Human flavin-containing monooxygenase isoform 3 (hFMO3) is a hepatic enzyme that catalyses the oxygenation of a large number of structurally diverse drugs and xenobiotics transforming them into benign and readily excretible products. Since the design and development of new therapeutic drugs may take advantage of these detoxifying properties, much interest has been focused on the development of novel techniques suitable for pharmacological research applied to hFMO3 catalytic properties. With this aim, an hFMO3 electrochemical sensor, developed by modifying a glassy carbon electrode with an entrapping gel obtained by glutaraldehyde co-crosslinking of hFMO3 with bovine serum albumin, is reported in this work. Redox properties of hFMO3 sensor have been compared with those of FAD entrapped gel electrode by cyclic voltammetry, revealing for the protein bound FAD, a significant shift of redox peaks towards negative potentials and a total reversibility of the redox reaction. The redox potential measured for the entrapped protein was -411±10 mV (vs. Ag/AgCl). The responsiveness of the sensor was investigated with four different substrates, trimethylamine, ammonia, triethylamine and benzydamine (nonsteroidal anti-inflammatory drug) by calculating kinetic parameters including the apparent Michaelis-Menten constant (82.3±4.3; 94.1±6.4; 120.7±11.2 and 115.9±6.8 µM, respectively), sensitivity (39-45 mAM-1cm-2) and response linearity from 2-80 µM. The data obtained confirm that the hFMO3 sensor has good characteristics in terms of substrate detection, reproducibility and stability, therefore can be employed for catalytic activity measurements of new chemical entities turned over by hFMO3.

catalysis of immobilised human flavin-containing monooxygenase

CASTRIGNANO', SILVIA;SADEGHI, JILA;GILARDI, Gianfranco
2010

Abstract

Human flavin-containing monooxygenase isoform 3 (hFMO3) is a hepatic enzyme that catalyses the oxygenation of a large number of structurally diverse drugs and xenobiotics transforming them into benign and readily excretible products. Since the design and development of new therapeutic drugs may take advantage of these detoxifying properties, much interest has been focused on the development of novel techniques suitable for pharmacological research applied to hFMO3 catalytic properties. With this aim, an hFMO3 electrochemical sensor, developed by modifying a glassy carbon electrode with an entrapping gel obtained by glutaraldehyde co-crosslinking of hFMO3 with bovine serum albumin, is reported in this work. Redox properties of hFMO3 sensor have been compared with those of FAD entrapped gel electrode by cyclic voltammetry, revealing for the protein bound FAD, a significant shift of redox peaks towards negative potentials and a total reversibility of the redox reaction. The redox potential measured for the entrapped protein was -411±10 mV (vs. Ag/AgCl). The responsiveness of the sensor was investigated with four different substrates, trimethylamine, ammonia, triethylamine and benzydamine (nonsteroidal anti-inflammatory drug) by calculating kinetic parameters including the apparent Michaelis-Menten constant (82.3±4.3; 94.1±6.4; 120.7±11.2 and 115.9±6.8 µM, respectively), sensitivity (39-45 mAM-1cm-2) and response linearity from 2-80 µM. The data obtained confirm that the hFMO3 sensor has good characteristics in terms of substrate detection, reproducibility and stability, therefore can be employed for catalytic activity measurements of new chemical entities turned over by hFMO3.
35th FEBS congess
Gothenburg-Sweden
26 June-1 July
277
262
263
Electrochemistry; Human FMO3; protein engineering; glassy carbon electrode; enzyme catalysis
S. Castrignano; S.J. Sadeghi; G. Gilardi
File in questo prodotto:
File Dimensione Formato  
Silvia FEBS-2010.pdf

Accesso aperto

Tipo di file: MATERIALE NON BIBLIOGRAFICO
Dimensione 57.64 kB
Formato Adobe PDF
57.64 kB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/131597
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 19
  • ???jsp.display-item.citation.isi??? 0
social impact