Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence

Capillary electrophoresis (CE) with multiphoton-excited fluorescence detection (CE-MPE) allows low-background analysis of spectrally distinct fluorophores using a single long-wavelength laser. Extracts were prepared from immortalized rat raphe nuclei neurons, and were analyzed by CE-MPE. Native fluorescence was detected from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), riboflavin, serotonin, and 5-hydroxytryptophan (5HTrp). Quantitation of exogenous serotonin (taken up by cells) and endogenous NADH and 5HTrp was possible using internal standards or standard addition. This system should be useful to study monamine oxidase inhibitors (MAOIs) and selective serotonin reuptake inhibitors (SSRIs).     

Ensemble and Single Molecule Studies on the Use of Metallic Nanostructures to Enhance the Intrinsic Emission of Enzyme Cofactors

We present a strategy for enhancing the intrinsic emission of the enzyme cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH). Ensemble studies show that silver island films (SIFs) are the optimal metal enhanced fluorescence (MEF) substrates for flavins and gave emission enhancements of over 10-fold for both FAD and FMN. A reduction in the lifetime of FAD and FMN on SIFs was also observed. Thermally evaporated aluminum films on quartz slides were found to be the optimal MEF substrate for NADH and gave a 5-fold increase in the emission intensity of NADH. We present finite-difference time-domain (FDTD) calculations that compute the enhancement in the radiated power emitting from an excited state dipole emitting in the wavelength range of NADH in close proximity to an aluminum nanoparticle, and a dipole emitting in the emission wavelength of flavins next to a silver nanoparticle. These calculations confirm that aluminum serves as the optimal MEF substrate for NADH and silver was the optimal MEF substrate for flavins. This is because the plasmon resonance properties of aluminum lie in the UV-blue regime and that of silver lie in the visible region. We also present the results of single molecule studies on FMN which show SIFs can both significantly enhance the intrinsic emission from single FMN molecules, significantly reduce their lifetimes and also significantly reduce FMN blinking. This is the first report of the observation of MEF from cofactors both at the ensemble and single molecule level. We hope this study will serve as a platform to encourage the future use of metallic nanostructures to study cofactors using their intrinsic fluorescence to directly monitor enzyme binding reactions without the need of extrinsic labeling of the molecules.     

Reductive metabolism of nitro-p-phenylenediamine by rat liver

Reductive metabolism of the hair dye constituent, nitro-p-phenylenediamine (2-nitro-1,4-diaminobenzene, NPDA), and its acetylated metabolite, NPDA N4-acetate, was investigated with rat liver subcellular fractions, microsomes and cytosol. Under anaerobic conditions, these compounds were reduced to their corresponding amines by these fractions. The microsomal nitro-reducing activity was retarded completely by air and strongly by carbon monoxide. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) functioned more effectively than reduced nicotinamide adenine dinucleotide (NADH) as an electron donor in the microsomal reduction of the nitro compounds, and flavin mononucleotide (FMN) gave rise to a marked enhancement in the microsomal activity, especially when added to an anaerobic incubation mixture containing both NADH and NADPH. The cytosolic nitro-reducing activity was attributed to xanthine oxidase, aldehyde oxidase and other unknown enzyme(s), based on the results of cofactor requirements and inhibition experiments.     

In vivo monitoring the changes of interstitial pH and FAD/NADH ratio by fluorescence spectroscopy in healing skin wounds

The aim of our study was to evaluate the changes of interstitial pH and flavin adenine dinucleotide (FAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio in healing skin wounds using fluorescence spectroscopy in Sprague Dawley rats. In the experiment, excisional and incisional models of wound healing were used. The florescein as the pH-sensitive probe using excitation spectra (lambda(Em) = 535 nm) was used for the measurement of pH changes, and synchronous fluorescence spectra (Deltalambda = 60 nm) for the monitoring of FAD/NADH ratio changes were measured from the surfaces of healing wounds. Increase of interstitial pH and FAD/NADH ratio was recorded during the time interval from the 15th to the 65th minute after surgery. The decrease of pH between the 48th and the 72nd hour after surgery as well as the increase of FAD/NADH ratio between the 72nd and the 96th hour of wound healing were recorded. The results indicate that the use of fluorescence spectroscopy may be considered as a valuable tool for noninvasive in vivo monitoring of selected redox parameters in the early phases of wound healing.     

Quantification of riboflavin,flavin mononucleotide,and flavin adenine dinucleotide in mammalian model cells by CE with LED‐induced fluorescence detection

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein‐bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED‐induced fluorescence with limit of detections (LODs 0.5–3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1–14 amol/cell) and FAD (2.2–17.0 amol/cell) were the predominant flavins, while FMN (0.46–3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.     

KINETICS OF STACKING INTERACTIONS IN FLAVIN ADENINE DINUCLEOTIDE FROM TIME-RESOLVED FLAVIN FLUORESCENCE

The time dependence of the fluorescence of flavin adenine dinucleotide (FAD) was measured with a subnanosecond-resolving fluorometer. In contrast to the fluorescence decay of FMN, the decay of FAD was proved to be nonexponential. The time-dependent fluorescence of FAD can be interpreted by assuming an equilibrium between closed and open conformers in the ground state. The rate constant for folding in the excited state and the fluorescence lifetime of the intramolecular complex could be evaluated from analysis of the observed fluorescence decay. The results on FAD were compared to those on NADH obtained earlier.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Experimental study on predicting skin flap necrosis by fluorescence in the FAD and NADH bands during surgery

The objective of the present study was to assess the feasibility of using endogenous fluorescence in intraoperative prediction of skin flap necrosis. The investigation was carried out in 10 Sprague-Dawley rats in which proximally based over-dimensioned random pattern skin flaps were dissected on the back and thereafter fixed into position. Immediately after surgery on each rat, synchronous fluorescence spectra (Deltalambda=90 nm) from five parts of the skin flap surface were measured. The presence of necrosis was evaluated on postoperative day 7. In flap parts designated as necrotic (n=18), a significantly lower (P<0.001) fluorescent signal from the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) bands (360-380 and 440-460 nm, respectively) was detected in comparison with the vital parts (n=32) (for FAD:1767+/-39 versus 2579+/-65 auxiliary units [A.U]. and for NADH:11724+/-340 versus 16810+/-473 A.U.). The results suggested a close relationship between the fluorescent signals from the FAD and NADH bands on one side and flap necrosis or survival on the other side. Thus, the use of fluorescence spectroscopy may be considered as a valuable noninvasive tool for the prediction of skin flap necrosis.     

Adsorption behavior of dinucleotides on bare and ru-modified glassy carbon electrode surfaces

The interactive behavior of flavin adenine dinucleotide (FAD) with a bare glassy carbon electrode (GCE) and a Ru-modified GCE was investigated. The reduction of FAD at a GCE/ruthenium-modified GCE surface is a quasi-reversible, surface-controlled process, and our data implied that the attachment of FAD onto the surface is caused by nonspecific adsorption instead of covalent linkage, in which the adenine ring of FAD adopts a flat orientation on the GCE surface in neutral and dilute solutions in order to maximize the pi-pi stacking with the carbon surface and reorients to a perpendicular orientation as the surface gets more crowded. FAD desorption during the exchange with nicotinamide adenine dinucleotide (NAD+) is one order of magnitude slower than desorption in the absence of NAD+, which indicates a strong interaction between FAD and NAD+. General knowledge of the interactive behavior of NAD+ on a FAD-adsorbed GCE provides useful information for the design of a modified electrode surface for the generation of NADH from NAD+.     

Raman spectra of flavins: avoidance of interference from fluorescence

Raman spectra of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in neutral aqueous solutions have been observed with excitations at 600.0, 363.8, 351.1, 337.1, and 257.3 nm. It has been suggested that, in general, an excitation in the absorption band of the second or the third longest waveleng (instead of the first) is an effective means for observing a resonance Raman spectrum of a chromophore without fluorescence disturbance.     

Delta-amino group hydroxylation of L-ornithine during coelichelin biosynthesis

The nonribosomally produced hydroxamate siderophore coelichelin from Streptomyces coelicolor contains the nonproteinogenic amino acids N(5)-hydroxyornithine and N(5)-hydroxyformylornithine that are important for iron assembly. The hydroxylation of the delta-amino group of L-ornithine is catalyzed by the flavin-dependent monooxygenase CchB. During the redox reaction nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen are consumed and flavin adenine dinucleotide (FAD) is needed as a cofactor. During this work the monooxygenase was biochemically characterized and it could be shown that the hydroxylation of l-ornithine is most likely the first step in the biosynthesis of the siderophore coelichelin.     

亲水作用加压毛细管电色谱-激光诱导荧光检测法分析痕量核黄素类物质

一种新型的亲水作用毛细管电色谱(HI-CEC)整体柱被应用于加压毛细管电色谱-激光诱导荧光检测(pCEC-LIF)联用法对核黄素类物质的分离分析。采用自组装的pCEC-LIF系统,实现了对痕量核黄素(RF)、黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)的快速分析。在最优的分离检测条件下,3种化合物在8.0 min内完全分离,RF、FMN和FAD的检出限(LOD, S/N=3)分别为5.0×10－11 mol/L、8.0×10－10mol/L和2.5×10－9mol/L,测定线性范围可达3个数量级,精密度良好。方法简便、全分析时间短、灵敏度和选择性高,血清样品分析实验结果良好,可望进一步应用于体液及细胞中核黄素类物质的痕量检测     

Endogenous fluorescence spectroscopy of cell suspensions for chemopreventive drug monitoring

Cancer chemopreventive agents such as N-4-(hydroxyphenyl)retinamide (4HPR) are thought to prevent cancers by suppressing growth or inducing apoptosis in precancerous cells. Mechanisms by which these drugs affect cells are often not known, and the means to monitor their effects is not available. In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment with 4HPR in ovarian and bladder cancer cell lines. Fluorescence signals consistent with nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan were measured to monitor cellular activity through redox status and protein content. Cells were treated with varying concentrations of 4HPR and measured in a stable environment with a sensitive fluorescence spectrometer. Results suggest that redox signal of all cells changed in a similar dose-dependant manner but started at different baseline levels. Redox signal changes depended primarily on changes consistent with NADH fluorescence, whereas the FAD fluorescence remained relatively constant. Similarly, tryptophan fluorescence decreased with increased drug treatment, suggesting a decrease in protein production. Given that each cell line has been shown to have a different apoptotic response to 4HPR, fluorescence redox values along with changes in tryptophan fluorescence may be a response as well as an endpoint marker for chemopreventive drugs.     

480—Redox and adsorption behaviour of some redox coenzymes involved in an electron-transport chain studied by the electro-optical technique

The redox and adsorption behaviour of some redox coenzymes involved in an electron-transport chain, i.e. ubiquinone-10 (CoQ), flavocoenzymes [flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)] and nicotinamide adenine dinucleotide (NAD⁺), has been studied at a gold electrode by cyclic voltammetry and specular reflectivity measurement. All the coenzymes investigated were found to participate in electron transport in adsorbed states on the electrode surface. Adsorbed CoQ and flavocoenzymes are reduced and the resulting products remain adsorbed at the surface. Contrary to them, adsorbed NAD⁺ is reduced and then desorbed immediately. Possible models for the surface orientation of adsorbed molecules were proposed based on the experimental data.Some analogies can be noted between the interfacial behaviour of these coenzymes at the electrode and that in mitochondria.     

Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage

The metabolic coenzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are the primary electron donor and acceptor respectively, participate in almost all biological metabolic pathways. This study develops a novel method for the quantitative kinetic analysis of the degradation reaction of NADH and the formation reaction of FAD in human plasma containing an uncalibrated interferent, by using three-way calibration based on multi-way fluorescence technique. In the three-way analysis, by using the calibration set in a static manner, we directly predicted the concentrations of both analytes in the mixture at any time after the start of their reactions, even in the presence of an uncalibrated spectral interferent and a varying background interferent. The satisfactory quantitative results indicate that the proposed method allows one to directly monitor the concentration of each analyte in the mixture as the function of time in real-time and nondestructively, instead of determining the concentration after the analytical separation. Thereafter, we fitted the first-order rate law to their concentration data throughout their reactions. Additionally, a four-way calibration procedure is developed as an alternative for highly collinear systems. The results of the four-way analysis confirmed the results of the three-way analysis and revealed that both the degradation reaction of NADH and the formation reaction of FAD in human plasma fit the first-order rate law. The proposed methods could be expected to provide promising tools for simultaneous kinetic analysis of multiple reactions in complex systems in real-time and nondestructively.     

Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors

High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action.     

Determination of picomolar levels of flavins in natural waters by solid-phase ion-pair extraction and liquid chromatography

A method is described for the rapid determination of flavins in sea water, based on solid-phase extraction followed by ion-pair high-performance liquid chromatography (HPLC) with fluorescence detection. Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) and their photochemical breakdown products, lumiflavin, formylmethylflavin, and lumichrome can be determined with subpicomolar detection limits. The method was used at sea in the analysis of coastal and open ocean waters. In both environments, riboflavin, lumiflavin and lumichrome were routinely observed at concentrations in the picomolar range; lumiflavin and lumichrome were generally confined to the photic zone while riboflavin was present throughout the water column. Formylmethylflavin, FMN, and FAD were only occasionally observed; when present, these flavins were observed at consistently higher concentrations than riboflavin, lumiflavin and lumichrome.     

Electrocatalytic reaction of hydrogen peroxide and NADH based on poly(neutral red) and FAD hybrid film

A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol.     

Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor—“a nicotinamide in flavin clothing”—needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) ¹H nuclear magnetic resonance (NMR) under biologically relevant conditions.     

Light-triggered proton and electron transfer in flavin cofactors

The pH dependent behavior of two flavin cofactors, flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN), has been characterized using femtosecond transient absorption spectroscopy for the first time. The flavin excited state was characterized in three states of protonation (Fl(-), Fl, and FlH(+)). We found that Fl and Fl(-) exhibit the same excited state absorption but that the lifetime of Fl(-) is much shorter than that of Fl. The transient absorption spectrum of FlH(+) is significantly different from Fl and Fl(-), suggesting that the electronic properties of the flavin chromophore become appreciably modified by protonation. We further studied the excited state protonation of the flavin and found that the protonation sites of the flavin in the ground and excited state are not equivalent. In the case of FAD, its excited state dynamics are controlled by the two conformations it adopts. At low and high pH, FAD adopts an "open" conformation and behaves the same as FMN. In a neutral pH range, FAD undergoes a fast excited state deactivation due to the "stacked" conformer. The transition from stacked to open conformer occurs at pH ～ 3 (because of adenine protonation) and pH ～ 10 (because of flavin deprotonation).