Date of Award
1995
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Metallothionein (MT) is a low molecular weight protein found in the kidneys and livers of all mammals. Twenty of its 61 or 62 amino acid residues are cysteinyl residues, allowing the protein to act as an impressive chelating agent. Although it has been well established that mammalian MT binds seven M(II) (M = Cd(II), Zn(II), Hg(II)) ions tetrahedrally in two metal-thiolate clusters of the form M{dollar}\sb4{dollar}(S{dollar}\rm\sb{lcub}cys{rcub})\sb{lcub}11{rcub},{dollar} (the {dollar}\alpha{dollar} domain) and {dollar}\rm M\sb3(S\sb{lcub}cys{rcub})\sb9,{dollar} (the {dollar}\beta{dollar} domain), little is known about the structure of copper-containing metallothioneins. In this thesis, absorption, circular dichroism (CD) and emission spectroscopies and excited state lifetime data are used to characterize the binding of Cu(I) to MT and to study the oxidative quenching reactions of Cu-MT.;Cu(I), in the form of {dollar}\rm Cu(CH\sb3CN)\sb4\sp+,{dollar} is used to replace Zn(II) in vitro in aqueous solutions of Zn{dollar}\sb7{dollar}-MT isolated from rabbit livers. The binding of Cu(I) to MT results in luminescence in the 600 nm region. Phosphorescence decay curves from this emission at room temperature are best fit to three-exponential curves. Changes in the ground state and excited state spectroscopic properties as Cu(I) is added to the Zn{dollar}\sb7{dollar}-MT indicate that 12 Cu(I) are needed to completely replace all 7 Zn(II). This has been confirmed by metal-displacement studies. The spectroscopic results are explained in terms of a Cu{dollar}\sb{lcub}12{rcub}{dollar}-MT structure in which the copper is trigonally bound in two copper-thiolate clusters of the form {dollar}\rm Cu\sb6(S\sb{lcub}cys{rcub})\sb{lcub}11{rcub},\ (\alpha){dollar} and {dollar}\rm Cu\sb6(S\sb{lcub}cys{rcub})\sb9\ (\beta).{dollar} Further changes in the spectroscopic properties as 12-20 Cu(I) are added indicate that the protein backbone begins to "unwind" from the binding site in order to incorporate the additional metal ions until there is one Cu(I) ion bound to each cysteinyl residue.;The temperature dependence of the spectroscopic properties indicate that the Cu(I) binds to Zn{dollar}\sb7{dollar}-MT in a random manner across both domains at all temperatures and then migrates at high temperatures to preferentially fill the {dollar}\beta{dollar} domain. At lower temperatures, this rearrangement occurs slowly enough to be monitored by luminesccnce spectroscopy and is analyzed in terms of first-order kinetics.;The addition of quenching affects to Cu-MT solutions agents the emission intensity and decay curves. The results have been analyzed using the Stern-Volmer approach and are interpreted in terms of solvent accessibility to the two copper-thiolate clusters.
Recommended Citation
Green, Anna Rae, "Spectroscopic Properties Of Copper Binding To The Protein Metallothionein" (1995). Digitized Theses. 2553.
https://ir.lib.uwo.ca/digitizedtheses/2553