Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes.
In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.
Limoges
Benoît
B
Laboratoire d'Electrochimie Moléculaire, Université de Paris 7-Denis Diderot, 2 place Jussieu,75251 Paris Cedex 05, France. limoges@paris7.jussieu.fr
Marchal
Damien
D
Mavré
François
F
Savéant
Jean-Michel
JM
D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction
D004800:Enzymes, Immobilized; D009243:NAD; D001710:Biotin; D008058:Dihydrolipoamide Dehydrogenase