Composition and production of thiol constituents induced by cadmium in the marine microalga Tetraselmis suecica.

Time course of intracellular levels of different thiols (phytochelatins, desglycyl-phytochelatins, glutathione, gamma-glutamylcysteine [gamma-Glu-Cys], and cysteine) were studied in the microalga Tetraselmis suecica exposed to different cadmium concentrations for 8 d. The cadmium concentrations assayed were 3, 7.9, 15, and 30 mg/L. Contents of thiol compounds synthesized by this microalga changed with cadmium concentration and with time of exposure. Cysteine concentrations increased significantly compared with those of gamma-Glu-Cys and glutathione in cultures containing 7.9, 15, and 30 mg/L. The increase in the amount of glutathione was significant only in cells exposed to the higher cadmium concentrations (15 and 30 mg/L). Nevertheless, in all the cadmium concentrations assayed, gamma-Glu-Cys levels were constant. A rapid increase in phytochelatins was observed with increased cadmium concentration during the first days of culture. The type of phytochelatins (number of subunits) also was dependent on the concentration of cadmium. The rate of (gamma-Glu-Cys)3-Gly accumulation was higher than those of other types of phytochelatins throughout the culture in cells exposed to 3, 15, and 30 mg/L. However, in cultures treated with 7.9 mg/L, (gamma-Glu-Cys)4-Gly increased substantially after 8 d of culture, exceeding the amount of (gamma-Glu-Cys)3-Gly. Cultures exposed to the higher cadmium concentrations (15 and 30 mg/L) showed a decrease in phytochelatins after 4 and 3 d of culture, respectively, whereas an increase in cysteine and glutathione occurred at the same time. A rapid decrease in phytochelatins also was measured when cells were placed into cadmium-free medium. This decrease is consistent with a degradation of the phytochelatins.

Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane.

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.