Fluorescence of flavins

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

Eley, M., et al. (1970). Bacterial bioluminescence. Comparisons of bioluminescence emission spectra, the fluorescence of luciferase reaction mixtures, and the fluorescence of flavin cations. Biochemistry 9 2902-2908. 

Flavins are vitamine B2 and bind to proteins as coenzyme. Some photoreceptors contain flavins which receive photons. Iso-alloxazine nucleus being chromophore of various flavins is yellow dye and intensely emits greenish fluorescence in organic and aqueous solutions. The fluorescence of flavins is remarkably quenched when they bind to protein moiety. Among amino acid... 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

Eley, M., j. Lee, J. M. Lhoste, C. Y. Lee, M. J. Cormier, and P. Hemmerich Bacterial Bioluminescence. Comparisons of Bioluminescence Emission Spectra, the Fluorescence of Luciferase Reaction Mixtures, and the Fluorescence of Flavin Cations. Biochemistry 9, 2902 (1970). 

A method has been reported [481] for the determination of flavins in seawater. The method is based on solid-phase extraction. With ion-pair HPLC using fluorescence detection, concentrations in the picomolar range can be measured. 

Vames AW, Dodson RB, Wehry EL (1972) Interactions of transition-metal ions with photoexcited states of flavins. Fluorescence quenching studies. J Am Chem Soc 94 946-950... 

The quantum yield of flavin fluorescence in proteins is very low in many cases, and the lifetimes are on the order of picoseconds. This is a result of the high electrophilicity of oxidized flavins, and their ability to quench fluorescence following electron transfer from the electron-rich groups of... 

Figure 6.4. Schematic representation of a fluorescent immunoassay for theophylline utilizing enzymatic hydrolysis of an intramolecularly quenched theophylline conjugate of flavin adenine dinucleotide. (Reprinted from Ref. 5, with permission from Academic Press.)...

Selected entries from Methods in Enzymology [vol, page(s)] Determination of FMN and FAD by fluorescence titration with apoflavodoxin, 66, 217 purification of flavin-adenine dinucleotide and coenzyme A on p-acetoxymercurianiline-agarose, 66, 221 a convenient biosynthetic method for the preparation of radioactive flavin nucleotides using Clostridium kluyveri, 66, 227 isolation, chemical synthesis, and properties of roseoflavin, 66, 235 isolation, synthesis, and properties of 8-hydroxyflavins, 66, 241 structure, properties and determination of covalently bound flavins, 66, 253 a two-step chemical synthesis of lumiflavin, 66, 265 syntheses of 5-deazaflavins, 66, 267 preparation, characterization, and coenzymic properties of 5-carba-5-deaza and 1-... 

The structure of the modified flavocoenzymes was elucidated by chemical synthesis and comparative physical studies 126, iso) (Scheme 2, (7), (S)). The compounds possess some unusual properties some of which are collected in Table 3. The most prominent difference between (7) and (S) is the fluorescence behaviour (7) is fluorescent, (5) does not fluoresce. Moreover, at pH > 10 the fluorescence quantum yield of (7) increases by a factor of about 2, in contrast to normal flavin the fluorescence of which is quenched. By this property (7) and (5) are easily distinguished (Table 3). From the visible absorption properties of analogs of (7) and (5) it was concluded that both compounds can exist in two tautomeric forms (proton on N(l) or C(8)O, C(6)O), leading to quinoid structures. 

The most prominent feature of the chemistry of flavin is its redox properties. These properties make flavin especially suitable for its broad involvement in biological reactions. In the following the pH-dependent species formed in one- and two-electron reductions will be dealt with first, including their visible absorption and fluorescence properties. These physical properties form the basis of many kinetical and analytical studies. In Scheme 3 the structures refer to the free and protein-bound prosthetic groups (cf. Scheme 1). To study the physical properties of the flavocoenzymes often N(3)-alkylated lumiflavin (R = CH3) is used which is better soluble in a variety of solvents. Other physical and chemical properties of these species will be discussed subsequently. 

W. Wei, G. Zue, and E. S. Yeung, One-Step Concentration of Analytes Based on Dynamic Change in pH in Capillary Zone Electrophoresis, Anal. Chem. 2002, 74, 934 P. Britz-McKibbin, K. Otsuka, and S. Terabe, On-Line Focusing of Flavin Derivatives Using Dynamic pH Junction-Sweeping Capillary Electrophoresis with Laser-Induced Fluorescence Detection, Anal. Chem. 2002, 74, 3736. 

Figure 3-5 Photograph of a two-dimensional thin layer (silica gel) chromatogram of a mixture of flavins formed by irradiation of 10 pg of the vitamin riboflavin. The photograph was made by the fluorescence of the compounds under ultraviolet light. Some riboflavin (RF) remains. The arrows indicate the location of the sample spot before chromatography. Chromatography solvents a mixture of acetic acid, 2-hutanone, methanol, and benzene in one direction and M-butanol, acetic acid, and water in the other. See Treadwell et al.H)2...

The attention of biochemists was first attracted to flavins as a result of their color and fluorescence. The study of spectral properties of flavins (Fig. 15-8) has been of importance in understanding these coenzymes. The biochemical role of the flavin coenzymes was first recognized through studies of the "old yellow enzyme"144 145 which was shown by Theorell to contain riboflavin 5 -phosphate. By 1938, FAD was recognized as the coenzyme of a different yellow protein, D-amino acid oxidase of kidney tissue. Like the pyridine nucleotides, the new flavin coenzymes were reduced by dithionite to nearly colorless dihydro forms (Figs. 15-7 and 15-8) revealing the chemical basis for their function as hydrogen carriers. 

The bright orange-yellow color and brilliant greenish fluorescence of riboflavin first attracted the attention of chemists. Blyth isolated the vitamin from whey in 1879 and others later obtained the same fluorescent, yellow compound from eggs, muscle, and urine. All of these substances, referred to as flavins because of their yellow color, were eventually recognized as identical. The structure of riboflavin was established in 1933 by R. Kuhn and associates, who had isolated 30 mg of the pure material from 30 kg of dried albumin from 10,000 eggs. The intense fluorescence assisted in the final stages of purification. The vitamin was synthesized in 1935 by R Karrer.3... 

Figure 10. Plots of log feel vs the oxidation peak potentials of methyl- and methoxy-substituted benzenes ( x) for the fluorescence quenching of flavin analogs (la,2a-c) by the quenchers in the absence (O) and presence of O.lOmol dm-3 Mg(C10J2 ( ) or Zn(C10J2 (3) in MeCN. Numbers refer to the quenchers (e.g., 1 = MeC6H5, 14 = p-(MeO)2C6H4) [154],...

Engelmann MD, Bobier RT, Hiatt T, Cheng IF (2003) Variability of the Fenton reaction characteristics of the EDTA, DTPAand citrate complexes of iron. BioMetals 16 519-527 Epsztejn S, Kakhlon O, Glickstein H, Breuer W, Cabantchik I (1997) Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem 248 31-40 Faraggi M, Hemmerich P, Pecht I (1975) Oj-affinity of flavin radical species as studied by pulse radiolysis. FEBS Lett 51 47-51... 

The Ksv value shows the importance of fluorophore accessibility to the quencher, while the value of A q gives an idea of the importance of the diffusion of the quencher within the medium. Figure 10.1 shows a Stern-Volmer plot of fluorescence intensity quenching with iodide of flavin free in solution and of flavin bound to flavocytochrome ba- The Ksv values found are 39 and 14.6 M-1 for free and bound flavins, respectively, i.e., values of kq equal to 8.3 x 109 and 3.33 x 109 M-1 s-1, respectively. Accessibility of flavin to KI is more important when it is free in solution, and the presence of protein matrix prevents frequent collisions between iodide and FMN thereby decreasing the fluorophore accessibility to the quencher. Also, as revealed by the Aq values, diffusion of iodide in solution is much more important than in flavocytochrome b2. The protein matrix inhibits iodide diffusion, thereby decreasing the A q value. 

Britz-McKibbin, P., Markuszewski, M.J., lyanagi, T., Matsuda, K., Nishioka, T., Terabe, S. Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection. Anal. Biochem. 313, 89-96 (2003)... 

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. 

Many enzymes use organic cofactors, such as flavin and porphyrin, at the active sites for catalysis. Some of these organic cofactors, especially flavin, have intrinsic fluorescence, and can be imaged readily at the singlemolecule level. If the fluorescence of these cofactors is coupled with the state of the active site in the catalytic cycle, monitoring the fluorescence of the active site can directly probe the catalysis. The classic example of this... 

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