Impedance spectroscopy and conductometric biosensing for probing catalase reaction with cyanide as ligand and inhibitor.
In this work, a new biosensor was prepared through immobilization of bovine liver catalase in a photoreticulated poly (vinyl alcohol) membrane at the surface of a conductometric transducer. This biosensor was used to study the kinetics of catalase-H(2)0(2) reaction and its inhibition by cyanide. Immobilized catalase exhibited a Michaelis-Menten behaviour at low H(2)0(2) concentrations (<100mM) with apparent constant K(M)(app)=84±3mM and maximal initial velocity V(M)(app)=13.4μS min(-1). Inhibition by cyanide was found to be non-competitive and inhibition binding constant K(i) was 13.9±0.3μM. The decrease of the biosensor response by increasing cyanide concentration was linear up to 50μM, with a cyanide detection limit of 6μM. In parallel, electrochemical characteristics of the catalase/PVA biomembrane and its interaction with cyanide were studied by cyclic voltammetry and impedance spectroscopy. Addition of the biomembrane onto the gold electrodes induced a significant increase of the interfacial polarization resistance R(P). On the contrary, cyanide binding resulted in a decrease of Rp proportional to KCN concentration in the 4 to 50μM range. Inhibition coefficient I(50) calculated by this powerful label-free and substrate-free technique (24.3μM) was in good agreement with that determined from the substrate-dependent conductometric biosensor (24.9μM).
Bouyahia
Naima
N
Université de Annaba, Laboratoire LPCI, BP 12, El Hadjar, 23000 Annaba, Algeria.
Hamlaoui
Mohamed Larbi
ML
Hnaien
Mouna
M
Lagarde
Florence
F
Jaffrezic-Renault
Nicole
N
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003217:Conductometry / Q000379:methods; D003486:Cyanides / Q000737:chemistry / Q000378:metabolism; D058266:Dielectric Spectroscopy / Q000379:methods*; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008024:Ligands; D057230:Limit of Detection
D003486:Cyanides; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008024:Ligands; D006861:Hydrogen Peroxide; D002374:Catalase