Fluorescence of the Flavin group in choline oxidase. Insights and analytical applications for the determination of choline and betaine aldehyde

Choline oxidase (ChOx) is a flavoenzyme catalysing the oxidation of choline (Ch) to betaine aldehyde (BA) and glycine betaine (GB). In this paper a fundamental study of the intrinsic fluorescence properties of ChOx due to Flavin Adenine Dinucleotide (FAD) is presented and some analytical applications are studied in detail. Firstly, an unusual alteration in the excitation spectra, in comparison with the absorption spectra, has been observed as a function of the pH. This is ascribed to a change of polarity in the excited state. Secondly, the evolution of the fluorescence spectra during the reaction seems to indicate that the reaction takes place in two consecutive, but partially overlapped, steps and each of them follows a different mechanism. Thirdly, the chemical system can be used to determine the Ch concentration in the range from 5×10-6 M to 5×10-5 M (univariate and multivariate calibration) in the presence of BA as interference, and the joint Ch+BA concentration in the range 5×10-6-5×10-4 M (multivariate calibration) with mean errors under 10%//a semiquantitative determination of the BA concentration can be deduced by difference. Finally, Ch has been successfully determined in an infant milk sample