Enhanced determination of As–phytochelatin complexes in Chlorella vulgaris using focused sonication for extraction of water-soluble species

Abstract

The most challenging areas in the analysis of As–GS/PC complexes are their extraction from small amounts of biological material and the maintenance of their stability during HPLC separation. Focused sonication was used to extract these complexes from Chlorella vulgaris and the integrity of such complexes was determined by HPLC online with simultaneous HR-ICP-MS and ES-MS/MS detection. Water soluble arsenic species were extracted with an improved 71.1% (SE 0.78) efficiency and much reduced extraction times (30 s) allowing the determination of unstable arsenic phytochelatin (PC) and glutathione (GS) species in small biomass making the method particularly well-suited for cell cultures. Here, it was found that C. vulgaris produces the following intact phytochelatins and homo-phytochelatins (with Ala and desGly instead of Gly) complexes when cells are exposed to As(III): As(III)–PC₂, GS–As(III)–PC₂, As(III)–(PC₂)₂, MMA(III)–PC₂, As(III)–PC₃, As(III)–PC₄, As(III)–γ-(Glu–Cys)₃–Ala, GS–As(III)–γ-(Glu–Cys)₂–Ala, As(III)–γ-((Glu–Cys)₂)₂–Ala, MMA(III)–γ-(Glu–Cys)₂–Ala, As(III)–γ-(Glu–Cys)₂, GS–As(III)–γ-(Glu–Cys)₂. When the alga was exposed to DMA, only DMAS^V–GS was found. In contrast, cells did not produce any complex when exposed to As(V). It is the first time that, as a result of the newly developed extraction method using sonication, such complexes have been identified in Chlorella vulgaris exposed to arsenic and their intact arsenic homo-phytochelatins have been reported in any organism.