A chemometric-assisted voltammetric analysis of free and Zn(II)-loaded metallothionein-3 states

Abstract

We focused on the application of mass spectrometry and electrochemical methods combined with a chemometric analysis for the characterization of partially Zn(II)-loaded metallothionein-3 species. The results showed decreased Cat1 and Cat2 signals for the Zn(II)-loaded MT3 species with respect to the metal-free protein, which might be explained by the arrangement of tetrahedral metal-thiolate coordination environments and the formation of metal clusters. Moreover, there was a decrease in the Cat1 and Cat2 signals, and a plateau was reached with 4–5 Zn(II) ions that corresponded to the formation of the C-terminal a-domain. Regarding the Zn7-xMT3 complexes, we observed three different electrochemical behaviours for the Zn1–2MT3, Zn3–6MT3 and Zn7MT3 species. The difference for Zn1–2MT3 might be explained by the formation of independent ZnS4 cores in this stage that differ with respect to the formation of ZnxCysy clusters with an increased Zn(II) loading. The binding of the third Zn(II) ion to MT3 resulted in high sample heterogeneity due the co-existence of Zn3–6MT3. Finally, the Zn7MT3 protein showed a third type of behaviour. The fact that there were no free Cys residues might explain this phenomenon. Thus, this research identifies the major proteins responsible for zinc buffering in the cell.We focused on the application of mass spectrometry and electrochemical methods combined with a chemometric analysis for the characterization of partially Zn(II)-loaded metallothionein-3 species. The results showed decreased Cat1 and Cat2 signals for the Zn(II)-loaded MT3 species with respect to the metal-free protein, which might be explained by the arrangement of tetrahedral metal-thiolate coordination environments and the formation of metal clusters. Moreover, there was a decrease in the Cat1 and Cat2 signals, and a plateau was reached with 4–5 Zn(II) ions that corresponded to the formation of the C-terminal a-domain. Regarding the Zn7-xMT3 complexes, we observed three different electrochemical behaviours for the Zn1–2MT3, Zn3–6MT3 and Zn7MT3 species. The difference for Zn1–2MT3 might be explained by the formation of independent ZnS4 cores in this stage that differ with respect to the formation of ZnxCysy clusters with an increased Zn(II) loading. The binding of the third Zn(II) ion to MT3 resulted in high sample heterogeneity due the co-existence of Zn3–6MT3. Finally, the Zn7MT3 protein showed a third type of behaviour. The fact that there were no free Cys residues might explain this phenomenon. Thus, this research identifies the major proteins responsible for zinc buffering in the cell.