Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex

Although thiourea has been used widely to study the role of hydroxyl radicals in metal-mediated biological damage, it is not a specific hydroxyl radical scavenger and may also exert antioxidant effects unrelated to hydroxyl radical scavenging. Thus, we investigated the effects of thiourea on copper-induced oxidative damage to bovine serum albumin (1 mg/ml) in three different copper-containing systems: Cu(II)/ascorbate, Cu(II)/H₂O₂, and Cu(II)/H₂O₂/ascorbate [Cu(II), 0.1 mM; ascorbate, 1 mM; H₂O₂, 1 mM]. Oxidative damage to albumin was measured as protein carbonyl formation. Thiourea (0.1–10 mM) provided marked and dose-dependent protection against protein oxidation in all three copper-containing systems. In contrast, only minor protection was observed with dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Strong protection was also observed with dimethylthiourea, but not with urea or dimethylurea. Thiourea also significantly inhibited copper-catalyzed oxidation of ascorbate, and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous, but not cupric, copper, as demonstrated by both UV-visible and low temperature electron spin resonance measurements. We conclude that the protection by thiourea against copper-mediated protein oxidation is not through scavenging of hydroxyl radicals, but rather through the chelation of cuprous copper and the formation of a redox-inactive thiourea-copper complex.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

A kinetic study on the distribution of Cu(II)-ions between albumin and transferrin

Serum albumin (human, bovine) has a specific Cu(II)-ion binding site, and is proposed to act as a copper transport protein in blood plasma. Human transferrin, normally about 30% saturated with iron in vivo, has two sites/molecule capable of complexing Cu(II); one more strongly than the other (Hirose et al. 1996). The present study shows that this binding site has a slightly stronger affinity for Cu(II) than that on the albumins. However, both human- and bovine albumin could take up part of the transferrin bound Cu(II), the second order rate constant for the reaction estimated to 12 mM(-1) min(-1) for both species. In vivo the albumin concentration is considerably higher than that of iron-free transferrin, and it seems unlikely that the latter can compete with albumin for non-ceruloplasmin cupric ions.     

Ascorbate-induced high-affinity binding of copper to cytosolic proteins

Copper chaperones are necessary for intracellular trafficking of copper to target proteins. This is probably because the milieu inside the cell has a large capacity for sequestering this metal. By fluorometry using a fluorescent Cu(II) chelator and by centrifugal ultrafiltration, we have studied copper binding of the whole cytosolic proteins from mouse brain and liver, and found that their binding capacity and affinity for copper were markedly increased by ascorbate. Brain cytosolic protein bound, with high affinity, 63 nmol of copper/mg, more than half of which was redox-inactive, as indicated by its inability to catalyze oxidation of ascorbate. Most of the bound copper was in the Cu(I) state, coordinating to thiol groups of protein. Cytosolic protein competed for copper more strongly than GSH when compared at their relative concentrations in tissues. The results taken together suggest that protein thiols of cytosol can strongly sequester copper.     

A kinetic study on the copper-albumin catalyzed oxidation of ascorbate

Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM^–1 min^–1 was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent K_m-value of 0.89 mM, and a catalytic constant of 0.066 M O₂/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H₂O₂ in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.     

An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes

alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetracycline-Cu(II) photo-induced fragmentation of serum albumin

The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 microM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0-1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals (*OH) in tetracycline-Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.     

Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): selectivity and conformational consequences of histidine modification.

We have studied the metal-catalyzed oxidation (MCO) of brain-derived neurotrophic factor (BDNF) with regard to target sites and potential conformational changes of the protein. The exposure of BDNF to three different levels of ascorbate/Cu(II)/O2 [20 microM Cu(II), 2 mM ascorbate (level 1); 20 microM Cu(II), 4 mM ascorbate (level 2); 40 microM Cu(II), 4 mM ascorbate (level 3)], chosen based on the extent of chemical modification of Met and His, respectively, resulted in the exclusive oxidation of a buried Met residue, Met92, at level 1 but in the predominant oxidation of His at level 3. His modification had a significant impact on the structure of BDNF, as quantified by CD and ANSA fluorescence measurements, while Met oxidation had not, also assessed through complementary oxidation of BDNF through hydrogen peroxide. Our ultimate objective was the correlation of the surface exposure of an oxidized His residue in a protein with potential effects on the conformational integrity of the oxidized protein. In a series of three proteins, human growth hormone (hGH), human relaxin (hR1x), and BDNF, we have now observed that His oxidation is paralleled by significant conformational changes when the target His residue is more surface exposed (hR1x, BDNF) while conformational consequences of His modification are less significant when the target His residues are more buried in the interior of the protein (hGH).     

Evaluation of peptide/metal ion interactions by UV laser desorption time-of-flight mass spectrometry.

A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), has been evaluated as a new means to investigate the metal ion-binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDa human plasma metal-binding transport protein histidine-rich glycoprotein (HRG) has been identified as a bioactive metal-binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF-MS before and after the addition of Cu(II) in solution with 2,5-dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)-binding capacity observed for the 26-residue peptide (5 g-atoms/mol) suggested that up to 1 Cu(II) may be bound per 5-residue internal repeat unit (GHHPH) within this peptide; several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)-binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF-MS.     

Copper (II) induced polymerization of human albumin, and its depolymerization by diglycyl-L-histidine: a pH static and ultracentrifugation study.

Copper (II) ions successively induce dimers and tetramers of human serum albumin (L) when the Cu (II) concentration is extended beyond that of 200 muM. This is shown by emf titrations and by ultracentrifugation experiments. The emf titrations, which involve a new pH static method, were performed at 25 degrees, in a 0.5 M NaCIO4 medium at pH 6.59, using glass and copper amalgam electrodes. The total concentration of Cu(II) varied from 0.14 to 2.2 mM and the albumin concentration from 0.05 to 0.7 mM. In order to evaluate the formula of the main complexes, without using any a priori assumptions regarding their compositions, a detailed graphic procedure was used. The results, in the form of equilibrium constants for the main species, were refined by the use of a general least squares computer program. The experimental data are found to be consistent with the formation of the monomeric CuL, Cu5L, and Cu6L species and the dimeric Cu3L2, Cu4L, Cu6L, and Cu8L2 species. In addition, there is some indication for a minor species, most probably the Cu12L4 tetramer. The pH static results qualitatively agree with the findings obtained by ultracentrifugation. As indicated by distinct bands and their S-values, ultracentrifugation experiments show not only monomeric and dimeric species of albumin, but also tetrameric species. The polymerization of the albumin is reversible, since diglycyl-L-histidine, a peptide designed to mimic the Cu (II) transport site of albumin, depolymerizes the Cu (II)-albumin polymers.     

Redox-changes associated with the glutathione-dependent ability of the Cu(II)-GSSG complex to generate superoxide

The intracellularly-occurring Cu(I)-glutathione complex (Cu(I)-[GSH](2)) has the ability to reduce molecular oxygen into superoxide. Removal of such radicals leads to the irreversible conversion of Cu(I)-[GSH](2) into the redox-inactive Cu(II)-GSSG complex. The present study addressed the potential of reduced glutathione, ascorbate and superoxide to reductively regenerate Cu(I)-[GSH](2) from Cu(II)-GSSG, and investigated the redox changes involved in such process. Results show that: (i) among the three tested reductants, only GSH is able to reduce the Cu(II) bound to GSSG; (ii) during the reduction of Cu(II)-GSSG, a Cu(I)-GSSG intermediate would be formed (supported here by Cu(I) and GSSG recovery data and by NMR studies); (iii) when GSH is present in a molar excess equal or greater than 1:3, the reduction of Cu(II)-GSSG into Cu(I)-[GSH](2) is quantitative and complete. Under such conditions, the Cu(II)-GSSG complex acquires a superoxide-generating capacity which is identical to that seen with the Cu(I)-[GSH](2) complex. Within cells, the concentrations of GSH are at least 2- to 3-fold order of magnitude higher than those expected for the Cu(II)-GSSG complex. Thus, we postulate that the interaction between GSH and Cu(II)-GSSG could be seen as a potential mechanism to regenerate continuously the Cu(I)-[GSH](2) complex and thereby affect the ability of the latter to generate superoxide.     

Uptake of copper from plasma proteins in cells where expression of CTR1 has been modulated

Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1?μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.     

Spectroscopic characterization of carbon monoxide complexes generated for copper/topa quinone-containing amine oxidases

Carbon monoxide complexes have been generated for copper/topa quinone (TPQ)-containing amine oxidases from Arthrobactor globiformis (AGAO) and Aspergillus niger (AO-I) and characterized by various spectroscopic measurements. Addition of CO to AGAO anaerobically reduced with its substrate 2-phenylethylamine led to a slight increase of absorption bands at 440 and 470 nm derived from the semiquinone form (TPQ(sq)) of the TPQ cofactor, concomitantly giving rise to new CO-related absorption bands at 334 and 434 nm. The intensity of the TPQ(sq) radical EPR signal at g = 2.004 also increased in the presence of CO, while its hyperfine coupling structure was affected insignificantly. FT-IR measurements revealed C-O stretching bands (nu(CO)) at 2063 and 2079 cm(-1) for the CO complex of the substrate-reduced AGAO (at 2085 cm(-1) for AO-I), which shifted nearly 100 cm(-1) to lower frequencies upon using (13)C(18)O. Collectively, these results suggest that CO is bound to the Cu(I) ion in the Cu(I)/TPQ(sq) species formed in the reductive half-reaction of amine oxidation, thereby shifting the Cu(II)/aminoresorcinol right arrow over left arrow Cu(I)/semiquinone equilibrium toward the latter. When AGAO was reduced with dithionite, an intermediary form of the enzyme with Cu(II) reduced to Cu(I) but TPQ still in the oxidized state (TPQ(ox)) was produced. Dithionite reduction of AGAO in the presence of CO resulted in the immediate formation of FT-IR bands at 2064 and 2083 cm(-1), which were assigned to the nu(CO) bands of the CO bound to the TPQ(ox) enzyme. The intense 2083 cm(-1) band was then displaced by a new band at 2077 cm(-1), corresponding to the formation of the fully reduced topa. Significant variation of these nu(CO) frequencies indicates that vibrational properties of CO bound to copper amine oxidases are sensitively influenced by the coordination structure of the Cu(I) ion, which may be modulated by the chemical and redox states of the TPQ cofactor.     

Use of amphotericin B as optical probe to study conformational changes and thermodynamic stability in human serum albumin

The binding of polyene antibiotic amphotericin B to serum albumin was studied using absorption, fluorescence, and circular dichroism techniques. A hypochromic effect was observed in the absorption spectrum of amphotericin B in the presence of albumin with maxima at 366 nm, 385 nm, and 408 nm, which correspond to the absorption of the monomeric form of amphotericin B. A modification on the circular dichroism spectrum of amphotericin B in the presence of albumin was observed at bands 329 nm and 351 nm (excitronic interaction), which suggests that only amphotericin B monomer is bound to the protein. Amphotericin B perturbs the specific markers for sites I, II, and fatty acid binding site bound to these sites, suggesting that amphotericin B interacts with a great binding area in albumin. Lysines 199 and 525 in albumin participate in the molecular interaction between amphotericin B and the protein. The absorption spectrum of amphotericin B bound to albumin was sensitive to the chemical and thermal treatment of the protein, to neutral-basic transition of albumin and to conformational changes induced by the binding of other ligands to this protein.     

Bathocuproine-assisted reduction of copper(II) by human albumin

Human albumin (studied here as the recombinant protein rHA), a copper-binding protein in blood plasma, is shown to reduce Cu(II) to Cu(I) in the presence of a Cu(I) chelator, bathocuproinedisulfonate (BD). This reaction was accelerated at low pH, when there was little binding of Cu(II) to rHA. The addition of a competitive metal ion, Ni(II), or an increase in the concentration of BD, enhanced the reduction of Cu(II) to Cu(I). It was concluded that the oxidant was the Cu(II) complex of BD, which is likely to bind strongly to albumin. The free thiol at Cys34 was ruled out as the sole reducing agent, since Cys34-blocked albumin also gave rise to Cu(I) in the presence of BD. Reactions with amino acids and peptides suggested that Tyr and possibly His side-chains are potential reductants. BD and its homologues are frequently used as Cu(I)-specific chelators in biological experiments, but the strong oxidant activity of [Cu(II)(BD)2]2- and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Different DNA damaging species as a result of oxidation of n-butyraldehyde and iso-butyraldehyde by Cu(II)

The isomers n- and iso-butyraldehyde (BuA) in combination with Cu(II) induced single and double strand breaks in PM2 DNA, whereas the aldehydes, or Cu(II) alone had only negligible effect. The DNA damage was the result of radical oxidations of the aldehydes under formation of Cu(I). Cu(I) formation was independent of molecular oxygen. Extensive DNA degradation was only observed in the presence of molecular oxygen. Characterization of DNA damage pointed to different ultimate DNA damaging species. While catalase and neocuproine inhibited strand break formation induced by iso-BuA/Cu(II) to a high degree, these inhibitors were less effective in the n-BuA/Cu(II) reaction. On the other hand, sodium azide showed a high strand break inhibition in the n-BuA/Cu(II) reaction, but low inhibition in the iso-BuA/Cu(II) reaction. 2-Deoxyguanosine was hydroxylated in the 8-position by iso-BuA/Cu(II) but little reaction occurred with n-BuA/Cu(II). Chemiluminescence was detected during both BuA/Cu(II) reactions, whereby the intensity of the luminescence signal was 3.5-fold higher for n-BuA/Cu(II) than for iso-BuA/Cu(II). We suppose that the copper(II)-driven oxidation of n- and iso-BuA proceeds via different pathways with different DNA damaging consequences. Whereas the oxidation of iso-BuA mainly results in damage by ^·OH-radicals, the oxidation of n-BuA may lead to a radical reaction chain whereby excited states are involved and the resulting DNA-damaging species are not ^·OH-radicals.