Separation of metalloproteins using a novel metal ion contaminant sweeping technique and detection of protein-bound copper by a metal ion probe in polyacrylamide gel electrophoresis: distribution of copper in human serum

Abstract

A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two types of gel electrophoresis, to obtain an accurate distribution of protein-bound metal ions in biological samples. First, proteins are separated by PAGE without the uptake of contaminant metal ions in the separation field and dissociation of metal ions from the proteins. This is followed by another PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using a fluorescent metal probe. The former is a new technique using blue-native (BN) PAGE to electrophoretically sweep all metal contaminants by employing two kinds of chelating agents. These agents form complexes with contaminants in the gel and the separation buffer solution, which migrate towards opposite pole directions, thus lowering the contaminants to below the ppt level during separation. This is termed "Metal Ion Contaminant Sweeping BN-PAGE (MICS-BN-PAGE)". After the separation of proteins under these first metal-free conditions, the metal ions in the gel fractions are eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxide dismutase were quantitatively determined, in addition to the exchangeable albumin-bound copper ions. This system successfully provided distribution maps of protein-copper in human serum. The precise distribution of copper in human serum was investigated, and found to be different from that which is widely accepted.

c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

“…Since CopI harbors a plastocyanin-like fold with a conserved copper binding motif and a cluster of 5 scattered histidines susceptible to bind transition metals like copper, we used the method of metal ion contaminant sweeping-blue native polyacrylamide gel electrophoresis (MICS-BN-PAGE) and a fluorescent probe ( 29 ) to map copper binding proteins in soluble fractions from WT cells grown microaerobically with 1.6 or 1,200 µM CuSO 4 compared with Δ copI mutant cells grown with 200 µM CuSO 4 . While no band was observed in the first BN-PAGE dimension for WT and Δ copI samples incubated, respectively, with 1.6 and 200 µM CuSO 4 ( Fig.…”

“…3B ). Subsequently, the copper content in each cut band was measured after acid extraction and addition of a fluorescence probe (FTC-ABDOTA); the resulting metal-probe complex was separated by PAGE ( 29 ). A strong copper signal was observed only in extracts of WT cells grown with 1,200 µM CuSO 4 ( Fig.…”

“…The procedure for metal ion contaminant sweeping (MICS)-BN-PAGE and copper detection PAGE was performed as reported previously by Saito et al ( 29 ). The procedure is described briefly as follows.…”

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye

“…To address the issue of contaminant metal ions, we have previously studied thermodynamically and kinetically stable metal chelates to exhaustively remove trace contaminant metal ions from the separation field in PAGE 21 . In this method, which we have called metal ion contaminant sweeping-blue native-PAGE (MICS-BN-PAGE), the cationic TPEN ( N,N,N ′, N ′-tetrakis (2-pyridylmethyl) ethylene-diamine) complexes and anionic EDTA complexes formed with contaminant metal ions migrate towards the cathodic and anodic directions, respectively.…”

“…Furthermore, MICS-BN-PAGE also avoids the possibility of metal-exchange reactions of holo-metalloproteins with contaminant metal ions M′’ 2+ (e.g. M 2+ -metalloprotein + M′ 2+ → M′ 2+ -metalloprotein + M 2+ ), which lead to the misidentification of metalloprotein species by conventional methods 21 . Still, the most difficult challenge remains, which is to isolate metalloproteins while simultaneously ensuring no dissociation of metal ions and the absence of contaminant metal ions in the separation field.…”