Doug Lloyd: The Separation, Identification, And Molecular Analysis Of Complex Mixtures Of Metalloproteins And Polypeptides By Tandemly linked Two-Dimensional HPLC-ICPMS. Recent evidence has indicated that the physiological function of MT may change in response to cellular redox status.  First, under reducing conditions in the presence of GSH the protein appears to play a role in metal detoxification.  It acts in this manner as a transcriptionally inducible intracellular ligand capable of sequestering metals.  By removing free metals from the cell cytoplasm, MT limits the potential for non-specific binding of metals to biomolecules susceptible to perturbation.  Second, the protein may also act to provide a physiologically available store of metal under oxidizing conditions in the presence of GSSG for the activation of Zn and Cu metalloenzymes. These, potentially rate limiting enzymes, modulate many important cellular processes (e.g. replication, respiration, transcription, protein synthesis and degradation and energy metabolism).  Two aspects of metallothionein function are not clear at this time.  First, it is not known whether the observed responses of MT to GSH and GSSG identify glutathione as a critical intermediary for metal transfer, or relate to a more general response to a localized or temporal change in cellular redox status.  Thus, the standard reduction potential of metallothionein (-366mv) provides for oxidation by cellular disulfides such as GSSG and other mild oxidants.  This destabilization of the zinc thiol clusters in a spatial manner lends itself to co-operative disulfide formation and metal release.  Second, it is not known whether glutathione or changes in redox status could cause the release of highly toxic, non-physiological metals such as Cd, Ag and Hg, which also bind with high affinity to MT.  The release of these metals from MT and their subsequent non-specific binding to macromolecules susceptible to perturbation may induce metal toxicity in cells experiencing stress.  To critically test the role of MT and glutathione in this context it has been necessary to develop techniques that can quantitatively monitor the kinetic transfer of metals between proteins in the cell.  Using a simplified model involving purified mixtures of proteins, we have shown that two dimensional HPLC-ICPMS can be used to quantify the metal content of proteins resolved on the basis of size and charge.  These initial studies have involved a mixture of MT, alkaline phosphatase and GSH and GSSG.  Identification of the eluant species has been accomplished using three independent approaches.  First, spectroscopically by their absorption spectra.  Second, by their enzymatic properties and third, by their elemental fingerprint.  Further refinements in the EC system will allow for precise control of the redox conditions in the reaction mixture and the pave the way for future studies involving the role of glutathione in mediating Zn and Cu transfer between MT and recipient apo-proteins.