ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 213, No. 1, January, pp. 109-117, 1982 Metal Binding Sites of Rat Liver Cu-Thionein’ BRUCE L. GELLER AND DENNIS R. WINGE’ Received June 1, 1981 Cu-thionein purified from rat liver contains 10 g atoms Cu per mole protein. EPR and NMR bulk susceptibility studies indicate that the copper ions are bound in a diamagnetic state. Purification of the metalloprotein anaerobically results in a sample in which 18 cysteines can be titrated by 2,2-dithi~ip~dine. only lo-12 cysteines could be titrated in aerobically prepared samples. The copper ions in aerobically prepared Cu-thionein are more easily removed by ethylenediaminetetraacetic acid and diethylenetriaminepenta- acetic acid than are the ions in the anaerobically purified protein. Likewise, the Cu ions in aerobically prepared Cu-thionein molecules have the ability to reactivate aposuperoxide dismutase and to bind to apocarbonic anhydrase whereas the metal ions in anaerobically prepared Cu-thionein molecules do not. A qualitative correlation was found between the extent of sul~ydryl oxidation in Cu-thionein and the reactivity of thionein-bound Cu ions with chelators such as the apometalloenzymes. The reconstitution assay system represents a sensitive indicator of the reactivity of Cu-thionein. The results suggest that rat liver Cu-thionein is very susceptible to oxidation and the Cu-binding affinity varies accordingly. Metallothionein is a protein capable of binding numerous cations including Cu’+, Zn’+, Cd’+, He, and Ag+ (l-9). These metal ions are capable of inducing for- mation of the protein in tissues and cul- tured cells (8-11). The protein induced by Cd” ions, referred to as Cd,Zn-thionein, has seven metal binding sites in the poly- peptide of 62 amino acids (5, 12, 13). The seven metal ions are bound by cysteinyl residues with chelation occurring in two metal-cysteine clusters (12-15). The eys- teinyl groups are fully titratable in Cd,Zn- thionein. Metallothionein induced in rat liver by CL? ions binds 9 to 11 Cu ions per molecule (16). In both yeast and rat liver cells, copper is primarily bound in an EPR nondetectable form presumably in cyste- ine dusters similar to those in Cd,Zn-thi- onein (16-19). Optical studies on yeast Cu- thionein suggested that both the metal ions and cysteinyl ligands were in the re- ’ Supported by Grant AM 20267-04 and predoctoral training Grant GM-07531 (B.L.G.). 2 To whom eorrepondences should be addressed. duced form, although limited Cu2’ para- magnetism of 1 to 12% was seen (19). Cu- thionein from fetal bovine liver was re- ported to be susceptible to oxidation which led to polymeric species and a loss of bound Cu (20). The purpose of this investigation was to investigate the &-binding site in rat liver Cu-thionein. Aerobic preparations of rat liver Cu-thionein revealed limited para- magnetism, but it was unclear whether the paramagnetism was a result of copper ox- idation (17). We were interested in know- ing how susceptible the redox state of the thiols and valence state of the copper ions were to oxygen. In addition, we wanted to find a sensitive assay to determine the in- tegrity of the Cu binding site. MATERIALS AND METHODS Male Sprague-Dawley rats weighing about 26Og were purchased from Simmonsen and were fed com- mercial rat chow ad 1~~~. Sephadex gels were purchased from Pharmacia. The chelators ethylene- 109 0003-9861/82/010109-09$02.00/O Copyright 8 1982 by Academic Press, Inc. All righta of reproduction in any form reserved.