Characterization of Copper Interactions with Alzheimer Amyloid  Peptides: Identification of an Attomolar-Affinity Copper Binding Site on Amyloid 1– 42 *²‡Craig S. Atwood, *²‡Richard C. Scarpa, *²‡Xudong Huang, ‡§Robert D. Moir, *²‡Walton D. Jones, David P. Fairlie, ‡§Rudolph E. Tanzi, and *²‡Ashley I. Bush *Laboratory for Oxidation Biology, Departments of ² Psychiatry and §Neurology, and ‡Genetics and Aging Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.; and Centre for Drug Design and Development, University of Queensland, Brisbane, Queensland, Australia Abstract: Cu and Zn have been shown to accumulate in the brains of Alzheimer’s disease patients. We have pre- viously reported that Cu 2+ and Zn 2+ bind amyloid  (A), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu 2+ -binding sites on synthetic A1– 40 and A1– 42. We have developed a ligand displacement tech- nique (competitive metal capture analysis) that uses met- al– chelator complexes to evaluate metal ion binding to A, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu 2+ -binding site on A1– 42 (log K app = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the pres- ence of trace Cu 2+ contamination (customarily 0.1 M). In contrast, A1– 40 has much lower affinity for Cu 2+ at this site (estimated log K app = 10.3), explaining why this peptide is less self-aggregating. The greater Cu 2+ -bind- ing affinity of A1– 42 compared with A1– 40 is associ- ated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing A precipitation was confirmed by the demonstration that A peptide (10 M) remained soluble for 5 days only in the presence of high-affinity Cu 2+ -selective chelators. Key Words: Human amyloid  peptide —Copper—Affinity— Stoichiometry—Alzheimer’s disease —Binding affinity method—Chelators. J. Neurochem. 75, 1219 –1233 (2000). Alzheimer’s disease (AD) is characterized pathologi- cally by the deposition of amyloid plaques and neurofi- brillary tangles and by neuronal degeneration in the brains of affected individuals. Amyloid deposits are composed primarily of the amyloid  (A) protein (Mas- ters et al., 1985; Roher et al., 1996) generated as a mixture of polypeptides manifesting carboxyl- and ami- no-terminal heterogeneity. The A1– 40 isoform is the predominant soluble species in biological fluids (Shoji et al., 1992; Vigo-Pelfrey et al., 1993). Although less abundant in biological fluids, A1– 42 is the predomi- nant species found in plaque deposits (Masters et al., 1985; Roher et al., 1996). Metal ion homeostasis is severely dysregulated in AD (Hershey et al., 1983; Ehmann et al., 1986; Thompson et al., 1988; Vance et al., 1990; Basun et al., 1991; Samudralwar et al., 1995; Deibel et al., 1996; Cornett et al., 1998; Lovell et al., 1998; Gonza ´lez et al., 1999). Although the transition metal ions Cu, Fe, and Zn are maintained at high concentrations within the healthy brain neocortical parenchyma (total dry weight concen- trations of 70, 340, and 350 M, respectively), increased concentrations of these metal ions are detected in the neuropil of the AD-affected brain, where they are highly concentrated within amyloid plaque deposits with total concentrations reaching 0.4 and 1mM for Cu and Fe/Zn, respectively (Lovell et al., 1998). An elevated Zn 2+ concentration also can be detected in plaque de- posits histologically (Suh et al., 2000). We previously found that A avidly binds Cu 2+ , Zn 2+ , and Fe 3+ (Bush et al., 1994a,b, 1995; Huang et al., 1997; Atwood et al., 1998), perhaps explaining the recruitment of these metals into amyloid plaque pathology. Evidence for an interaction between Cu 2+ and A1– 40 was first observed by the stabilization of an Resubmitted manuscript received April 4, 2000; accepted April 18, 2000. Address correspondence and reprint requests to Dr. A. I. Bush at Laboratory for Oxidation Biology, Genetics and Aging Unit, Neuro- science Center, Massachusetts General Hospital East, Building 149, 13th Street, Charlestown, MA 02129-9142, U.S.A. E-mail: bush@helix.mgh.harvard.edu Drs. C. S. Atwood and R. C. Scarpa contributed equally to this article. Abbreviations used: A, amyloid ; AD, Alzheimer’s disease; CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; CMCA, competitive metal capture analysis; DSA, dog serum albumin; DTPA, diethylenetriaminepentaacetic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate. 1219 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry