Metalation and demetalation of human metallothionein-2A (MT) with Cd 2+ is investigated by using chemical labeling and "bottom-up" and "top-down" proteomics approaches. Both metalation and demetalation of MT-2A by Cd2+ are shown to be domain specific and occur as two distinct processes. Metalation involves sequential addition of Cd2+ to the α-domain resulting in formation of an intermediate, Cd4MT. Chemical labeling with N-ethylmaleimide (NEM) and tandem mass spectrometry experiments clearly show that the four metal ions are located in the α-domain. In the presence of excess Cd2+, the Cd4MT intermediate reacts to add Cd2+ to the β-domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd2+ is removed (by EDTA) first from the β-domain followed by Cd2+ removal from the α-domain. Metalation of human MT-2A is shown to be metal ion specific by comparing relative metal ion binding constants for Cd2+ and Zn2+.
ASJC Scopus subject areas
- Analytical Chemistry