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

Leonardo Pantoja Munoz, Diane Purchase, Huw Jones, Jörg Feldmann, Hemda Garelick

Research output: Contribution to journalArticle

9 Citations (Scopus)

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)-PC 3, 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 DMASV-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.
Original languageEnglish
Pages (from-to)791-797
Number of pages7
JournalAnalytical Methods
Volume6
Issue number3
Early online date5 Dec 2013
DOIs
Publication statusPublished - 7 Feb 2014

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Phytochelatins
Sonication
Arsenic
Water
Dynamic mechanical analysis
Algae
Complex Mixtures
Cell culture
Biological materials
Glutathione
Biomass

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Enhanced determination of As-phytochelatin complexes in Chlorella vulgaris using focused sonication for extraction of water-soluble species. / Munoz, Leonardo Pantoja; Purchase, Diane; Jones, Huw; Feldmann, Jörg; Garelick, Hemda.

In: Analytical Methods, Vol. 6, No. 3, 07.02.2014, p. 791-797.

Research output: Contribution to journalArticle

Munoz, Leonardo Pantoja ; Purchase, Diane ; Jones, Huw ; Feldmann, Jörg ; Garelick, Hemda. / Enhanced determination of As-phytochelatin complexes in Chlorella vulgaris using focused sonication for extraction of water-soluble species. In: Analytical Methods. 2014 ; Vol. 6, No. 3. pp. 791-797.
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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)-PC 3, 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 DMASV-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.",
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AB - 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)-PC 3, 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 DMASV-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.

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