Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.     

Altered flavin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Flavins were extracted from sporangiophores of the lower fungus Phycomyces blakesleeanus and identified by HPLC with fluorescence detection. In the wild-type strain NRRL1555 they were found to be present at the following concentrations: riboflavin (5.5 x 10(-6) M), flavin mononucleotide (FMN) (4.0 x 10(-6) M) and flavin adenine dinucleotide (1.4 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of behavioral mutants (genotype mad) with specific defects in their light-transduction pathway. The photoreceptor mutants C109 (madB), C111 (madB) and L1 (madC) had normal amounts of flavins. The most prominent changes were found in single mutants with a defective madA gene which contained about 25% of riboflavin and about 10% of FMN and FAD normally found in the wild type. A hypertropic mutant with a defective madH gene contained instead 80% of riboflavin and 120% of FMN and FAD. The double mutant L52 (madA madC) and the triple mutant L72 (madA madB madC) had normal amounts of FAD and FMN. This indicates that the madC mutation, which itself causes loss of light sensitivity and which affects the near-UV/blue-light receptor (Galland and Lipson, 1985, Photochem. Photobiol. 41, 331-335) functions as a restorer of the flavin content in a genetic madA background. The double mutant L51 (madA madB) had about 40% of FMN and FAD, suggesting that the madB mutation functions as a partial restorer of flavin content. The photogravitropic thresholds (450 nm) reported for the wild type and the madA and madH mutants were positively correlated to the endogeneous concentrations of FMN and FAD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subsecond separation of cellular flavin coenzymes by microchip capillary electrophoresis with laser-induced fluorescence detection

In this article, it was demonstrated that a subsecond separation of cellular metabolites such as riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) was achieved using microchip capillary electrophoresis with laser-induced fluorescence detection. The influences of crucial parameters that governed analysis time (e.g., channel length and electric field for separation) and separation resolution (e.g., sample size) were investigated, both in theoretical aspects and experimental practice. Quantitative analyses were performed that exhibited linear dynamic range of two orders of magnitude, with calculated detection limits of 34, 201, and 127 nM for RF, FAD, and FMN, respectively. To test the validity of the method, it was successfully applied to characterize several recombinant flavin-binding domains in a human neuronal nitric oxide synthase.     

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.     

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.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

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.     

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (-460 to -480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl(2), Na(2)SO(4) at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.     

Infrared spectrum and absorption coefficient of the cofactor flavin in water

Flavins play a key role as redox cofactors of enzymes involved in important metabolic processes. Moreover, they undergo photochemical reactions as chromophores in sensors of blue light or magnetic field in many organisms. The reaction mechanisms of flavoproteins have been investigated by infrared spectroscopy and theoretical studies. However, basic information on flavins in the infrared spectral range has been missing, such as absorption spectra in water and absorption coefficients. Here, the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were investigated in aqueous medium by Fourier transform infrared spectroscopy. Transmission and attenuated total reflection (ATR) configuration were employed in direct comparison. Absorption spectra in the range of 920–1800 cm⁻¹ were determined after accurate subtraction of the contributions from the water vibrations. The important carbonyl vibrations were resolved at 1661 and 1712 cm⁻¹. The absorption spectra may serve as a reference for theoretical and experimental studies on the effect of the microenvironment on the flavin cofactor. Furthermore, the molar absorption coefficient of FAD at 1547 cm⁻¹ was determined to 2200 L mol⁻¹ cm⁻¹ with an integral absorption coefficient of ∼50,000 L mol⁻¹ cm⁻². These values are prerequisite for the determination of reaction yields in flavoproteins from reaction-induced difference spectra.     

Fluorescence quenching of flavins by reductive agents

The fluorescence behaviour of the flavins riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and lumiflavin in aqueous solution at pH 8 in the presence of the reducing agents β-mercaptoethanol (β-ME), dithiothreitol (DTT), and sodium nitrite (NaNO₂) is studied under aerobic conditions. The fluorescence quantum yields and fluorescence lifetimes are determined as a function of the reducing agent concentration. For all three reducing agents diffusion controlled dynamic fluorescence quenching is observed which is thought to be due to photo-induced reductive electron transfer. For DTT additionally static fluorescence quenching occurs.     

FLUORESCENCE QUENCHING FOR FLAVINS INTERACTING WITH EGG WHITE RIBOFLAVIN-BINDING PROTEIN

Abstract— The effect of flavin structure variation upon the binding process of flavin to hen egg white riboflavin was studied using fluorescence methods for formylmethylflavin (FMF), riboflavin (RF) and flavomononucleotide (FMN).
Measurements of flavin fluorescence intensities (steady state and phase-sensitive) and lifetimes were performed in a variety of RBP concentrations and temperatures (4 to 40°C). No fluorescence of flavoproteins was detected, while the fluorescence of flavins was found to be quenched by RBP. The overall quenching process is dominated by the static quenching (about 88%) due to the flavoprotein complex formation in the ground state, presumably a charge transfer complex.     

Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.     

Reduced Flavin: NMR investigation of N(5)-H exchange mechanism,estimation of ionisation constants and assessment of properties as biological catalyst

Background  

The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context.     

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     

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).     

MO study of flavin reduction

MINDO /3 calculations have been performed on semiquinone and fully reduced lumiflavins and on hydroperoxy adducts of lumiflavin. Frontier orbital indices were calculated. Reduction of the flavin was studied in bent and planar flavin rings. The results suggest that the planar reduced flavin has a smaller ionization potential than the bent reduced flavin. This could account for the low redox potential of protein-bound reduced flavins.     

Light‐emitting‐diode‐induced chemiluminescence detection for capillary electrophoresis

In this work, an LED‐induced‐chemiluminescence (LED‐CL) system was developed to extend the application of CL detection in CE. In the LED‐CL, the analyte photooxidizes luminol under the irradiation of LEDs and generates CL. Taking the advantage of the small size nature of LEDs, the constructed photoreactor is greatly miniaturized, and especially suitable as a CE detector. The feasibility of the proposed detector was evaluated by detection of riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) after CE separation. Under the optimized conditions, the LODs for RF, FMN and FAD were 0.007, 0.02 and 0.1 μg/mL, respectively, better than those by UV detection. The RSDs were 3.4, 3.6 and 4.1% for 0.5 μg/mL RF, 2 μg/mL FMN and 5 μg/mL FAD, respectively. The LED‐CL detector features low cost, miniaturization, fast response, high sensitivity and good reproducibility.     

Preparation and redox properties of N,N,N-1,3,5-trialkylated flavin derivatives and their activity as redox catalysts

Eight different flavin derivatives have been synthesized and the electronic effects of substituents in various positions on the flavin redox chemistry were investigated. The redox potentials of the flavins, determined by cyclic voltammetry, correlated with their efficiency as catalysts in the H2O2 oxidation of methyl p-tolyl sulfide. Introduction of electron-withdrawing groups increased the stability of the reduced catalyst precursor.     

Oxygen uptake after electron transfer from amines, amino acids and ascorbic acid to triplet flavins in air-saturated aqueous solution

The photolysis of lumichrome, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) was studied in air-saturated aqueous solution at room temperature in the presence of appropriate electron donors: ascorbic acid, aromatic amino acids or amines, e.g. ethylenediaminetetraacetate (EDTA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide radical into hydrogen peroxide. The quantum yield of oxygen uptake increases with the donor concentration, e.g. up to 0.3 for riboflavin, FMN or FAD in the presence of EDTA or ascorbic acid (0.3-10mM). The formation of H(2)O(2) is initiated by quenching of the acceptor triplet state by the electron donor and subsequent reaction of the semiquinone radical with oxygen. Specific properties of flavins are discussed including the radicals involved and the pH and concentration dependences. The quantum yield of photodegradation is low under air, but substantial under argon, where the major product absorbing in the visible spectral range is the corresponding hydroquinone.     

Synthesis and redox behavior of flavin mononucleotide-functionalized single-walled carbon nanotubes

In this contribution, we describe the synthesis and covalent attachment of an analogue of the flavin mononucleotide (FMN) cofactor onto carboxylic functionalities of single-walled carbon nanotubes (SWNTs). The synthesis of FMN derivative (12) was possible by coupling flavin H-phosphonate (9) with an aliphatic alcohol, using a previously unreported N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride coupling. We found that the flavin moiety of 12-SWNT shows a strong pi-pi interaction with the nanotube side-walls. This leads to a collapsed FMN configuration that quenches flavin photoluminescence (PL). The treatment of 12-SWNT with sodium dodecyl sulfate (SDS) overcomes this strong nanotube/isoalloxazine interaction and restores the FMN into extended conformation that recovers its luminescence. In addition, redox cycling as well as extended sonication were proven capable to temporally restore PL as well. Cyclic voltammetry of FMN onto SWNT forests indicated profound differences for the extended and collapsed FMN configurations in relation to oxygenated nanotube functionalities that act as mediators. These findings provide a fundamental understanding for flavin-related SWNT nanostructures that could ultimately find a number of usages in nanotube-mediated biosensing devices.