Background: Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes. Methods: In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry. Results: Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in KM values of 52 μM and 27 μM, with Vmax of 8 nmol min- 1 mg- 1 and 4 nmol min- 1 mg- 1, respectively, which are in agreement with data obtained with the microsomal enzyme. Conclusions: The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme. General significance: This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM, FTIR and electrocatalysis

S. Castrignanò;SADEGHI, JILA;GILARDI, Gianfranco
2012

Abstract

Background: Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes. Methods: In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry. Results: Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in KM values of 52 μM and 27 μM, with Vmax of 8 nmol min- 1 mg- 1 and 4 nmol min- 1 mg- 1, respectively, which are in agreement with data obtained with the microsomal enzyme. Conclusions: The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme. General significance: This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.
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2078
enzyme catalysis; Glassy carbon electrode; Human flavin-containing monooxygenase isoform 3; Stabilized gold nanoparticle
S. Castrignanò; S. J. Sadeghi; G. Gilardi
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/130188
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