A differential assay for the reduced and oxidized states of metallothionein and thionein.
In the cellular environment, the sulfur ligands in zinc/thiolate coordination sites of proteins can be oxidized with concomitant mobilization of zinc. The characterization of such "redox zinc switches" requires the determination of three species, i.e., the zinc-containing complex and the zinc-free complex with the thiolate ligands either reduced or oxidized. Differential chemical modification of thiol groups in the presence and absence of either reducing or chelating agents allows the analytical speciation of such systems as demonstrated here for the characterization of the redox and metal-binding states of mammalian metallothionein. Thiol derivatization with 6-iodoacetamidofluorescein in the presence and absence of the reducing agent tris(2-carboxyethyl)phosphine, high-performance liquid chromatographic separation, and photometric detection are employed to determine the reduced and oxidized protein. Because the holoprotein reacts only in the presence of a chelating agent such as ethylenediaminetetraacetate (EDTA) its amount can be determined as the difference between measurements in the presence and the absence of EDTA. This method is applied to the study of the chemical and enzymatic oxidation of metallothionein/thionein. It should also greatly facilitate the characterization of the redox and metal-binding properties of zinc/thiolate coordination environments of other proteins such as zinc finger proteins.
Haase
Hajo
H
Department of Pathology, Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, One Kendall Square, Bldg. 600, 3rd floor, Cambridge, MA 02139, USA.
Maret
Wolfgang
W
D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry*; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D004492:Edetic Acid; D005452:Fluoresceins; D008668:Metallothionein; D010720:Phosphines; D013438:Sulfhydryl Compounds; D015032:Zinc; C080938:tris(2-carboxyethyl)phosphine; C050046:6-iodoacetamidofluorescein