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PubMed1997

Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions.

To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia coli as maltose binding protein fusions. The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme. A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158 enzyme. The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes. N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively. It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity. Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion. Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities. Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.

ATP-Binding Cassette Transporters↗

PubMed1997

Direct electrochemistry of the flavin domain of assimilatory nitrate reductase: effects of NAD+ and NAD+ analogs.

Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.

Adenosine Diphosphate↗

PubMed1980

Metal-flavin complexation. A resonance Raman investigation.

Several groups have recently shown that high quality resonance Raman spectra can be obtained for flavin species in spite of their intense fluorescence. We are interested in obtaining the resonance Raman spectra of flavins in various chemical environments in order to determine whether the spectra are useful in probing the chemical interaction between flavins and protein in flavoenzymes. We have obtained the resonance Raman spectrum of a nonfluorescent Ag+ complex of FMN. Several large changes occur in the FMN resonance Raman spectrum upon Ag+ complexation; among these are changes in the 1580 cm-1 region of the FMN spectrum (assigned to nu C=N at N-5 and C-4a), the 1410 cm-1 region and the 1260 cm-1 region (associated with a vibration having some delta N-N-H character at N-3). Similar changes are observed in the same region of a Ru2+-FMN complex. Since these spectral changes occur in two metal flavin complexes with very different electronic spectra, they would seem to be due to vibrational changes induced by metal complexation at N-5 and the oxygen at C-4 of flavin rather than the details of the vibronic interactions which give rise to the resonance enhancement of the spectrum. A structure for the Ag+-FMN complex is suggested. This study has potential physiological significance, because it illustrates the possible role of resonance Raman spectroscopy as a tool for the determination of direct flavin metal interaction in dilute aqueous solution of metalloflavoproteins.

Chemical Phenomena↗

PubMed1995

2'-fluoro-2'-deoxy-D-arabinoflavin: characterization of a novel flavin and its effects on the formation and stability of two-electron-reduced mercuric ion reductase.

With the goal of generating a novel fluorine-containing flavin analogue with a reduction potential the same as normal flavin, 2'-fluoro-2'-deoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD forms, UV-visible spectral properties are similar to those of normal flavins, and tight binding to riboflavin binding protein and mercuric ion reductase occurs with very similar spectral changes. The reduction potential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1'-methylene group insulates the redox-active isoalloxazine from the 2'-fluorine. With the intent of using the analogue as a fluorine NMR probe of the active site environments of two-electron-reduced mercuric ion reductase, apoenzyme was reconstituted and its behavior under reducing conditions examined. Whereas with normal enzyme, addition of two electrons gives rapid formation of a charge-transfer species where FAD remains oxidized and a disulfide is reduced to a thiol/thiolate pair, with the 2'-FaFAD enzyme, addition of two electrons gives rapid reduction of the flavin followed by slow transfer of electrons to the disulfide with very little development of the typical charge-transfer absorption. Analysis of crystal structure data suggests that having the fluorine in the alternate arabino stereochemistry places it much nearer the flavin-proximal cysteine/cystine sulfur, where it may inhibit both electron transfer from reduced flavin and the charge-transfer interaction between reduced thiolate and FAD.

Apoenzymes↗

PubMed2001

Insight into the chemistry of flavin reduction and oxidation in Escherichia coli dihydroorotate dehydrogenase obtained by rapid reaction studies.

Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichia coli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate. The chemistry of the reduction of the flavin in DHOD from E. coli by the substrate dihydroorotate (DHO) was studied at 4 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 1 ms) upon mixing with DHO. The flavin of the intermediate was reduced by DHO, forming a reduced flavin-orotate charge-transfer complex. The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s(-1) at pH 6.5 to approximately 360 s(-1) at pH values greater than an observed pK(a) of 9.5 which was ascribed to Ser175, the active-site base. At all pH values, the reduced flavin-orotate charge-transfer complex dissociated too slowly to be catalytically relevant. Therefore, the oxidizing quinone substrate must bind to the reduced enzyme-orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies [Björnberg, O., Grüner, A.-C., Roepstorff, P., and Jensen, K. F. (1999) Biochemistry 38, 2899-2908]. Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD. The reduced enzyme-orotate complex reacted rapidly with menadione (180 s(-1)), demonstrating that the reduced enzyme-orotate complex is a catalytically competent intermediate.

Anaerobiosis↗

PubMed1978

Light-mediated reduction of flavoproteins with flavins as catalysts.

It has been found that small amounts of free flavins greatly accelerate the photochemical reduction of flavoproteins both to the radical and fully reduced oxidation states. This catalytic effect has been shown to be due to the rapid photochemical reduction of the free flavin to its fully reduced state, followed by its reaction with the flavoprotein to yield flavoprotein radical and by its reaction with flavoprotein radical to yield fully reduced flavoprotein. Evidence is presented that the same route may occur with flavoproteins in the absence of added flavins. In this case the photoreduction is mediated by the small equilibrium concentration of free flavin coenzyme present in a flavorprotein solution. Hence, it is suggested that flavoprotein reduction with EDTA as photosubstrate does not involve an excited state of the holoprotein, nor contact of EDTA with the enzyme, but exchange of electrons between enzyme flavin and free reduced flavin.

Dose-Response Relationship, Radiation↗

PubMed1996

Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys.

The isoalloxazine ring system of the FAD cofactor of p-hydroxybenzoate hydroxylase must be secluded from solvent at specific stages of catalysis in order to form and stabilize a flavin C4a-hydroperoxide. This species may then react with the activated phenolate of p-hydroxybenzoate. A number of crystal structures of the enzyme with alterations to active site substituents or complexes with analogue benzoates have revealed an alternate position for the isoalloxazine (Gatti et al. (1994) Science 266, 110-114; Schreuder et al. (1994) Biochemistry 33, 10161-10170). This new flavin conformation is 7 A "out" toward solvent and may open a passage for substrate entry to the active site. Arginine 220 is one of the few residues in the structure to demonstrate conformational changes when the flavin is "out". In this study we have made the Arg220Lys mutant to test the significance of this residue in flavin movement. The R220K mutation has brought about dramatic alterations to all aspects of catalysis. Stopped flow kinetic characterization of the mutant has revealed that, while the effector role for the substrate is maintained, there exists an order of magnitude decrease in the limiting rate of reduction, even though there is 40-fold increase in association with NADPH. The mutant enzyme has only a fraction of its reductive half-reaction coupled to product formation, and the hydroxylation process is slow. This occurs despite a higher proportion of the more activated substrate phenolate in the active site. Many of the observed changes can be attributed to a decrease in the stability of the "in" conformation of the flavin during the catalysis and indicate a role for flavin conformational states in many of the catalytic processes of the enzyme.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1999

Using Raman spectroscopy to monitor the solvent-exposed and "buried" forms of flavin in p-hydroxybenzoate hydroxylase.

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1990

Flavin composition of human milk.

The identity and quantity of greater than 95% of the flavins present in human milk were assessed by acid-phenol extractions followed by high-performance liquid chromatography. Flavin adenine dinucleotide (FAD) and riboflavin were the predominant flavins, followed by 10-(2'-hydroxyethyl)-flavin. In addition, traces of 7 alpha- and 8 alpha-hydroxyriboflavins (7-hydroxymethylriboflavin and 8-hydroxymethylriboflavin, respectively) were detected. The flavin content of human milk samples in this study was higher than contents reported in earlier studies where no correction for the internal fluorescence quenching of FAD was made. This finding may have implications for dietary recommendations concerning both lactating women and infants. In practical terms, the types and amounts of flavins in human milk are very similar to those recently reported for cow milk.

Animals↗

PubMed1990

Qualitative and quantitative assessment of flavins in cow's milk.

Although milk is one of the primary sources of riboflavin in the North American diet, the amounts of riboflavin and other flavin derivatives in milk have not been accurately quantified. In this study, a comprehensive assessment of milk was performed to identify and quantify greater than 95% of the flavins in this food. Riboflavin and flavin adenine dinucleotide (FAD) were the predominant flavins in all milk samples; 10-(2'-hydroxyethyl)flavin was next most common. The latter is potentially an antivitamin that may exert its effect at either the absorption or utilization level. Therefore, the presence of this analog should be considered when evaluating the riboflavin efficiency of milk. The large quantity of FAD indicates that precautions against underestimating FAD are warranted when determining total flavin content by fluorescence. This is particularly important because the fluorescence of FAD at pH 7.0 is only 10-20% of that for riboflavin. The presence of 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) in milk was ascertained by extensive testing. Traces of 8 alpha-hydroxyriboflavin (8-hydroxymethylriboflavin) were also detected. These metabolites may serve as indicators of cellular biochemical activity.

Animals↗

PubMed1995

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.

Amino Acid Sequence↗

PubMed1979

On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by fluorescence.

The incorporation of four amphiphilic flavins ("amphiflavins") as fluorescence markers bearing C18-hydrocarbon chains at various positions of the chromophore into artificial membrane vesicles has been investigated. The vesicles utilized were made from three different saturated phospholipids. The stability of the flavin-charged vesicles was found to be good over several days, depending somewhat on the temperature, the pH, and their concentration. A marked increase of the fluorescence quantum yield near the vesicle phase transition (crystalline leads to liquid crystalline) was found which was taken to indicate that the flavin nuclei are imbedded more deeply into the hydrophobic portion of the membranes. This is further supported by a hypsochromic shift of the near flavin UV-peak and the increase of absorbance at 450 nm upon melting. Rotational relaxation times of the various amphiflavins bound to the different vesicles are obtained from measurements of the fluorescence polarizations as a function of temperature. From these data, the microviscosities in the region of the chromophors are calculated. Measurements of the fluorescence polarization as a function of the solvent viscosity and vesicle phase (crystalline-liquid crystalline) indicate that below the phase transition the flavin nucleus is protected from the suspension medium by a lipid-water interphase, which softens above phase transition. The dependence of the flavin orientation and microenvironment on the position of the substitution of the aliphatic chain is reflected in the differences of the fluorescence yields and the shape of the emission spectra.

Flavins↗

PubMed2006

Flavin-dependent halogenases involved in secondary metabolism in bacteria.

The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme FeII alpha-ketoglutarate- and O2-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.

Bacteria↗

PubMed1990

Evidence for the presence of distinct flavin-containing monooxygenases in human tissues.

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.

Animals↗

PubMed1982

Limited rotational motion of amphiphilic flavins in dipalmitoylphosphatidylcholine vesicles.

The rotational motion of amphiphilic flavins in dipalmitoyl phospholipid bilayers was investigated with fluorescence anisotropy decay measurements. At temperatures between 10 and 50 degrees C the rotation proved to be anisotropic, which indicated composite motion of both the aliphatic side-chain and the isoalloxazine moiety of the octadecyllumiflavin derivatives. Above the phase transition temperature (crystalline leads to liquid-crystalline state) the depolarization is complete within the average flavin fluorescence lifetime, implicating unrestricted motion and resulting in a non-ordered microenvironment. In the gel or crystalline state the flavin motion can best be characterized as a limited rotation or librational motion. The fluorescence decay of the flavins is heterogeneous at temperatures between 10 and 50 degrees C, which is explained by assuming nanosecond relaxation of the polar phosphatidyl head-groups around the excited flavin. The lack of a significant cholesterol effect suggests that the isoalloxazine is located at the interphase of the bilayer and not in the hydrocarbon region. The microstructure is fluid-like, not in agreement with a preferred static localization of the flavins in the bilayer.

Flavins↗

PubMed2003

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

Basidiomycota↗

PubMed2003

Monoamine oxidase A inhibitory potency and flavin perturbation are influenced by different aspects of pirlindole inhibitor structure.

Reversible inhibitors of monoamine oxidase A (MAO A) are used as antidepressants. The influence of inhibitors such as pirlindole (pyrazinocarbazole) on the redox co-factor (flavin adenine dinucleotide, FAD) is a key factor in the inhibition. The kinetic, spectral, and thermodynamic changes induced by four closely related pirlindole analogues have been determined to investigate their interaction with the FAD in the active site of MAO A. For a model of flavin-inhibitor stacking, more favourable association would be expected between lumiflavin and the flatter analogues with a double bond at N3, and indeed lower K(i) values were found. However, the spectral changes induced by inhibitor binding to MAO A were 45% less for inhibitors with a double bond. Both in the absence and presence of the double bond, compounds with cyclohexyl at C8 induced 85% larger decrease in absorbance at 500nm than did those with a methyl substituent. In contrast, the K(i) values for the cyclohexyl compounds were lower, indicating greater affinity despite the lower perturbation of the flavin spectrum. All inhibitors stabilised the semiquinone of the FAD when MAO A was titrated with dithionite and prevented further reduction. These results indicate that the active site of MAO A is far more sensitive to structural variation than would be predicted by the simple flavin stacking model. Further, the independent changes in inhibitory potency and flavin perturbation preclude direct interaction with the flavin as a mode of binding and indicate that inhibitor-protein interactions must be important for inhibition.

Binding Sites↗

PubMed2002

On-line focusing of flavin derivatives using Dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection.

Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.

Coenzymes↗