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.
Highlights
The samples were stacked in thin bands, less than 1 mm wide based on the isotachophoresis mechanism,[52] the stacking mode was not canceled a er migration into the separation gel, i.e., the separation mode did not commence like that of the protein
Using the Metal ion contaminant sweeping (MICS)-BN-polyacrylamide gel electrophoresis (PAGE)/metal detection PAGE system, the accurate distribution of Cu in human serum was investigated by quantitative determination of exchangeable HSA–Cu
Metal detection PAGE is suitable for determination of ultratrace metal ions in many cut gel fractions a er slab gel electrophoresis, including two dimensional gels, because simultaneous analysis of many samples is accessible with re-concentration of the diluted elutes.[35]
Summary
HPLC-based methods[24,25,26] are disadvantageous in that the interaction between the proteins and the hydrophobic stationary phase frequently causes denaturing of the proteins, which leads to dissociation of the metal ions. While size-exclusion HPLC (SEC) results in less denaturing of proteins due to weak interactions between proteins and the stationary phase, the resolution between the proteins is insufficient, frequently resulting in coelution of various proteins.[26,27,28] The CE-based method, which allows high resolution among proteins, has rather poor detection sensitivity for metal ions due to an innate and very small injection volume. Several research groups have developed PAGE-based techniques combined with laser-ablation (LA)-ICP-MS.[29,30,31,32] Paper
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