HEAVY-METAL INDUCED CHANGES IN NONPROTEINACEOUS THIOL LEVELS AND HEAVY-METAL BINDING PEPTIDE IN TETRASELMIS TETRATHELE (PRASINOPHYCEAE)

The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular green alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5-chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols. The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl₂. Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl₂: a normal state, a thiol-depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h. Most cells died after 24 h of exposure to more than 3.0 μM HgCl₂, whereas exposure up to 1.0 mM CdCl₂ did not cause depletion of nonproteinaceous thiols or cell death within 48 h.   HPLC analyses revealed that glutathione was a major nonprotein thiol in T. tetrathele and that it was oxidized by exposing the cells to HgCl₂. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg-Arg-Glu, was found to be abundant, and it showed ability to bind Hg²⁺, suggesting that it functions to scavenge heavy metals as well as thiol molecules.