科研之友
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摘要
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 Cu-I ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu-I affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu-I to yield distinct 1:2 chromatophoric complexes [(CuL2)-L-I](3-) with formation constants beta(2) = 10(17.2) and 10(19.8) 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 Cu-I with K-D similar to 10(-15) M at pH 7, but it is air-sensitive, and its Cu-I affinity varies with pH. The Cu-I 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 Cu-I binding and the individual quantitative affinities (K-D 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 Cu-I with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.
- 出版日期2011-4-1
- 单位迪肯大学
