Flavins, determination

Bacterial concentrations have also been determined by using the enzyme-catalyzed chemiluminescent reaction of reduced flavin mononucleotide (FMN) with oxygen and aldehydes. The detection limit was reported to be 10 ceUs of E. coli, which contains 7 x 10 g of FMN per ceU (303). 

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. 

Walsh and coworkers oxidized ethyl p-tolyl sulfide on an analytical scale to the S-sulfoxide of 64% enantiomeric purity using a bacterial flavoenzyme cyclohexanone monooxygenase derived from Adnetobacter . Using a flavin adenine dinucleotide containing monooxygenase purified from hog liver microsomes yielded the R-sulfoxide of 90% enantiomeric purity. HPLC on a column containing a 3,5-dinitrobenzoyl-D-phenylglycine chiral stationary phase was used to determine the optical purity of the sulfoxides. 

The varions flavin phosphates and their acetyl derivatives were identified by pH titration, electrophoresis, and H-NMR, which permit direct analysis of crude reaction prodncts as well as rapid purity check of commercial flavin mononucleotide or riboflavin 5 -monophosphate (FMN or 5 -FMN) [7]. Riboflavin 4 -monophosphate was determined as the main by-product of commercial FMN by preparative TLC on cellulose with n-butanol/acetic add/water (5 2 3, v/v) as a solvent [7]. 

It must be realised however (as indeed it was at the time of these experiments (53)) that the use of such studies to determine the sequence, has certain limitations. The first limitation is that nothing can be said about the role of species not detectable by EPR, such as fully reduced flavin. Secondly and more generally, it has to be assumed for both the oxidizing and reducing substrates, that each reacts with only one of the... 

Based on the previous publications, azo dye can be reduced by azoreductase-catalyzed reduction under anaerobic conditions. But still there is a speculation whether bacterial flavin reductases are responsible for the azo reductase activity observed with bacterial cell extracts. In a published report, it is reported that flavin reductases are indeed able to act as azo reductases [24]. Bacteria produce extracellular oxidative enzymes, which are relatively nonspecific enzymes catalyzing the oxidation of a variety of dyes. It was reported that so many diverse groups of bacteria play a role in decolorization. It has been also reported that mixed microbial community could reduce various azo dyes, and members of the y-proteabacteria and sulfate reducing bacteria (SRB) were found to be prominent members of mixed bacterial population by using molecular methods to determine the microbial population dynamics [1],... 

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. 

Flavoprotein dehydrogenases usually accept electrons from reduced pyridine nucleotides and donate them to a suitable electron acceptor. The oxidation-reduction midpoint potential of the FAD of the oxidase has been determined by ESR spectroscopy and shown to be -280 mV. The NADP+/ NADPH redox potential is -320 mV and that of the cytochrome b is -245 mV hence, the flavin is thermodynamically capable of accepting electrons from NADPH and transferring them to cytochrome b. As two electrons are transferred from NADPH, although O2 reduction requires only one electron, the scheme of electron transfer shown in Figure 5.8 has been proposed by Cross and Jones (1991). 

The conversion (19) of thiols to disulphides coupled with reduction of flavin (vitamin B2 family) is a topic of import in connection with coenzyme reactivity in flavoenzymes. Since flavin oxidation of thiols involves nucleophilic attack of thiolate ion in the rate-determining step (Loechler and Hollocher, 1975 Yokoe and Bruice, 1975), this biologically important reaction would be markedly affected by hydrophobic environments. 

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. 

Schneider K, Schlegel HG. 1978. Identification and quantitative determination of the flavin component of soluble hydrogenase from Alcaligenes eutrophus. Biochem Biophys Res Commun 84 564-71. 

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-... 

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