Flavin mononucleotide fluorescence

Flavin mononucleotide (Na, 2H2O salt, FMN) [ 130-40-5JM 514.4, pKj 2.1 (PO4H2), pK2 6.5 (PO4H ), pKj 10.3 (CONH), fluorescence Xmax 530nm (870nm for reduced form). 

Riboflavin is heat-stable in the absence of light, but extremely photosensitive. It has a high degree of natural fluorescence when excited by UV light. This property can be used for detection and determination. Two coenzymes (Fig. 2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are derived from riboflavin. 

HPLC with fluorescence detection was employed for the analysis of riboflavin (RF), flavin mononucleotide (FMN) and flavin-adenin dinucleotide (FAD) in beer, wine and other beverages. The investigation was motivated by the finding that these compounds are responsible for the so-called taste of light which develops in beverages exposed to light. Samples were filtered and injected in to the analytical column without any other pretreatment. Separations were carried out in an ODS column (200 X 2.1mm i.d. particle size 5 pm). Solvents A and B were 0.05 M phosphate buffer (pH 3) and ACN, respectively. The... 

C. Andres-Lacueva, F. Mattivi and D. Tonon, Determination of riboflavin, flavin mononucleotide and flavin-adenin dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection. J. Chromatogr.A 823 (1998) 355-363. 

Using blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage sensing domains, flavin mononucleotide-based fluorescent proteins were produced that can be used as fluorescent reporters in both aerobic and anaerobic biological systems. 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

The molecule consists of a d-ribitol unit attached to an isoalloxazine ring (Figure 9-15). Anything more than a minor change in the molecule results in a loss of vitamin activity. Aqueous solutions of riboflavin are yellow with a yellowish-green fluorescence. The vitamin is a constituent of two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN is... 

Enzymatic cofactors, such as nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (EAD), flavin mononucleotide (EMN), and pyridoxal phosphate, are fluorescent and commonly found associated with various proteins where they are responsible for electron transport (see Fig. lb and Table 1). NADH and NADPH in the oxidized form are nonfluorescent, whereas conversely the flavins, FAD and EMN, are fluorescent only in the oxidized form. Both NADH and FAD fluorescence is quenched by the adenine found within their cofactor structures, whereas NADH-based cofactors generally remain fluorescent when interacting with protein structures. The fluorescence of these cofactors is often used to study the cofactors interaction with proteins as well as with related enzymatic kinetics (1, 9-12). However, their complex fluorescent characteristics have not led to widespread applications beyond their own intrinsic function. 

Some proteins contain other native fluorophores in addition to fluorescent amino acids. These include cofactors such as nicotinamide adenine dinucleotide (fluorescent in its reduced, NADH state) and flavin adenine dinucleotide (FAD). NADH is weakly fluorescent in water, but its fluorescence yield increases markedly on binding to a protein-binding site with an emission peak around 470 nm (3). FAD and flavin mononucleotide (FMN) are also fluorescent with an emission maximum around 520 nm, but fluorescence is quenched on binding to many flavoproteins (4). 

Earlier, we found that heavy-atom effect can also be observed in bioluminescent systems 3,4 bioluminescence inhibition coefficients were found to decrease in the series potassium halides KC1, KBr, and KI. Two mechanisms can be responsible for the change of the intensity of bioiuminescence in the presence of heavy ions the physicochemical effect of external heavy atom mentioned above, and the biochemical effect, i.e. interactions with the enzymes resulting in changes in enzymatic activity. A series of model experiments was conducted to evaluate the contribution of the physicochemical mechanism. These involved the photoexcitation of model fluorescent compounds close to bioiuminescence emitters in chemical nature and fluorescent properties - flavin mononucleotide, firefly luciferin and coelenteramide. These results are clear evidence of the smaller contribution of the physicochemical mechanism to the decrease in the bioiuminescence intensity for the three bioluminescent systems under study.4... 

Flavin molecule by its redox properties plays an important role in energy providing reactions. Flavin occurs as riboflavin or as a nucleotide in flavin mononucleotide (FMN) and combined to adenine nucleotide in flavin adenine dinucleotide (FAD) Very recently it was shown by Spiro et al. that free fluorescence SERRS spectra from flavoproteins adsorbed at silver colloids (average size of 7.5 nm) can be obtained... 

ACE, affinity capillary electrophoresis CEC, capillary electrochromatography CL, chemiluminescence EC, electrochemistry ECL, electrochemiluminescence FAD, flavin adenine dinucleotide FMN, flavin mononucleotide HCV, hepatitis C vims HIV-1 RTase, reverse transcriptase of human immunodeficiency vims type 1 LC, liquid chromatrography LIE, laser-induced fluorescence MALDI-TOF, matrix-assisted laser desorption ionization-time-of-ilight mass spectrometry UV, UV-visible spectram. 

Several other analytical procedures are available for enzyme activity determination. Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less flexible and the equipment not so common in a standard research laboratory. 

Table 11.2. Flavin Mononucleotide Intensity and Anisotropy Decays in the Presence of Yellow Fluorescent Protein (YFP) ...

Riboflavin (vitamin B2) Riboflavin exists primarily as the coenzyme forms flavin mononucleotide and flavin adenine dinucleotide. Not commonly measured, both can be measured by their natural fluorescence at 530 nm following reversed-phase... 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Riboflavin and other flavinoids are found in dairy produce and meat and to a lesser extent in cereals. The RDA is 1.6-2.0 mg. Flavins are stable to heat and acid but destroyed by exposure to light. UV irradiation of riboflavin in acid or neutral solution gives rise to the fluorescent compound lumichrome, whereas in alkaline solutions irradiation produces lumiflavin. Flavins are required in the body as their coenzymes flavin mononucleotide and flavin adenine dinucleotide, which are involved in redox reactions involving one- and two-electron transfers and linked to many energy-dependent processes in the body. 

More than 100 years ago a fluorescent compound was isolated first fi om whey, and later from different biological materials. When it Ijecame clear that the isolated yellow pigments, named lactochrome, ovoflavin, or lactoflavin, had a common structure, the new compound was named riboflavin (vitamin B2) (for historical review see 2). In the years between 1933 and 1935 the structure and the main chemical reactions of riboflavin were studied and the chemical synthesis was performed. Soon afterward, the coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), were isolated in pure form, and the structures were determined. In the last 50 years many flavoproteins were isolated and their physicochemical properties were studied. Succinate dehydrogenase was the first enzyme found with the prosthetic group (FAD) covalently bound to the protein. About 20 flavoproteins are now known to contain covalently bound coenzyme (mainly via carbon atom 8a) (3). In mammalian tissue, the number of covalently bound flavoproteins appears to be limited. 

Riboflavin (vitamin B2) also acts as a cofactor and is a precursor for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes are used in metabolism and catalyze numerous oxidation—reduction reactions. Among the good dietary sources for riboflavin, most animal-derived products, milk and dairy products, are pointed out. Foods are usually pretreated before analysis of riboflavin following similar procedures to those described for vitamin Bi. Similarly, fluorescence detection is mostly employed (370 nm ex., 520 ran em.) after RP separation. 

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