A fluorescence assay for tetradecyltrimethylammonium mono-oxygenase activity that catalyzes the cleavage of the C–N bond with the production of trimethylamine

Abstract

This article describes a simple fluorescent method for the determination of tetradecyltrimethylammonium monooxygenase (TTAB-monooxygenase) activity involving N‑dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminium (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA, and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB-monooxygenase reaction and the amount of protein used in the reaction. The kinetic behaviour is characterized by K0.5 = 4.26 x 10-4 M, and the apparent Hill coefficient (napp) = 2.24. These values are both comparable to those determined by GC-MS (K0.5 = 4.41 x 10-4 M and napp = 2.35). Advantages of this assay include rapid and efficient implementation and potential employment for routine accurate determinations of TTAB-monooxygenase activity over a wide range of substrate concentrations.