Abstract
Solvent perturbation studies in 100% dimethyl sulfoxide (Me2SO) have been carried out on rabbit liver metallothionein (MT) in an effort to learn more about the factors stabilizing the three-dimensional structure and the mechanism of cluster formation. As indicated by the electronic absorption spectra of Co7-metallothionein, the reconstituted protein preserves its structural integrity in this solvent. Minor spectral differences between water and Me2SO were fully reversible. The titration of apoMT with cobalt(II) in Me2SO, followed by UV-visible-near-infrared electronic absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopy, indicate that the protein can refold in this solvent. A comparison with the previous titration data in water reveals that the first four titration steps in both solvents are identical, indicating a thermodynamically controlled folding process. However, the reversed order of the cluster completion between Me2SO and water may suggest the involvement of a kinetically controlled folding process in the last three titration steps. A new cluster form developed with approximately nine Co(II) equivalents.
Highlights
An understanding of the biological function of M T as a metal storage protein icslosely related to the cluster stability, as well as with their formation anddegradation
Since the biosynthesis of M T is induced by these metals andby certain hormones, it is believed that they play a crucial role in the Preparation of apoMT-Rabbit liver MT used in this study was isolated andpurified as described previously [14,15]
Comparison of Co7-MTStructures in Aqueous Solution and of the low energy S + Co(I1) charge-transfer transition and in Me2SO-In order to show that the reconstitution of MT an increased absorption at about 500 nm
Summary
An understanding of the biological function of M T as a metal storage protein icslosely related to the cluster stability, as well as with their formation anddegradation. To determine the relative spin concentrations (la), the MT above 7 cobalt eq, not seen in the previous studies in size of the EPR signals of the MT (0.5 mM) samples in MezSOwith water [5],is discussed below.
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