Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein.
The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.
Yang
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Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115, USA.
Maret
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Vallee
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D000818:Animals; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D005456:Fluorescent Dyes; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D005456:Fluorescent Dyes; D008668:Metallothionein; D003545:Cysteine