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A study of cytochrome bo3 in a tethered bilayer lipid membrane.

ABSTRACT: An assay has been developed in which the activity of an ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), is determined as a function of the hydrophobic substrate ubiquinol-10 (UQ-10) in tethered bilayer lipid membranes (tBLMs). UQ-10 was added in situ, while the enzyme activity and the UQ-10 concentration in the membrane have been determined by cyclic voltammetry. Cbo(3) is inhibited by UQ-10 at concentrations above 5-10 pmol/cm(2), while product inhibition is absent. Cyclic voltammetry has also been used to characterise the effects of three inhibitors; cyanide, inhibiting oxygen reduction; 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibiting the quinone oxidation and Zn(II), thought to block the proton channels required for oxygen reduction and proton pumping activity. The electrochemical behaviour of cbo(3) inhibited with HQNO and Zn(II) is almost identical, suggesting that Zn(II) ions inhibit the enzyme reduction by quinol, rather than oxygen reduction. This suggests that at Zn(II) concentration below 50µM the proton release of cbo(3) is inhibited, but not the proton uptake required to reduce oxygen to water.

SUBMITTER: Weiss SA

PROVIDER: S-EPMC3827738 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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A study of cytochrome bo3 in a tethered bilayer lipid membrane.

Weiss Sophie A SA Bushby Richard J RJ Evans Stephen D SD Jeuken Lars J C LJ

Biochimica et biophysica acta 20100121 12

An assay has been developed in which the activity of an ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), is determined as a function of the hydrophobic substrate ubiquinol-10 (UQ-10) in tethered bilayer lipid membranes (tBLMs). UQ-10 was added in situ, while the enzyme activity and the UQ-10 concentration in the membrane have been determined by cyclic voltammetry. Cbo(3) is inhibited by UQ-10 at concentrations above 5-10 pmol/cm(2), while product inhibition is absent. Cyclic v ...[more]

PMID: 20096262

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Project description:Interfacing nanoelectronic devices with cell membranes can enable multiplexed detection of fundamental biological processes (such as signal transduction, electrophysiology, and import/export control) even down to the single ion channel level, which can lead to a variety of applications in pharmacology and clinical diagnosis. Therefore, it is necessary to understand and control the chemical and electrical interface between the device and the lipid bilayer membrane. Here, we develop a simple bottom-up approach to assemble tethered bilayer lipid membranes (tBLMs) on silicon wafers and glass slides, using a covalent tether attachment chemistry based on silane functionalization, followed by step-by-step stacking of two other functional molecular building blocks (oligo-poly(ethylene glycol) (PEG) and lipid). A standard vesicle fusion process was used to complete the bilayer formation. The monolayer synthetic scheme includes three well-established chemical reactions: self-assembly, epoxy-amine reaction, and EDC/NHS cross-linking reaction. All three reactions are facile and simple and can be easily implemented in many research labs, on the basis of common, commercially available precursors using mild reaction conditions. The oligo-PEG acts as the hydrophilic spacer, a key role in the formation of a homogeneous bilayer membrane. To explore the broad applicability of this approach, we have further demonstrated the formation of tBLMs on three common classes of (nano)electronic biosensor devices: indium-tin oxide-coated glass, silicon nanoribbon devices, and high-density single-walled carbon nanotubes (SWNT) networks on glass. More importantly, we incorporated alemethicin into tBLMs and realized the real-time recording of single ion channel activity with high sensitivity and high temporal resolution using the tBLMs/SWNT network transistor hybrid platform. This approach can provide a covalently bonded lipid coating on the oxide layer of nanoelectronic devices, which will enable a variety of applications in the emerging field of nanoelectronic interfaces to electrophysiology.

Dataset Information

A study of cytochrome bo3 in a tethered bilayer lipid membrane.

Publications

A study of cytochrome bo3 in a tethered bilayer lipid membrane.

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