ABSTRACT
The comparative kinetic study of two glucose oxidizing enzymes, FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase, is presented in the artificial electron transfer mediator system based on ruthenium(III) compounds. It is demonstrated that FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase follow Michaelis kinetics in the D-glucose/ruthenium(III) system. PQQ-dependent glucose dehydrogenase is more active than FAD-dependent glucose oxidase in the process of D-glucose oxidation by ruthenium(III) compounds, this being due to the different catalytic mechanisms of these enzymes.
Subject(s)
Glucose Dehydrogenases/chemistry , Glucose Oxidase/chemistry , Glucose/chemistry , Ruthenium/chemistry , Catalysis , Glucose/metabolism , Glucose Dehydrogenases/metabolism , Glucose Oxidase/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Structure , Oxidation-Reduction/drug effects , Quinolones/chemistry , Quinones/chemistry , Ruthenium/pharmacology , Stereoisomerism , Structure-Activity Relationship , Time FactorsABSTRACT
Based on previous results which showed that quinohemo-protein alcohol dehydrogenase (QH-ADH) entrapped within polypyrrole is able to directly transfer electrons via the conducting polymer to the electrode surface, the electron-transfer properties of this multi-cofactor enzyme adsorbed and covalently-bound to self-assembled thiol monolayers and bare electrode surfaces has been investigated more closely. While the dissolved enzyme is able to transfer electrons to the electrode via heme c as well as via the more deeply buried PQQ (fast adsorption-chemical reaction-desorption mechanism), an orientation of adsorbed QH-ADH on hydrophobic electrode surfaces, as well as of adsorbed and covalently bound QH-ADH on negatively-charged thiol monolayers could be observed. In these cases the heme c units are pointing towards the electrode surfaces resulting in an optimised direct ET rate.
Subject(s)
Alcohol Dehydrogenase/metabolism , Biosensing Techniques , Electron Transport , Oxidoreductases/metabolism , Alcohol Dehydrogenase/chemistry , Electrochemistry , Electrodes , Enzymes, Immobilized , Membranes, Artificial , Surface PropertiesABSTRACT
Partially hydrolyzed polyarbutin-containing benzoquinone groups were synthesized by using chemoenzymatic methods. This polymer was used as a mediator for the oxidation of pyrroloquinoline quinone-dependent glucose dehydrogenase. Polymer was covalently attached to the enzyme through the glucose moiety of the polymer and amine residues in the protein. Electrochemical studies show that the oxidized benzoquinone attached to the enzyme can act as a mediator for the reoxidation of the enzyme at carbon electrode surfaces. The apparent Michaelis constant and inactivation rate constant of the coupled enzyme were found to be similar to these parameters of the native enzyme.
ABSTRACT
It is reported for the first time that direct electron-transfer processes between a polypyrrole (PPY) entrapped quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 (QH-ADH) and a platinum electrode take place via the conducting-polymer network. The cooperative action of the enzyme-integrated prosthetic groups--pyrroloquinoline-quinone and hemes--is assumed to allow this electron-transfer pathway from the enzyme's active site to the conducting-polymer backbone. A hypothetical model of the electron transfer is proposed which is supported by the influence of various parameters, such as, e.g., ionic strength and nature of the buffer salts. This unusual electron-transfer pathway leads to an accentuated increase of the K M app value (102 mM) and hence to a significantly increased linear detection range of an ethanol sensor based on this enzyme.
Subject(s)
Alcohol Oxidoreductases , Polymers , Pyrroles , Acetobacteraceae/enzymology , Electron Transport , Ethanol , Models, Chemical , Models, Molecular , Platinum , Spectrophotometry, UltravioletABSTRACT
Amperometric glucose sensors were constructed based on solid graphite electrodes, surface-modified with NAD+ dependent glucose dehydrogenase (GDH), Toluidine Blue O (TBO), and protective ionic polymers. The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with TBO dissolved, adsorbed, and electrostatically or covalently bound to polymers. The NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl using single potential step chronoamperometry. The operational stability of the glucose sensors was limited by leakage of NAD+. A glucose sensitivity much higher than carbon paste electrode was found. A sensitivity as high as 25 microA cm-2 mM-1 was achieved.
Subject(s)
Biosensing Techniques , Glucose Dehydrogenases , Glucose/analysis , NAD/analysis , Tolonium Chloride , Enzymes, Immobilized , Glucose 1-Dehydrogenase , Oxidation-ReductionABSTRACT
Flow-through and catheter glucose sensors and glucose-, lactate-, ethanol- and uric acid-sensitive catheter biosensors possessing a linear region of calibration curves over the physiological range of metabolite concentrations in blood are designed.
Subject(s)
Biosensing Techniques , Blood Glucose/analysis , Calibration , Catheterization/methods , Diffusion , Ethanol , Glucose Oxidase , Lactates , Membranes, Artificial , Uric AcidABSTRACT
Urea-sensitive sensors, based on immobilized urease and the differential antimony electrode, have been constructed. The calibration curve of the sensor is linear up to 1.6-2.0 mmol litre-1 in the stationary or kinetic mode of operation. The sensors retain 50% of their sensitivity for 16-17 days. The analyser for urea, constructed on the basis of the sensor, ensures quick and precise urea determination in pure blood.
