Metalloproteins, speciation analysis

The combination of XRF with electrophoresis can constitute a quasi-on-line coupling technique. Great efforts have been made to develop on-line analysis techniques with XRF. Mann et reported a technique of on-line SRXRF detection of metal ions, such as Fe, Co, Cu, and Zn, in their high binding-constant complexes for capillary electrophoresis (CE) separation. An X-ray transparent polymer coupling is used to create a window for the on-line X-ray detection. In contrast to ICP-MS, this detection technique is not limited by sample or the buffer volatility or atomization efficiency. Simultaneous XRF and UV absorbance detection can be used to provide on-line determination of metal/chelate ratios. A bench-top energy-dispersive micro X-ray fluorescence system was also combined with the CE apparatus constructed by a thin-walled fused-silica capillary for elemental analysis of the species containing Fe, Co, and Cu, for example. This coupled technique used for metalloprotein speciation analysis can avoid the compromise between optimal separation and sensitive detection, which must be taken into account in the HPLC-ICP-MS procedure. In addition, this detection scheme is non-destructive, so the separated material can be recovered for additional characterization. 

The application of LC online with ESTMS is especially attractive for the speciation analysis of Cd and Zn complexes. Metallated complexes such as MTs may suffer from several artifacts in their chromatographic separation due to the presence of those metals. For instance, the presence of other metals may modify the retention of the complex Cd-Zn-MTs on a RP column. As an alternative, ESI-MS with pH control can help in the analysis of metals in native and reconstituted metalloproteins, in order to exactly elucidate which biocompoimd binds Cd, how many atoms of Cd per bioHgand molecule are boimd, and what is the combination with other metals (e.g., Zn and Cu) in such molecules. In this line, samples of rabbit liver and horse kidney MTs (previously isolated by SEC and anionic-exchange chromatography) were analyzed imder pH selected conditions. In moderately acidic (pH 4.0) and neutral (pH 6-7) media, the complexes of MTs with Cd and Zn retain... 

The ease of sample introduction by nebulization and the rapid multielement detection capabilities of ICP-MS lend the technique to online detection for metal and metalloprotein speciation by liquid chromatographic separations. This approach of LC/HPLC-ICP-MS has been applied to speciation of organo-Sn [50], organo-Pb [51], and organo-As [52], separation of metalloproteins such as metallothionein and ferritin [53,54], analysis of Au metabolites in blood from patients undergoing chrysotherapy for arthritis [55], and separation and quantitation of macromolecular forms of Fe in human liver biopsies [56]. 

Example The function of metalloproteins critically depends on their interaction with a metal, e.g., Cu, Zn, Fe, Mo, or metalloid such as Se or As. The living cell not only depends on its genome and proteome, but also on its metallome, i.e., the distribution of those elements among different biomolecules. The complexity of speciation analysis demands for a combined approach of separation techniques and different methods of mass spectrometry. This is best illustrated by the metallomics toolbox that reflects the current understanding of how the metallome can be explored (Fig. 15.1) [21]. 

The information on the chemical speciation of trace elements in biological systems is much needed to evaluate their biological significance. Although a number of analytical techniques based on atomic behavior are available for the analysis of chemical speciation of trace elements, neutron activation analysis, as a nuclear analytical technique, can be successfully used in chemical speciation studies, after appropriate fractionation steps. Table 2.5 lists some typical applications of NAA in chemical speciation analysis of metalloproteins. The main advantages of NAA are of its high sensitivity and the absence of matrix effects inherited from the conventional neutron activation analysis. It can, therefore, be used to analyze the chemical species of trace elements in very small samples or complicated matrices, which is often impossible for non-nuclear techniques. 

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