| dc.description.abstract | Eukaryotic cells contain low-molecular-mass metal complexes (LMMMCs), defined as having masses between 200 – 10,000 Da, but these so-called labile or chelatable metal pools are poorly defined in terms of structures and functions. LMMMCs are thought to participate in metal-ion regulation, trafficking, storage and/or signaling in cells. These cellular processes are often dysfunctional in metal-associated diseases. The objective of these studies was to detect and characterize LMMMCs in eukaryotic cells, organelles and tissues. A novel liquid chromatography system in a cold inert-atmosphere glove box was interfaced with an in-line inductively coupled plasma mass spectrometer, and this LC-ICP-MS system was used to detect LMMMCs in yeast cells, mitochondria, and vacuoles as well as in mouse brain and liver cells and mitochondria. In each biological system, this separations technique was applied to detect numerous LMMMCs. The molecular mass and concentration of such species were estimated.
In yeast, the previously reported mismetallation of MnSOD2 was examined in the mutant strain Δmtm1. A combination of SEC and AEX chromatography revealed that the degree of mismetallation of the SOD2 protein, in which Fe replace Mn in the active site, was no greater in Δmtm1 cells than in WT cells. The mitochondria of such mutant cells did exhibit an intense chromatography peak of Mn corresponding to at mass of 2000 – 3000 Da. Mitochondria from WT cells exhibited a similar species, but at much lower intensity. This was the only Mn species present, suggesting that it was the used to metallate apo-SOD2.
Mitochondria isolated from WT yeast cells contained 6 Co, 3 Cu, 2 Mn, 5 Fe and 3 Zn LMMMCs and approximately 6 P- and S- LLM species. Some of the P- and S- LMMCs probably arose from compounds like ATP, ADP, etc. Molecular masses of the LMM Cu peaks were higher (> 5 kDa) than for the LMM complexes of other transition metals. Zinc, Mn, and Fe had multiple species of interest which demonstrate the presence and labiality of the metals in pools.
The same separation system was utilized to examine mice brain LMM extracts were found to contain > 30 LMMMCs. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn LLM complexes were detected. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4–20 kDa. In these systems, Co was the only metal for which the aqueous complex was reproducibly observed.
A second mouse study used the LC-ICP-MS system to examine the forms of iron present in mouse plasma. Chromatograms exhibited ~6 Fe-associated peaks that were assigned to ferritin, transferrin, and hemopexin, respectively; the other 3 peaks could not be assigned. The LC-ICP-MS experiment demonstrates that numerous Fe-containing species coexist with transferrin in healthy WT mouse plasma. | en |
