Measurement of the interaction of aqueous copper(II) with a model amyloid-β protein fragment — Interference from buffers

For the formation of a complex of Cu2+ with the amyloid-β (Aβ) proxy N-α-dihydrourocanylhistamine (L) in unbuffered aqueous solution (pH ∼ 5.7, 25.0 °C), UV spectrophotometric measurements give a stability constant of 3.8 × 105 L mol–1. This stability constant is within the lower limit of the range of stability constants reported in the literature for complexes of Aβ with Cu2+ — as expected, in view of the smaller number of coordination sites in L. Computer modeling indicates that the Cu2+–L complex is CuL(H2O)22+, with terdentate L bound to Cu2+ via two Nπ atoms and the O atom of the peptide link. Attempts to make stability constant measurements for Cu2+ with L in aqueous solution buffered with Tris/TrisH+/ClO4– to pH near 7.2 were unsuccessful because the Tris base when in large excess over CuL2+ promoted its dissociation to Cu2+ + L by scavenging free Cu2+ as Cu(Tris)(TrisH–1)+, or when in roughly equimolar concentrations formed a ternary adduct, CuL(Tris)2+. The interactions of Cu2+ with Tris buffer were re-examined spectrophotometrically and with the aid of computations that show that the most stable Cu2+–Tris complexes are the syn- and anti-isomers of Cu(Tris)22+, but in the experimental pH ranges these are present as Cu(Tris)(TrisH–1)+. Since Cu2+(aq) is strongly complexed by almost any base capable of forming a buffer system with near-physiological pH, stability constants reported for Cu2+–Aβ complexes in any buffer solution should be regarded with skepticism unless interactions of the buffer with Cu2+ and with CuAβ2+ are taken quantitatively into account. Moreover, in vivo, biological buffers will reduce the physiological importance of Aβ–Cu2+ complexes by competing with Aβ for Cu2+.