Metalloproteins electrophoresis

Characterization of Metalloproteins Using Capillary Electrophoresis Mark P. Richards... 

Capillary electrophoresis has proven to be useful in characterizing different molecular forms of various metalloproteins like metallothionein, transferrin, and conalbumin [2-5]. Molecular forms arise from differences in the amino acid sequence of proteins (isoforms), differences in the amount or type of metal bound (metalloforms), or from differences in the type and amount of carbohydrate side chains linked to the protein (glycoforms). CZE was used to follow the formation of the oligomeric iron core and its incorpora-... 

In the context of chemical speciation, a new hyphenated technique, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), in combination with gel electrophoresis, appears as an emerging and powerful tool for metal complexation studies of proteins, giving multielement information about metals bound to the previously separated metalloproteins. 

A combined use of PIXE with polyacrylamide gel electrophoresis (PAGE) provides a new biochemical method in studying separated groups of metalloproteins by permitting in situ... 

Chang, I-J., Gray, H. B., and Albin, M. (1993) Characterization of protein modification sites in metalloproteins using capillary electrophoresis. Anal. Biochem. 212, 24-27. 

As mentioned above, EDXRF is a non-destructive multielemental analytical technique. Because the beam size of the primary X-ray can be monitored with a slit or focusing system, XRF is capable of microscopic analysis, supplying information about the 2D distribution of trace elements. The technique can, thus, be used for imaging of trace elements in a biological specimen, also for direct elemental determination in protein bands after slab-gel electrophoresis. Reviews concerning analysis of metalloproteins with XRF after electrophoresis separation have been published. ... 

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. 

Table 3.4 The main analytical techniques for direct metalloprotein analysis on electrophoresis gels.

Prange et al. analyzed the isoforms of metallothioneins in human brain cytosol with capillary electrophoresis (CE) coupled to ICP-SFMS (inductively coupled plasma-sector field mass spectrometry). In the study, three metal-lothionein isoforms were separated by CE, and the enriched isotopes of Cu, Zn, Cd, and were mixed as a specific-unspecific spiking solution finally, the Cu, Zn, Cd, and S in metallothioneins were quantified with IDA. They found that the levels of MT-3 and MT-1 were lower in the brain samples from the patients with Alzheimer s disease in comparison with the controls (see Figure 4.8). The results indicated that quantification of metalloproteins gave additional information on its relationship with Alzheimer s disease. ... 

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