported by both superoxide dismutase activity measurements of the haemolysate and EPR-quantification studies throughout the course of the isolation.

Enzymatic and immunological activity of 4000 years aged bone alkaline phosphatase.

Structurally intact and functionally active human bone alkaline phosphatase was isolated from clavicle fragments of IDU, an Egyptian mummy of the Old Kingdom (2150 +/- 50 BC). Both anion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme resulting in a specific activity of 180 +/- 30 mU/mg. The intactness of the bone enzyme fractions of the wheat-germ lectin affinity chromatography was successfully demonstrated in an ELISA using the monoclonal antibody BAP A. Fortunately, the mummified bone was not contaminated by fungi or bacteria.

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

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

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

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

Monoclonal antibodies to monomeric rat liver metallothionein-I: the immunoreactivity of lysine residues in metallothionein.

Regardless of the weak immunological response against the low-Mr 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 NH2-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.

Monoclonal antibodies recognize 2300 years aged alkaline phosphatase.

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.