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) (k_s = 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 k_cat/K_M revealed two ionisations with pK_a values of 7.5 and 10. The pK_a of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pK_a 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.