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

Abstract

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.

The intrinsic fluorescence of FAD and its application in analytical chemistry: a review

“…As has been indicated above, we have use the FAD fluorescence of ChOx for the determination of Ch and BA, obtaining longer linear response range than for Glu [36]. In this case, the fluorescence of the FAD, as expected, decreased during the enzymatic reaction and the evolution of the fluorescence spectrum during the enzymatic reaction indicated that the reaction takes place in two consecutive, but partially overlapping, steps and each of them follows a different mechanism.…”

“…We have also studied the spectroscopic properties of choline oxidase (ChOx, from both Alcaligenes sp and Arthrobacter globiformis) [36] during the enzymatic oxidation reaction of both, BA and Ch to GB, and also we find a remarkable behaviour of the ChOx fluorescence. Figure 13(A) shows the 3D spectra obtained with ChOx at pH = 6 before the reaction (FAD oxidized) and figure 13(B) shows the 3D spectra after the addition of a very high excess of BA (or Ch), when the enzyme is in the reduced form.…”

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

“…The N-terminal 37 amino acids of FRDg that include the Dxx(s/t)(s/g)AS consensus motif [32] are sufficient as substrate for efficient flavinylation when fused to different proteins localised in the cytoplasm or in the glycosome. We also confirmed that Ser9 in this motif is essential for flavinylation [64], in agreement with Serebryakova et al [68,69], a broad functional pH range (4)(5)(6)(7)(8)(9)(10)(11) [70] and their small size -for iLOV approximately 10 kDa [71]. Other variants, such as miniSOG, can generate reactive singlet oxygen upon illumination, which is exploited for electron microscopy [72].…”

“…To trace a prominent endogenous auto-fluorescent band in T. brucei cell lines upon in-gel fluorescence analysis [49], we enriched the respective band by cell fractionation and identified it by mass spectrometry ( Figure S2) as the flavoprotein fumarate reductase (FRD). This explained the optical properties [8,9] and resistance of the fluorescence to denaturing conditions, as covalent flavin attachment to FRD had been reported in the closely related kinetoplastid L. pyrrhocoris [32]. The presence of an ApbE-like domain at the N-terminus of FRDg led to the hypothesis of covalent FMN attachment catalysed by a eukaryotic ApbE flavin transferase activity [32].…”

“…Blue-light photoreceptors and bacterial luciferases [5,6] utilise the distinct fluorescent properties of flavins for their function [7]. They typically feature excitation/emission maxima of the oxidised form at around 450/520 nm, depending on environmental conditions and binding partners [8,9]. The optical properties do not only allow characterisation of specific flavoproteins and their state transitions [10,11], but also visualisation of flavin-rich cell populations or subcellular structures such as mitochondria [12,13].…”