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 nonprotein aceous thiols in a unicellular preen alga Tetraselmis tetrathele (West) But cher (Prasinophyceae) were investigated by using a fluorescent dye, 5-chlor omethylfluorescein (5CMF), as a probe for nonproteinaceous thiols, The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluoresce nce was decreased by exposure of the cells to HgCl2, Analysis of the fluore scent intensity of 5CMF by flow cytometry made it possible to distinguish c ells in three states during the dying process caused by HgCl2: a normal sta te, 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 mu M HgCl2, whereas exp osure 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. Phytoch elatins, 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 tri peptide, Arg-Arg-Glu, was found to be abundant, and it showed ability to bi nd Hg2+, suggesting that it functions to scavenge heavy metals as well as t hiol molecules.