Characterization of lead induced metal–phytochelatin complexes in Chlamydomonas reinhardtii

Abstract

Accumulation of Pb and induction of phytochelatin synthesis were observed in Chlamydomonas reinhardtii upon Pb(II) exposure. Our aim was to examine whether Pb(II) is bound by phytochelatins (PCs) in C. reinhardtii and to examine formed complexes for their stoichiometry and composition. Metal-phytochelatin (Me-PC) complexes induced by Pb were isolated by size-exclusion chromatography in 13 collected fractions, which were analyzed for their PC and metal content by high-performance liquid chromatography and inductively coupled plasma mass spectrometry. A recovery of more than 90% of Pb from standard Pb-PC₂ complexes within the total volume of the size-exclusion column indicated the adequacy of the method for Pb-PC(n) complex separation and characterization. Phytochelatins were detected mainly in a molecular weight ranging from 1,000 to 5,300 daltons (Da), indicating the formation of complexes with various stoichiometries. Approximately 72% of total PC₂ eluted in the range from 1,000 to 1,600 Da, and 80% of total PC₃ eluted in the molecular weight range from 1,600 to 2,300 Da. The distribution of Cu, Zn, and Pb showed that more than 70% of these metals were associated with the high-molecular-weight fractions. Copper, zinc, and lead were also observed in PC-containing fractions, suggesting the formation of various Me-PC complexes. The results of the present study indicate that the role of PCs in Pb detoxification is minor, because only 13% of total Pb was associated with PCs.