Date of Award
Doctor of Philosophy
Metallothioneins (MT) are a class of cysteine-rich proteins that bind a wide variety of metal ions. Although it has been suggested that the structure of the metal binding sites in Cd,Zn-MT involves two metal clusters (alpha and beta) which coordinate the metals through a tetrahedral arrangement of cysteine residues, considerably less information is available concerning the metal binding sites in MT containing Hg('2+) and Cu('+). In this work, absorption, circular dichroism and magnetic circular dichroism spectroscopies have been used to characterize metal binding to metallothioneins. Three species of MT (rat, guinea pig and crab) have been studied. In addition to the metal binding properties of MT itself, changes in the spectroscopic properties when the Cd('2+) and Zn('2+) of the native protein are replaced by Hg('2+) and Cu('+) in vitro have also been investigated. Although the MCD spectra of the Cd,Zn-MT from all three species resemble each other closely, the CD spectra indicate a difference in the protein conformation in the crab protein. However, all three species behave in a similar manner to protonation and metal loading with Cd('2+). A comparison of the MCD spectra of Cd,Zn-MT with that obtained for a Cd-BAL (BAL = 2,3-dimercaptopropanol) model complex suggests that the Cd('2+) bound in MT has a tetrahedral symmetry.;Spectral changes observed during metal replacement studies with Hg('2+) and Cu('+) clearly demonstrate that first Zn('2+) and then Cd('2+) ions are displaced from the MT. The following order of binding in the two clusters can be concluded from this study: Alpha cluster: Hg > Cd > Cu > Zn; Beta cluster: Cu,Hg > Cd > Zn.;Titration studies of Cd,Zn-MT with Hg('2+) show the isomorphous replacement of Cd('2+) and Zn('2+). A comparison of the MCD spectra obtained from the Hg-substituted MT and a Hg-BAL model complex suggests that the Hg('2+) ions bound to the protein are not in a tetrahedral environment.;Titration studies with Cu('+) show that at least four Cu('+) ions can be bound to the alpha fragment, while a total of six Cu('+) ions are associated with the beta domain. The MCD data indicate that the Cu('+) is not bound with a tetrahedral symmetry.
Law, Annie Yiu-chu, "Spectroscopic Studies Of Metal Binding To Metallothioneins" (1984). Digitized Theses. 1374.