Issue 2, 2003

Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode

Part 1. Wild-type enzyme redox mediation

Abstract

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe–4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe–4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (ks = 0.93 × 105 M−1 s−1) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0–11.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA–N per 100g wet fish muscle). The obtained results compared well with a reference method based on picric acid.

Article information

Article type
Paper
Submitted
29 Nov 2002
Accepted
20 Dec 2002
First published
08 Jan 2003

Analyst, 2003,128, 166-172

Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode

C. Loechel, A. Basran, J. Basran, N. S. Scrutton and E. A. H. Hall, Analyst, 2003, 128, 166 DOI: 10.1039/B211895E

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