Cadmium toxicity on the freshwater microalga Chlamydomonas moewusii Gerloff: Biosynthesis of thiol compounds.

Cadmium (Cd) toxicity and production of different thiols (phytochelatins, glutathione, gamma-Glu-Cys and cysteine) were studied in the microalga Chlamydomonas moewusii exposed to different concentrations of this metal (1, 2, 4, 6, 8, and 10 mg/L) for 96 h. The inhibitory effect of Cd on growth was demonstrated. The value of EC50 (metal concentration which reduces the population growth to 50% of the control) obtained for this microalga was estimated at 4.1 +/- 0.8 mg/L of Cd after 96 h of exposure. The amount of thiol compounds synthesized by C. moewusii changed with Cd concentration. Cysteine concentrations were significantly higher compared to those of gamma-Glu-Cys and glutathione in all the Cd concentrations assayed. The amino acid cysteine reached its higher levels in those cultures in which a decrease in the concentration of phytochelatins (PCs) was observed. Both cysteine and glutathione concentrations showed significant differences along the Cd concentrations assayed, while the amount of gamma-Glu-Cys detected remained stable. The PCs detected were of two, three, and four subunits. The level of PC(2) was higher than that of PC(3) and PC(4). PC(4) was detected only in the cultures exposed to the Cd concentrations of 1 and 2 mg/L, in which the synthesis of phytochelatins was higher. A rapid increase in the production of PC(2) and PC(3) was observed up to a Cd concentration of 2 mg/L, after which their levels began to decrease. Phytochelatins were not detected in cultures without Cd (controls) and in those exposed to the maximum Cd concentration (10 mg/L), in which cell growth was completely inhibited.

Structure and metal loading of a soluble periplasm cuproprotein.

A copper-trafficking pathway was found to enable Cu(2+) occupancy of a soluble periplasm protein, CucA, even when competing Zn(2+) is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu(2+), but not Zn(2+), quenches the fluorescence of Trp(165), which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn(2+) following exposure to equimolar Zn(2+) and Cu(2+). Cu(2+)-CucA is more thermodynamically stable than Zn(2+)-CucA but k((Zn→Cu)exchange) is slow, raising questions about how the periplasm contains solely the Cu(2+) form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu(2+)-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, ΔctaA cells overaccumulate low M(r) copper complexes in the periplasm, and purified apoCucA can readily acquire Cu(2+) from ΔctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.

A capillary zone electrophoresis for determination of thiolic peptides in biological samples.

A new method to improve the analyses of thiolic peptides (cysteine, gammaGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 microm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 microM respectively.

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.

Cadmium removal by living cells of the marine microalga Tetraselmis suecica.

Cadmium removal by living cells of the marine microalga Tetraselmis suecica was tested in cultures exposed to different cadmium concentrations (0.6, 3, 6, 15, 30 and 45 mg/l). The EC50 for growth was 7.9 mg Cd/l after six days of exposure. The cadmium removed was proportional to the concentration of this metal in the medium and it was dependent on the time of exposure; cultures with higher cadmium concentration removed a higher amount of this metal. In cultures exposed to 0.6 mg/l, T suecica cells removed 98.1% of added cadmium with 0.392 x 10(-6) microg Cd/cell, whereas in cultures with 45 mg/l only 7.7% was removed with 16.052 x 10(-6) microg Cd/cell. The highest amount of cadmium removed per liter of culture was observed in cultures exposed to 6 mg/l, with 3.577 mg/l of cadmium. After six days of incubation, the higher proportion of cadmium was bioaccumulated intracellularly in all cultures except in 45 mg/l cultures, the percentage of intracellular cadmium being always more than 50%. The highest percentage of bioadsorbed cadmium (60.1%) was found in cells of cultures with the highest cadmium concentration (45 mg/l). Furthermore, a relation between intracellular cadmium and the concentration of sulfhydryl groups was observed.