@article{el kasmi_leopold_galligan_robertson_saavedra_el kacemi_bowden_2002, title={Adsorptive immobilization of cytochrome c on indium/tin oxide (ITO): electrochemical evidence for electron transfer-induced conformational changes}, volume={4}, ISSN={["1388-2481"]}, DOI={10.1016/S1388-2481(01)00299-5}, abstractNote={The adsorptive immobilization and electrochemistry of horse and yeast cytochrome c on indium/tin oxide (ITO) electrodes is reported. Near-monolayer coverage was achieved in pH 7 phosphate buffers of ionic strength equal to 10 and 50 mM, respectively, for the horse and yeast species. The layers exhibit very well-behaved voltammetry and are stable on the timescale of hours to days. Cyclic voltammetry revealed quasireversible behavior that is a product of both electron transfer (ET) kinetics and ET-induced conformational changes. A square scheme mechanism linking the redox states and the conformational states is proposed. Using a simple ET kinetic model that adequately describes the voltammetry at higher scan rates, a standard ET rate constant of 18s−1 was determined for adsorbed horse cytochrome c. With decreasing scan rate, we observed a limiting peak separation of approximately 10 mV, an example of unusual quasireversibility (UQR) that we attribute to the effect of conformational changes. Finally we note that the intrinsic cytochrome c ET rate on ITO is some 6 orders of magnitude less than for gold.}, number={2}, journal={ELECTROCHEMISTRY COMMUNICATIONS}, author={El Kasmi, A and Leopold, MC and Galligan, R and Robertson, RT and Saavedra, SS and El Kacemi, K and Bowden, EF}, year={2002}, month={Feb}, pages={177–181} }
 @article{leopold_bowden_2002, title={Influence of gold substrate topography on the voltammetry of cytochrome c adsorbed on carboxylic acid terminated self-assembled monolayers}, volume={18}, ISSN={["0743-7463"]}, DOI={10.1021/la011456c}, abstractNote={Interfacial investigations of a protein monolayer electrochemical system, equine cytochrome c (cyt c) adsorbed to carboxylic acid terminated self-assembled monolayer (COOH SAM) modified gold electrodes, were performed. Electrochemical, spectroscopic, and scanning probe microscopy techniques were utilized to explore the influence of gold topography in the cyt c/COOH SAM/gold system. COOH SAMs were prepared from 14-mercaptotetradecanoic acid and 11-mercaptoundecanoic acid on a variety of substrates including evaporated, bulk, single crystal, and epitaxially grown gold on mica. These substrates encompassed a wide range of surface roughness. As the topography of the gold became smoother, SAMs exhibited an increased ability to block a solution probe molecule, indicative of a lower level of defectiveness. At the same time, after exposure to equine cyt c deposition solutions, the extent of adsorption and the magnitude of the electrochemical response of the adsorbed cyt c decreased significantly with increasingly smooth substrates. The results show cyt c adsorption and electrochemistry to be intimately related to the density of defects in the SAM, which in turn are largely dictated by the topography of the gold substrate. This hypothesis is supported by experiments in which the density of defects in the SAMs was controlled on each type of gold substrate using intentional roughening/smoothing procedures as well as through the use of mixed SAMs. Results are interpreted in terms of the topographically dependent acid/base properties of the COOH SAMs, which can limit the electrostatically driven adsorption of cyt c and the effectiveness of the protein's electronic coupling at the differently textured SAM surfaces.}, number={6}, journal={LANGMUIR}, author={Leopold, MC and Bowden, EF}, year={2002}, month={Mar}, pages={2239–2245} }
 @article{leopold_black_bowden_2002, title={Influence of gold topography on carboxylic acid terminated self-assembled monolayers}, volume={18}, ISSN={["0743-7463"]}, DOI={10.1021/la011683e}, abstractNote={Film permeability and double layer capacitance (Cdl) results are reported for mercaptotetradecanoic acid self-assembled monolayers (C13COOH SAMs) on gold substrates spanning a range of topography. Whereas film permeability was observed to decrease with increasing substrate smoothness, indicative of lower defect density in the SAMs, the capacitance, unexpectedly, was observed to increase as the topography became smoother. To explain these results, a simple structural model is proposed in which the extent of hydrogen bonding (H-bonding) among carboxylic acid endgroups is related to the underlying gold substrate topography. The model predicts that more extensive H-bonding will occur as substrates become smoother which, in turn, impacts the dielectric properties of the film. The model also provides an explanation for the discordant pKa results that have heretofore been reported for COOH SAMs. Altered surface acidity of up to 3 pKa units appears to be attributable to substrate topography.}, number={4}, journal={LANGMUIR}, author={Leopold, MC and Black, JA and Bowden, EF}, year={2002}, month={Feb}, pages={978–980} }
 @article{goldstein_leopold_huang_atwood_saunders_hartshorn_lim_faget_muffat_scarpa_et al._2000, title={3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction}, volume={39}, ISSN={["0006-2960"]}, DOI={10.1021/bi992997s}, abstractNote={The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the α-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent α-crystallin cross-linking. 3HK- or 3HAA-modifed α-crystallin showed enhanced redox activity in comparison to unmodified α-crystallin or ascorbate-modified α-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as α-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.}, number={24}, journal={BIOCHEMISTRY}, author={Goldstein, LE and Leopold, MC and Huang, XD and Atwood, CS and Saunders, AJ and Hartshorn, M and Lim, JT and Faget, KY and Muffat, JA and Scarpa, RC and et al.}, year={2000}, month={Jun}, pages={7266–7275} }
 @article{huang_cuajungco_atwood_hartshorn_tyndall_hanson_stokes_leopold_multhaup_goldstein_et al._1999, title={Cu(II) potentiation of Alzheimer A beta neurotoxicity - Correlation with cell-free hydrogen peroxide production and metal reduction}, volume={274}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.274.52.37111}, abstractNote={Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Aβ amyloid deposits in Alzheimer's disease. The neurotoxicity of Aβ in cell culture has been linked to H2O2generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Aβ in cell culture. The potentiation of toxicity is greatest for Aβ1–42 > Aβ1–40 ≫ mouse/rat Aβ1–40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H2O2 in cell-free assays and to exhibit amyloid pathology. The copper complex of Aβ1–42 has a highly positive formal reduction potential (≈+500–550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Aβ-mediated oxidative damage in Alzheimer's disease. Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Aβ amyloid deposits in Alzheimer's disease. The neurotoxicity of Aβ in cell culture has been linked to H2O2generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Aβ in cell culture. The potentiation of toxicity is greatest for Aβ1–42 > Aβ1–40 ≫ mouse/rat Aβ1–40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H2O2 in cell-free assays and to exhibit amyloid pathology. The copper complex of Aβ1–42 has a highly positive formal reduction potential (≈+500–550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Aβ-mediated oxidative damage in Alzheimer's disease. Alzheimer's disease thiobarbituric acid-reactive substance reactive oxygen species dichlorofluorescein bathocuproine disulfonic acid bicinchoninic acid phosphate-buffered saline Oxidative damage in the neocortex coincides with Aβ accumulation both in Alzheimer's disease (AD)1 (1Hensley K. Hall N. Subramanian R. Cole P. Harris M. Aksenov M. Aksenova M. Gabbita S.P. Wu J.F. Carney J.M. Lovell M. Markesbery W.R. Butterfield D.A. J. Neurochem. 1995; 65: 2146-2156Crossref PubMed Scopus (689) Google Scholar) and in Aβ amyloid-bearing transgenic mice (2Smith M.A. Hirai K. Hsiao K. Pappolla M.A. Harris P. Siedlak S. Tabaton M. Perry G. J. Neurochem. 1998; 70: 2212-2215Crossref PubMed Scopus (522) Google Scholar), but the mechanisms of oxidation are unknown. The possibility that Aβ accumulation causes oxidation, perhaps by radical formation (3Hensley K. Carney J.M. Mattson M.P. Aksenova M. Harris M. Wu J.F. Floyd R.A. Butterfield D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3270-3274Crossref PubMed Scopus (1093) Google Scholar), has been explored, but the nature of the chemistry involved in generating Aβ-associated oxidation products such as lipid peroxides (4Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar) remains to be elaborated. In culture, Aβ-induced neurotoxicity is characterized by elevated cellular H2O2 and is combated by antioxidants such as vitamin E and catalase (5Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2052) Google Scholar). The origin of the toxic H2O2 is unknown. Recently, we reported that Fe(III) interacts directly with Aβ1–42 and Aβ1–40 to produce H2O2 and TBARS formation in a cell-free manner in vitro, through reduction of the metal ion (6Huang X. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Crossref PubMed Scopus (1036) Google Scholar), suggesting that a source of the H2O2 that mediates toxicity in cell cultures exposed to Aβ is extracellular. Cu(II)and Fe(III) have been found in abnormally high concentrations in amyloid plaques (≈0.4 and ≈1 mm, respectively) and AD-affected neuropil (7Lovell M.A. Robertson J.D. Teesdale W.J. Campbell J.L. Markesbery W.R. J. Neurol. Sci. 1998; 158: 47-52Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar), and copper-selective chelators have been shown to dissolve Aβ deposits extracted from AD post-mortem brain specimens (8Cherny R.A. Legg J.T. McLean C.A. Fairlie D. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Therefore, these metal ions may be important cofactors in Aβ-associated oxidative damage. Importantly, we have also reported that the generation of both Cu(II) and Fe(III)-mediated TBARS is greatest for Aβ1–42 > Aβ1–40 ≫ rat Aβ1–40 (6Huang X. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Crossref PubMed Scopus (1036) Google Scholar). This rank order is of interest because it mirrors the relative participation of the peptides in amyloid neuropathology, and because the most active one (Aβ1–42) is overproduced in familial AD (9Scheuner D. Eckman C. Jensen M. Song X. Citron M. Suzuki N. Bird T.D. Hardy J. Hutton M. Kukull W. Larson E. Levy-Lahad E. Viitanen M. Peskind E. Poorkaj P. Schellenberg G. Tanzi R. Wasco W. Lannfelt L. Selkoe D. Younkin S. Nat. Med. 1996; 2: 864-870Crossref PubMed Scopus (2282) Google Scholar). Rats and mice do not develop amyloid (10Vaughan D.W. Peters A. J. Neuropathol. Exp. Neurol. 1981; 40: 472-487Crossref PubMed Scopus (69) Google Scholar), even in mice transgenic for familial-AD linked mutant presenilin that overexpress endogenous mouse Aβ1–42 (11Duff K. Eckman C. Zehr C. Yu X. Prada C.M. Perez-tur J. Hutton M. Buee L. Harigaya Y. Yager D. Morgan D. Gordon M.N. Holcomb L. Refolo L. Zenk B. Hardy J. Younkin S. Nature. 1996; 383: 710-713Crossref PubMed Scopus (1325) Google Scholar), probably due to the three amino acid substitutions in their homologue of Aβ (Arg5 → Gly, Tyr10 → Phe, and His13 → Arg) (12Shivers B.D. Hilbich C. Multhaup G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (388) Google Scholar). Although Fe(III) mediates and potentiates Aβ1–40 toxicity in cell culture (13Schubert D. Chevion M. Biochem. Biophys. Res. Commun. 1995; 216: 702-707Crossref PubMed Scopus (143) Google Scholar), it is not clear whether this is due to metal interaction with the peptide or due to a nonspecific increase in reactive oxygen species (ROS) generation within the cell. Redox active metal ions, such as Cu(II) and Fe(III), play an obligatory role in generating ROS, and in mediating ROS-induced damage (e.g. the Fenton reaction) (14Halliwell B. Gutteridge J.M.C. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4577) Google Scholar, 15Stadtman E.R. Oliver C.N. J. Biol. Chem. 1991; 266: 2005-2008Abstract Full Text PDF PubMed Google Scholar). Similarly, the Cu(II) and Fe(III) enhancement of dichlorofluorescein (DCF)-reactive oxygen species generated by Aβ25–35 treatment of post-mitochondrial rat cerebrocortex (16Bondy S.C. Guo-Ross S.X. Truong A.T. Brain Res. 1998; 799: 91-96Crossref PubMed Scopus (126) Google Scholar) could be due to catalytically enhanced ROS generation within the tissue, rather than due to metal interaction with the peptide. Cu(II) causes the peptide to aggregate to a greater extent than Fe(III) (Aβ1–42 > Aβ1–40 > rat Aβ1–40) (17Atwood C.S. Moir R.D. Huang X. Bacarra N.M.E. Scarpa R.C. Romano D.M. Hartshorn M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. 1998; 273: 12817-12826Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar), a property that may be related to the relative affinities of the metal ions for Aβ. We hypothesize that if such redox active metal ions bind to Aβ peptides with high affinity and become more oxidizing, they may potentiate Aβ-induced cytotoxicity. Furthermore, if the redox competence of Aβ is responsible for its neurotoxicity, then toxicity should be greatest for Aβ1–42 > Aβ1–40 > rat Aβ1–40. Aβ1–42 has been reported to be more neurotoxic than Aβ1–40 (18Doré S. Kar S. Quirion R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4772-4777Crossref PubMed Scopus (294) Google Scholar), but a comparison of the neurotoxicity of these three peptides, or the effects of Cu(II) upon the potentiation of their respective toxicities in culture, has not yet been reported to our knowledge. Here we report that, in the presence of Cu(II), Aβ is indeed redox-competent (Aβ1–42 > Aβ1–40 ≫ rat Aβ1–40), and that a series of electron transfer reactions occur when Cu(II) binds to Aβ, including reduction to Cu(I) and consequent O2-dependent, cell-free peroxide formation. These changes correlate with a striking potentiation in the neurotoxicities of the respective Aβ species in cell culture, supporting an extracellular origin for the H2O2that mediates Aβ-induced toxicity. These data suggest that formation of an Aβ-copper complex may be a pathophysiological interaction, and a new target for therapeutic interdiction in AD. Aβ peptides 1–40 and 1–42 were synthesized by the W. Keck Laboratory, Yale University, New Haven, CT. Confirmatory data were obtained by reproducing experiments with Aβ peptides synthesized and obtained from other sources: Glabe Laboratory, University of California, Irvine, CA; Multhaup Laboratory, University of Heidelberg; U.S. Peptides, Bachem (Torrance, CA); and Sigma. Rat Aβ1–40 was synthesized and purified by the Multhaup Laboratory. For the EPR experiments, Aβ1–40 was synthesized at the University of Queensland. Aβ1–28 and Aβ25–35 were purchased from U.S. Peptides, Bachem, and Sigma. Aβ40–1 was purchased from Bachem, and also synthesized by the Multhaup Laboratory (giving corroborating results). Aβ peptide stock solutions were prepared in water treated with Chelex-100 resin (Bio-Rad) and quantified, according to established procedures (17Atwood C.S. Moir R.D. Huang X. Bacarra N.M.E. Scarpa R.C. Romano D.M. Hartshorn M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. 1998; 273: 12817-12826Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). Cu(II)-Gly stock solutions were used to prevent metal-hydroxy and metal-oxy polymers that form in neutral metal ion solutions and were prepared by mixing National Institute of Standards and Technology (NIST) standard copper with glycine at metal/ligand molar ratio of 1:6. Other reagents are from Sigma, unless otherwise mentioned. Assays were performed using a 96-well microtiter plate (Costar), based upon a modification of established protocols (19Landers J.W. Zak B. Am. J. Clin. Pathol. 1958; 29: 590-592Crossref PubMed Scopus (68) Google Scholar). Polypeptides (10 μm) or vitamin C (10 μm), Cu(II)-glycine and Cu(I) indicator (250 μm), either bathocuproine disulfonic acid (BC) or bicinchoninic acid (BCA, 4,4′-dicarboxy-2,2′-biquinoline), were coincubated in Dulbecco's phosphate-buffered saline (PBS: 1.19 mm CaCl2, 0.6 mm MgCl2, 2.7 mm KCl, 1.4 mmKH2PO4, 137 mm NaCl, 7.68 mm Na2HPO4, pH 7.4), at 37 °C. Absorbances were then measured using a plate reader (SPECTRAmax Plus, Molecular Devices). In control samples, both metal ion and indicator were present to determine the background buffer signal. Absorbance of metal ion and peptide present in the absence of indicator were taken to estimate the contribution of light scattering due to turbidity. The net absorbances (A) were obtained by deducting the absorbances from these controls from the absorbances generated by the peptide and metal in the presence of the indicator. Cu(I) concentrations (μm) were calculated as A × 106/M, where M is the known molar absorption coefficient (m−1cm−1). For Cu(I)-BC, M = 12,250 at 483 nm; and for Cu(I)-BCA, M = 7700 at 562 nm. Cyclic voltammograms were obtained at room temperature (22 ± 2 °C) on air-equilibrated solutions using an EG&G PARC potentiostat, model 263A. The electrochemical cell incorporated an indium/tin oxide working electrode (Donnelly Corp.) of 0.32-cm2 area, a platinum auxiliary electrode, and a Ag/AgCl (1 m KCl) reference electrode. Indium/tin oxide electrodes were pretreated by successive 30 min sonications in Alconox solution (8 g/liter), 95% ethanol, Milli-Q water, and PBS, pH 7.3, followed by overnight equilibration in PBS. The peptide solution was prepared by first dissolving Aβ1–42 with sonication in double-distilled water to 300 μm, after which the peptide solution was added to Dulbecco's phosphate-buffered saline without calcium or magnesium (Sigma) to a final concentration of 100 μm. Background voltammograms were first acquired in buffer on each new electrode used, followed by examination of Aβ1–42 (100 μm) in buffer, CuCl2 (17 μm) in buffer, and Aβ1–42 (100 μm) with added CuCl2 (17 μm) in buffer. The possibility that the formal potentials measured were for surface-adsorbed complexes cannot be excluded, and, in fact, the wave shapes (Fig. 3, lines c and d) are suggestive of involvement by adsorbed species. Further investigation is planned to resolve this issue. Aβ1–40 (0.42 mg) was dissolved in 300 μl of PBS buffer (20 mmNa2HPO4, 20 mmNaH2PO4, 150 mm NaCl, pH 7.4) to which CuCl2 (as indicated) was later added. Q-, X-, and S- band EPR spectra were recorded on a Bruker ESP300E EPR spectrometer; the magnetic field and microwave frequency were calibrated with a Bruker ER-035M gaussmeter and an EIP 548B microwave frequency counter. Quantitation of the Cu(II)-Aβ1–40 EPR resonance was performed by a comparison method (20Pilbrow J.R. Hanson G.R. Methods Enzymol. 1993; 227: 330-353Crossref Scopus (17) Google Scholar) using an X-band dual TE104rectangular cavity with TEMPO as reference sample. The individual cavities have different modulation amplitudes, which can be calibrated, and different microwave magnetic strengths (B1), which was overcome by measuring standard and reference samples in each cavity. The doubly integrated areas are proportional to the spin concentration and were normalized for the anisotropic probability <g 12> and instrument settings. The relative areas can then be used to calculate the concentration of Cu(II)-peptide that produces the residual spectrum. The anisotropic spectra of the Cu(II)-AB1–40 complexes were measured at 130 K using a flow-through cryostat. Spin Hamiltonian parameters were extracted from the spectra with the XSophe/Sophe computer simulation software suite (21Wang D.M. Hanson G.R. J. Magn. Reson. A. 1995; 117: 1-8Crossref Scopus (118) Google Scholar). Buffers were demetallated with Chelex 100 resin. Rat embryonic day 17 forebrain neuronal cultures were grown at 95% O2, 5% CO2, 85% humidity for 4 days in serum-free NeurobasalTM medium with B-27 supplement (Life Technologies, Inc.), 20 μml-glutamate, 100 units/ml penicillin, 0.1 g/ml streptomycin, and 2 mml-glutamine. On the fifth day (treatment day), the medium was replaced with serum-free NeurobasalTM plus l-glutamine without B-27 supplement (vehicle medium). Stock solutions were mixed in vehicle medium to a final concentration of peptide or copper-glycine. Experimental trials were done in triplicate wells. Viable cells were counted manually using a 1-mm2 grid (10× objective) stained with either calcein-AM (Live/DeadTM assay, Molecular Probes) or trypan blue. Data were analyzed using one-way analysis of variance followed by post-hocStudent-Newman-Keuls method and/or Student's t test. Significance level was set at p < 0.05. The colorimetric H2O2 assay was performed in a 96-well microtiter plate (SpectraMax Plus, Molecular Devices), according to a modification of an existing protocol (22Han J. Yen S. Han G. Han P. Anal. Biochem. 1996; 234: 107-109Crossref PubMed Scopus (21) Google Scholar). Polypeptides (10 μm) or vitamin C (10 μm), Cu(II) (1 μm), and a H2O2 scavenging agent, tris(2-carboxyethyl)phosphine hydrochloride (Pierce, 50 μm), were co-incubated in PBS buffer (300 μl), pH 7.4, for 1 h at 37 °C. Following incubation, the unreacted tris(2-carboxyethyl)phosphine hydrochloride was detected by 5,5′-dithiobis(2-nitrobenzoic acid) (Sigma, 50 μm). The amount of H2O2 produced was quantified based on the formula: H2O2 (μm) =A × 106/(2 × L ×M), where A is the absolute absorbance difference between a sample and catalase-only (Sigma, 100 units/ml) control at 412 nm; L = the vertical pathlength, corrected automatically by the plate reader to 1 cm; M is the molecular absorbance for 2-nitro-5-thiobenzoate (14,150m−1 cm−1 at 412 nm). For the DCF assay, 5 mm of 2′,7′-dichlorofluorescin diacetate (Molecular Probes) in 100% ethanol was de-acetylated by 0.01m NaOH for 0.5 h, 200 units/ml horseradish peroxidase was then added, and the DCF solution was neutralized and diluted to 200 μm by PBS before use. Then 20 μm DCF, 10 μm Aβ1–42, and 1 μm Cu(II)-glycine were co-incubated at 37 °C for 20 min in PBS. Catalase (1000 units/ml) with or without heat inactivation (100 °C for 30 min) was used to validate the signal. The fluorescent readings were recorded by a Packard 96-well fluorocounter (485 nm excitation; 530 nm emission). Where the O2 tension of the buffers were manipulated, the buffer vehicle was continuously bubbled for 2 h at 20 °C with 100% O2 to create conditions of increased O2tension, or purged with argon (Ar) to create anerobic conditions, prior to the addition of vitamin C or polypeptide. To establish whether Aβ reduces Cu(II) to Cu(I), we used three independent methods. In the first approach various Aβ peptides, vitamin C (as a positive control), and other control polypeptides were incubated with Cu(II)-glycine chelate. Cu(I) formation was monitored using a chromogenic Cu(I)-trapping agent, BC. Aβ1–42 and Aβ1–40 were the only tested peptides found to reduce significant amounts of Cu(II) to Cu(I) (generating 6 and 3 μm, respectively) during the 1-h incubation period (Fig. 1). Cu(II) was not significantly reduced (<1 μm) by rat/mouse Aβ1–40, reverse sequence human peptide (Aβ40–1), amylin, insulin, Aβ1–28, or Aβ25–35 (Fig. 1). Since it has been pointed out that BC could also bind to Cu(II), potentially altering its reduction potential and cause artifactual estimates of Cu(I) (23Sayre L.M. Science. 1996; 274: 1933-1934Crossref PubMed Scopus (35) Google Scholar), we corroborated the apparent Cu(II)-reducing properties of Aβ1–40/2 by comparing the assay results using BC to those obtained with another Cu(I) detection agent, BCA. The apparent amounts of Cu(I) generated by Aβ1–42 and Aβ1–40 using either BC or BCA were in excellent agreement (Fig. 1, inset). Secondly, EPR spectroscopy was used to measure residual Cu(II) remaining after incubating stoichiometric ratios of CuCl2with Aβ1–40. This peptide caused a loss of the Cu(II) signal (76%), in relative agreement with the corresponding Cu(I) detected in the bioassays above. The detection of Cu(I) by BC assay, at levels roughly comparable to those estimated from loss of the EPR signal for Cu(II), suggests that the EPR signal was not disappearing due to formation of antiferromagnetically coupled (S = 0) dicopper species. Experimental EPR S-band spectra (Fig. 2) and X- and Q-band spectra (data not shown) establish that copper binds very tightly to these peptides. Fig. 2 A shows that an approximately equimolar mixture of Aβ1–40 and CuCl2 produces a single Cu(II)-peptide complex. The multiple resonance signal shown in the EPR spectrum for copper-peptide (Fig. 2 A) is for a single Aβ-bound Cu(II) species that is paramagnetic. Spin quantitation employing a dual mode TE104 X-band rectangular cavity revealed that this EPR signal accounted for 24% of the added Cu(II), consistent with 76% of the Cu(II) being converted to EPR-silent species, very likely Cu(I). There was no evidence of free, uncomplexed Cu(II) remaining after addition of the peptide, since unbound Cu(II) itself gives a different multiple resonance signal. The loss of 76% of the Cu(II) signal upon incubation with Aβ1–40 is compatible with peptide-mediated reduction of Cu(II) to diamagnetic Cu(I), which is undetectable. Computer simulation of the experimental spectrum of Aβ1–40 with an axially symmetric spin Hamiltonian and the g andA matrices (g ∥, 2.295;g ⊥, 2.073; A ∥, 163.60, A ⊥, 10.0 × 10−4cm−1) yielded the spectrum shown in Fig. 2 B. Expansion of the M I = −1/2 resonance revealed nitrogen ligand hyperfine coupling. Computer simulation of these resonances indicated the presence of at least three nitrogen atoms. The magnitude of the g ∥ andA ∥ values also suggest a tetragonally distorted geometry, which is commonly found in type 2 copper proteins (24Frausto da Silva J.J.R. Williams R.J.P. The Biological Chemistry of the Elements. Clarendon Press, Oxford1991Google Scholar), and together with the Blumberg Peisach plots, are consistent with a fourth equatorial ligand binding to copper via an oxygen rather than a sulfur donor atom. Thus, the coordination sphere for the copper-peptide complex is CuN3O1. The third line of independent evidence to support Aβ-dependent reduction of Cu(II) was the electrochemical behavior of Cu(II), assessed in the presence and absence of Aβ by cyclic voltammetry (Fig. 3). This revealed that Aβ1–42 (line c) gives rise to a voltammetric response with a formal reduction potential of approximately +500–550 mV (versus Ag/AgCl) in phosphate-buffered saline alone. This is an extraordinarily high positive potential, which suggests a Cu(I) oxidation state that is highly stabilized by the peptide. Our buffers, even after careful preparation and filtration through Chelex-100 resin, were found routinely to possess ≈0.1 μm copper background contamination, as assessed by inductively coupled plasma analysis (8Cherny R.A. Legg J.T. McLean C.A. Fairlie D. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). To determine whether interaction with this trace quantity of metal ion was responsible for the electrochemical response of Aβ, we added CuCl2 (17 μm) to the solution. This increased the magnitude of the response from the Aβ1–42 solution (line d), consistent with this potential being characteristic of a copper-Aβ complex. Background voltammograms of the PBS vehicle did not produce such peaks (line a), and Cu(II) in PBS showed only a Cu(II/I) peak in the reduction wave at ≈−80 mV (line b) along with the return oxidation wave. This reduction process was not observed when Cu(II) was added to Aβ1–42, consistent with the complete reaction of Cu(II) with Aβ. Since Cu(I) can in principle reduce O2, we tested Aβ peptides in the presence of Cu(II) (1 μm) for direct production of H2O2. We found that peroxide was indeed formed in these solutions, the amount of H2O2 produced in 1 h by the various Aβ and control peptides was greatest for Aβ1–42 (10 μm) > Aβ1–40 (7.5 μm) ≫ rat Aβ1–40, Aβ40–1, Aβ25–35, Aβ1–28, insulin, and amylin (≈0 μm) (Fig. 4 A), paralleling the amounts of metal reduction by the same peptides (Fig. 1). Validation of these results was achieved by coincubating Aβ with catalase, which abolished the H2O2 signal in a dose-dependent manner, and also by performing a corroborating assay using dichlorofluorescein (data not shown). To investigate whether the formation of H2O2 by Aβ was due to the specific reduction of O2, we studied the generation of H2O2 by Aβ1–42, Aβ1–40, and vitamin C under different O2 tensions in the presence of 1 μm Cu(II) (Fig. 4 B). The presence of vitamin C was used as a control measure to estimate the maximum amount of H2O2 that could be detected in the buffer vehicle by the non-protein generation of Cu(I). This experiment confirmed that there was a significant dependence of H2O2 production upon the O2tension. The presence of either Aβ1–42 and Aβ1–40 generated significantly more H2O2 (Aβ1–42 > Aβ1–40) than vitamin C under any O2 tension studied, and generated H2O2 under low O2 tension where vitamin C produced none. Under ambient and argon-purged conditions in this system, the reduction of Cu(II) alone by the positive control, vitamin C, was insufficient to produce detectable H2O2. Therefore, Aβ facilitated the reduction of O2 more than would be expected by the interaction of the Cu(I) reduced by Aβ with passively dissolved O2. Hence, Aβ acts not only to reduce metal ions, but also to trap molecular O2 to form H2O2. These data also suggest that copper cycles between the oxidized and reduced forms when bound to Aβ, since the presence of 1 μm Cu(II) was sufficient to produce a catalytic amount (10 μm) of H2O2 in 1 h, consistent with redox cycling and multiple electron donation events from Cu(I) to molecular O2. We examined Aβ1–42 (10 μm) in Cu(II) (1 μm in PBS, pH 7.4) for evidence of O2− formation over a 1 h incubation. Neither of the O2−-selective detection reagents hydroethidium (20 μm, Molecular Probes), nor nitro blue tetrazolium (NBT, 0.1 mm) detected O2− formation from Aβ, using xanthine (1 mm) with xanthine oxidase (0.015 units/ml) in PBS as a positive control. To prove that Aβ-mediated H2O2 formation is metal-ion dependent, H2O2 production by Aβ1–42 in the presence of copper-selective chelators was assayed (Fig. 4 C). The presence of 200 μm BC or diethylenetriaminepentaacetic acid abolished Aβ-mediated H2O2 formation in the presence of 1 μm Cu(II). Triethylenetetramine dihydrochloride only decreased the formation of H2O2 by ≈50%, indicating that high affinity copper chelators may only be able to interrupt these interactions if they have sufficient stereochemical access to the bound copper atom. We tested the consequences of Cu(II)-Aβ interaction upon the survival of primary neuronal cultures. We found that the combination of Aβ1–42 with Cu(II)-glycine (each at 10 μm) significantly potentiated Aβ neurotoxicity (70% cell death in the presence of copper, 40% in its absence) (Fig. 5 A). The presence of catalase slightly rescued the toxicity of the peptide alone (30% cell death), but entirely rescued the fraction of Aβ1–42 toxicity that was enhanced by Cu(II), indicating that the potentiation of toxicity induced by the presence of Cu(II) was mediated by H2O2. Catalase could not completely rescue the toxicity of Aβ1–42, even when catalase was used at higher concentrations (2000 and 3000 IU, data not shown). Since Cu(II)-glycine alone was not neurotoxic, these results strongly support the possibility that Cu(II) interaction modifies Aβ leading to enhanced H2O2-mediated neurotoxicity. The inability of catalase to completely rescue the neurotoxicity caused by Aβ1–42 suggests that other neurotoxic mechanisms that are not mediated by H2O2 may contribute up to 25% of the lethality observed. To confirm that Cu(II)-enhanced toxicity of Aβ was mediated by extracellular H2O2 production, we studied the effects of Cu(II)-glycine supplementation upon the toxicity of the other biologically occurring Aβ species in primary neuronal culture, comparing human Aβ1–42 and Aβ1–40 to rat Aβ1–40 (Fig. 5 B). In the absence of additional Cu(II), Aβ1–42 was observed to be more neurotoxic than both human and rat Aβ1–40, whose lethal effects were indistinguishable and marginal at the concentrations tested (10–25 μm). However, whereas additional Cu(II) (10 μm) dramatically increased the toxicities of human Aβ1–42 and Aβ1–40, additional Cu(II) did not enhance the toxicity of rat Aβ1–40. The Cu(II)-induced potentiation of Aβ toxicity therefore followed the relationship of Aβ1–42 > Aβ1–40 ≫ rat Aβ1–40, which parallels both Cu(II) reduction, and the Cu(II)-mediated generation of H2O2, by the same peptides. Taken together, these data argue that Cu(II) enhances the neurotoxicity of Aβ substantially through the cell-free generation of H2O2. The experiments described above establish that Cu(II) is reduced by Aβ peptides, that Cu(I) mediates O2-dependent cell-free H2O2 generation, and that these properties directly correlate with the Cu(II)-mediated potentiation of Aβ neurotoxicity in cell culture. Previously, we showed that concentrations of Cu(II) at = 1 μm induce the aggregation of Aβ (17Atwood C.S. Moir R.D. Huang X. Bacarra N.M.E. Scarpa R.C. Romano D.M. Hartshorn M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. 1998; 273: 12817-12826Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar) and generation of TBARS reactivity (6Huang X. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Crossref PubMed Scopus (1036) Google Scholar). The amounts of aggregation (17Atwood C.S. 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