Date of Award
1990
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Metallothioneins (MT) are a class of low molecular weight, cysteine rich proteins, which bind a wide range of metals both in vivo and in vitro. This protein has become the subject of intensive interest in many branches of the life sciences over the past two decades.;In this thesis, the manner in which mercury and cadmium bind to metallothionein has been studied using the techniques of circular dichroism (CD), magnetic circular dichroism (MCD), UV-visible absorption, and X-ray near edge absorption structure (XANES) spectroscopy.;At neutral pH, the Hg{dollar}\sb7{dollar}-MT and Hg{dollar}\sb4{dollar}-{dollar}\alpha{dollar} MT species formed from metal-free MT (apo-MT), or Zn-MT adopt a mercury-thiolate cluster structure. This Hg{dollar}\sb{lcub}\rm m{rcub}{dollar}S{dollar}\sb{lcub}\rm n{rcub}{dollar} cluster has a different arrangement compared with the cadmium thiolate cluster formed between MT and Cd{dollar}\sp{lcub}2+{rcub}{dollar}. When Hg{dollar}\sp{lcub}2+{rcub}{dollar} binds to apo-MT, two different coordination geometries are involved (linear and tetrahedral). Both solvent and temperature affect the formation of the Hg{dollar}\sb{lcub}\rm m{rcub}{dollar}S{dollar}\sb{lcub}\rm n{rcub}{dollar} clusters. Strikingly, Hg{dollar}\sb7{dollar}-MT formed by adding Hg{dollar}\sp{lcub}2+{rcub}{dollar} to Cd{dollar}\sb7{dollar}-MT has a very different Hg{dollar}\sb{lcub}\rm m{rcub}{dollar}S{dollar}\sb{lcub}\rm n{rcub}{dollar} arrangement compared with the Hg{dollar}\sb{lcub}\rm m{rcub}{dollar}S{dollar}\sb{lcub}\rm n{rcub}{dollar} clusters formed from apo-MT and Zn-MT. The spectroscopic properties of Hg{dollar}\sb4{dollar}-{dollar}\alpha{dollar} MT formed by adding Hg{dollar}\sp{lcub}2+{rcub}{dollar} to Cd{dollar}\sb4{dollar}-{dollar}\alpha{dollar} MT are different from proteins formed between not only apo-MT and Hg{dollar}\sp{lcub}2+{rcub}{dollar}, and Zn-MT and Hg{dollar}\sp{lcub}2+{rcub}{dollar}, but also Cd{dollar}\sb7{dollar}-MT and Hg{dollar}\sp{lcub}2+{rcub}{dollar}.;The formation of a novel Hg{dollar}\sb{lcub}18{rcub}{dollar}-MT species under acidic conditions is reported. The discovery of this highly structured Hg{dollar}\sb{lcub}18{rcub}{dollar}-MT species adds a new type of structure to those described previously for metallothionein. Dramatic spectroscopic differences in the formation of Hg{dollar}\sb{lcub}18{rcub}{dollar}-MT from apo-MT 1 or 2 indicates a new discriminatory property between different metallothionein isoforms.;Cadmium binds to Zn-MT in a "distributed manner", but binds to apo-MT in a "domain specific manner". The onset of cluster formation in the {dollar}\alpha{dollar} domain is associated with the appearance of a derivative feature in the CD spectrum. This feature has been attributed to exciton coupling arising from adjacent Cd-SR units (S-Cd-SR-Cd-S) in the {dollar}\alpha{dollar} domain.;Results from a XANES study suggest that while Zn{dollar}\sb7{dollar}-MT, Cd{dollar}\sb7{dollar}-MT, and Hg{dollar}\sb7{dollar}-MT have similar structures from the perspective of the coordinating sulfurs, the binding sites in Hg{dollar}\sb7{dollar}-MT are not exactly the same. Hg{dollar}\sb{lcub}18{rcub}{dollar}-MT adopts a completely different structure when compared with the M{dollar}\sb7{dollar}-MT complexes.;The conditions for the use of Chelex-100 in metallothionein systems are presented. Use of Chelex-100 greatly simplifies the procedures of removing free metals.
Recommended Citation
Lu, Wuhua, "Spectroscopic Studies Of Mercury And Cadmium Binding To The Protein Metallothionein" (1990). Digitized Theses. 1993.
https://ir.lib.uwo.ca/digitizedtheses/1993