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Extracting kinetic parameters for homogeneous [Os(bpy)2ClPyCOOH]+ mediated enzyme reactions from cyclic voltammetry and simulations.

Flexer V, Ielmini MV, Calvo EJ, Bartlett PN - Bioelectrochemistry (2008)

Bottom Line: Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1).The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface.At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

View Article: PubMed Central - PubMed

Affiliation: INQUIMAE, Departamento de Quimica Inorganica, Analitica y Quimica Fisica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA, Argentina.

ABSTRACT
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

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Comparison of experimental (full line) and numerical simulations (dotted line) of cyclic voltammograms for mΣ = 1.0 mM; in 0.1 M NaH2PO4/Na2HPO4 + 0.1 M NaCl buffer solution of pH 7.0, scan rate 5 mV s− 1. From a to e same enzyme concentration eΣ = 1.6 μM and different d-glucose concentrations (mM): a) 0.7; b) 1.4; c) 2.8; d) 7.0; e) 33.6; f) eΣ = 4.72 μM and s∞ = 50.0 mM.
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fig8: Comparison of experimental (full line) and numerical simulations (dotted line) of cyclic voltammograms for mΣ = 1.0 mM; in 0.1 M NaH2PO4/Na2HPO4 + 0.1 M NaCl buffer solution of pH 7.0, scan rate 5 mV s− 1. From a to e same enzyme concentration eΣ = 1.6 μM and different d-glucose concentrations (mM): a) 0.7; b) 1.4; c) 2.8; d) 7.0; e) 33.6; f) eΣ = 4.72 μM and s∞ = 50.0 mM.

Mentions: Fig. 8 shows a comparison of typical experimental cyclic voltammetric data and simulation results. Panels a to e in Fig. 8 correspond to the same mediator and enzyme concentration and increasing substrate concentration (see figure caption). Panel f corresponds to a different enzyme concentration and glucose saturation.


Extracting kinetic parameters for homogeneous [Os(bpy)2ClPyCOOH]+ mediated enzyme reactions from cyclic voltammetry and simulations.

Flexer V, Ielmini MV, Calvo EJ, Bartlett PN - Bioelectrochemistry (2008)

Comparison of experimental (full line) and numerical simulations (dotted line) of cyclic voltammograms for mΣ = 1.0 mM; in 0.1 M NaH2PO4/Na2HPO4 + 0.1 M NaCl buffer solution of pH 7.0, scan rate 5 mV s− 1. From a to e same enzyme concentration eΣ = 1.6 μM and different d-glucose concentrations (mM): a) 0.7; b) 1.4; c) 2.8; d) 7.0; e) 33.6; f) eΣ = 4.72 μM and s∞ = 50.0 mM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2742741&req=5

fig8: Comparison of experimental (full line) and numerical simulations (dotted line) of cyclic voltammograms for mΣ = 1.0 mM; in 0.1 M NaH2PO4/Na2HPO4 + 0.1 M NaCl buffer solution of pH 7.0, scan rate 5 mV s− 1. From a to e same enzyme concentration eΣ = 1.6 μM and different d-glucose concentrations (mM): a) 0.7; b) 1.4; c) 2.8; d) 7.0; e) 33.6; f) eΣ = 4.72 μM and s∞ = 50.0 mM.
Mentions: Fig. 8 shows a comparison of typical experimental cyclic voltammetric data and simulation results. Panels a to e in Fig. 8 correspond to the same mediator and enzyme concentration and increasing substrate concentration (see figure caption). Panel f corresponds to a different enzyme concentration and glucose saturation.

Bottom Line: Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1).The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface.At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

View Article: PubMed Central - PubMed

Affiliation: INQUIMAE, Departamento de Quimica Inorganica, Analitica y Quimica Fisica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA, Argentina.

ABSTRACT
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.

Show MeSH
Related in: MedlinePlus