Evaluation of quantitative probes for weaker Cu(i) binding sites completes a set of four capable of detecting Cu(i) affinities from nanomolar to attomolar

Abstract

Copper plays essential roles in biology, but abnormal interactions are damaging. Reliable quantification of copper-protein interactions will underpin the molecular understanding of copper nutrition and toxicity. We have previously established two high affinity probes, Bathocuproine disulfonate (Bcs) and Bicinchoninate (Bca) anions, that are capable of in vitro quantification of Cu(i) binding with affinities from pico- to atto-molar concentrations. Quantitative probes are required for Cu(i) binding of lower affinity for proteins and peptides typically associated with neurodegenerative diseases. The present work evaluates two classic Fe(ii) ligands Ferene S (Fs) and Ferrozine (Fz) as quantitative probes for Cu(i). Both react with Cu(i) quantitatively to yield well-defined complex anions [Cu(I)(Fs)2](3-) (λmax = 484 nm, ε = 6700 cm(-1) M(-1)) and [Cu(I)(Fz)2](3-) (λmax = 470 nm, ε = 4320 cm(-1) M(-1)). These complexes are sensitive to aerial oxidation (E1/2∼ +0.36 V vs. SHE) and to substitution by other ligands (e.g., Cl(-), MeCN). However, they can be protected effectively under anaerobic conditions by suitable reductants and an excess of the free probe ligands. Formation constants β2 were determined by two approaches: direct metal ion titration and ligand competition. They provided estimates which differed by ∼3 orders of magnitude. The sources of these differences were examined carefully to consolidate the affinities of the two probes to a unified standard (10(15.1) M(-2) for Fz and 10(13.7) M(-2) for Fs). It is apparent that application of direct metal ion titrations to quantification of Cu(i) binding affinities is problematical and should be avoided. The four ligands Bcs, Bca, Fz and Fs in combination form a set of versatile probes for ligand competition experiments and are capable of detecting and differentiating an extended spectrum of Cu(i) binding affinities from nano- to atto-molar concentrations. Selected examples of quantification of weaker Cu(i) binding in proteins and peptides are provided, including that of an amyloid-β peptide.

Chemical and functional properties of metal chelators that mobilize copper to elicit fungal killing of Cryptococcus neoformans

“…In contrast, the harder O, O, donor ligands (PyO, maltol, Dfp) did not compete for Cu(I) with either BCA or the weaker ferrozine (Fz), which forms a Cu(I) complex with an absorption centered at 470 nm (ε = 4320 M −1 cm −1 ) and a log K Fz2Cu of 15.1. 35 The absence of competition for Cu(I) from these indicators by PyO, Dfp and maltol reinforces the absence of any change in spectrum of the free ligands in the presence of Cu(I).…”

“…34, 35 The conditional Cu(II) binding affinities at pH 7.4 for select agents are shown in Table 2 and representative titrations are shown in ESI Figures S5–S6. The data were best fit using a 2:1 L:Cu(II) stoichiometry in all cases.…”

Copper(i) targeting in the Alzheimer's disease context: a first example using the biocompatible PTA ligand

“…Note also that quantitative formation of [Cu I (PTA) 4 ] + complex observed here by spectroscopy is in agreement with the respective affinity constants of Aβ and PTA ligands for Cu(I). [20][21][22] Then, the impact of Cu removal from Aβ and sequestration as the air-stable [Cu I (PTA) 4 ] + complex on ROS production was investigated by established methods. 23 Briefly, the ROS production can be seen as the reduction of dioxygen by ascorbate catalysed by the Cu I/II (Aβ) complex leading to O 2°-, H 2 O 2 and HO°.…”

Tripodal amine ligands for accelerating Cu-catalyzed azide–alkyne cycloaddition: efficiency and stability against oxidation and dissociation

“…The dissociation constants ( K D ) of the ligand-Cu(I) complexes were determined using bicinchoninic anion ( Bca ) as a competing ligand (Scheme 1C), 48, 49 which forms a 2:1 complex with Cu(I) (formation constant β 2 = 1.58 × 10 17 M −2 ) that is essentially pH-independent at pH > 7.0. 48, 50 Lower affinity probes such as Ferene would give better accuracy for ligands with K D > 10 −9 M, 49 but all ligands in Fig.…”

“…48, 50 Lower affinity probes such as Ferene would give better accuracy for ligands with K D > 10 −9 M, 49 but all ligands in Fig. 2 possess sufficient binding affinity (Fig.…”