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Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

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Typical linear sweep voltammograms obtained for solutions of 20 mM [Fe(CN)6]3− in the absence (A) and presence (B) of S.cerevisiae. Scan (C) was obtained from a solution containing cells, [Fe(CN)6]3− (20 mM) and MD (100 μM). Standard incubation conditions were used.
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f2: Typical linear sweep voltammograms obtained for solutions of 20 mM [Fe(CN)6]3− in the absence (A) and presence (B) of S.cerevisiae. Scan (C) was obtained from a solution containing cells, [Fe(CN)6]3− (20 mM) and MD (100 μM). Standard incubation conditions were used.

Mentions: Steady-state voltammetry is a convenient method for determining the amounts of oxidised and reduced forms of an electroactive species in solution. The position of the voltammogram on the current axis gives an immediate indication of the proportions of each oxidation state, and the anodic and cathodic plateau currents allow quantitation of each redox form. The linear sweep voltammogram (LSV) of [Fe(CN)6]3− (Figure 2 scan A) shows only cathodic current, which arises from the reduction of [Fe(CN)6]3− to [Fe(CN)6]4−. The absence of anodic current indicates, as expected, that there is no [Fe(CN)6]4− in the bulk solution. When [Fe(CN)6]3− was incubated with cells, a relatively small proportion of the [Fe(CN)6]3− was reduced to [Fe(CN)6]4− as evidenced by the small anodic current at potentials positive of 0.3 V (Fig. 2 scan B). [Fe(CN)6]3− is hydrophilic and can only interact with redox sites that are embedded in the cell membrane and exposed to the periplasm. These tPMETs only transfer a small proportion of cellular electrons to the periplasm resulting in the relatively small signal. After incubation for 1 h with cells, glucose, [Fe(CN)6]3− and the lipophilic mediator, MD, the voltammogram (Fig. 2 scan C) has shifted up the current axis and there is mainly anodic current which arises from the oxidation of [Fe(CN)6]4− and a small cathodic current which arises from reduction of unreacted [Fe(CN)6]3−. The large amount of reduced mediator is attributed to the lipophilicity of MD which allows it to cross the cell membrane, enter the cell and accept electrons from a large number of redox molecules3. MD, in the reduced form, returns to the extracellular environment and transfers its electrons to [Fe(CN)6]3− generating [Fe(CN)6]4−, which is oxidised at the electrode.


Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Typical linear sweep voltammograms obtained for solutions of 20 mM [Fe(CN)6]3− in the absence (A) and presence (B) of S.cerevisiae. Scan (C) was obtained from a solution containing cells, [Fe(CN)6]3− (20 mM) and MD (100 μM). Standard incubation conditions were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048887&req=5

f2: Typical linear sweep voltammograms obtained for solutions of 20 mM [Fe(CN)6]3− in the absence (A) and presence (B) of S.cerevisiae. Scan (C) was obtained from a solution containing cells, [Fe(CN)6]3− (20 mM) and MD (100 μM). Standard incubation conditions were used.
Mentions: Steady-state voltammetry is a convenient method for determining the amounts of oxidised and reduced forms of an electroactive species in solution. The position of the voltammogram on the current axis gives an immediate indication of the proportions of each oxidation state, and the anodic and cathodic plateau currents allow quantitation of each redox form. The linear sweep voltammogram (LSV) of [Fe(CN)6]3− (Figure 2 scan A) shows only cathodic current, which arises from the reduction of [Fe(CN)6]3− to [Fe(CN)6]4−. The absence of anodic current indicates, as expected, that there is no [Fe(CN)6]4− in the bulk solution. When [Fe(CN)6]3− was incubated with cells, a relatively small proportion of the [Fe(CN)6]3− was reduced to [Fe(CN)6]4− as evidenced by the small anodic current at potentials positive of 0.3 V (Fig. 2 scan B). [Fe(CN)6]3− is hydrophilic and can only interact with redox sites that are embedded in the cell membrane and exposed to the periplasm. These tPMETs only transfer a small proportion of cellular electrons to the periplasm resulting in the relatively small signal. After incubation for 1 h with cells, glucose, [Fe(CN)6]3− and the lipophilic mediator, MD, the voltammogram (Fig. 2 scan C) has shifted up the current axis and there is mainly anodic current which arises from the oxidation of [Fe(CN)6]4− and a small cathodic current which arises from reduction of unreacted [Fe(CN)6]3−. The large amount of reduced mediator is attributed to the lipophilicity of MD which allows it to cross the cell membrane, enter the cell and accept electrons from a large number of redox molecules3. MD, in the reduced form, returns to the extracellular environment and transfers its electrons to [Fe(CN)6]3− generating [Fe(CN)6]4−, which is oxidised at the electrode.

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

Show MeSH
Related in: MedlinePlus