Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Graphene/tri-block copolymer composites prepared via RAFT polymerizations for dual controlled drug delivery via pH stimulation and biodegradation

A novel tri-block copolymer poly(oxopentanoate ethyl methacrylate)-block-poly(pyridyl disulfide ethyl acrylate)-block-poly(ethylene glycol acrylate) [poly(OEMA-b-PDEA-b-PEGA)], retaining active keto groups and pyridyl disulfide (PDS) side functionalities, was synthesized as a drug delivery vehicle using reversible addition-fragmentation chain transfer (RAFT) polymerization method. One mimic drug pyridine-2-thione (PT) was introduced into the monomer, PDEA for copolymerization. The other mimic drug O-benzylhydroxylamine (BHA) was conjugated with tri-block copolymer via efficient oxime coupling chemistry, followed by the attachment onto graphene via π-π stacking interaction to obtain a graphene/tri-block copolymer composite. 1H NMR, UV-vis absorption spectroscopy, fluorescence spectroscopy, gel permeation chromatography (GPC), atomic force microscope (AFM) and transmission electron microscope (TEM) were used to verify the successful step-wise preparation of the tri-block copolymer and drug loaded composite. In vitro release behaviors of BHA and PT from graphene/tri-block copolymer composite via dual drug release mechanisms were investigated. BHA can be released under acid environment, while PT will be released in the presence of reducing agents, such as dithiothreitol (DTT) or glutathione (GSH). It can be envisioned that this novel composite could be exploited as a novel intracellular drug delivery system via dual release mechanisms.