Abstract
Publisher Summary This chapter discusses the determination of flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD), by fluorescence titration, with apoflavodoxin. In assay method, the apoenzymes of flavodoxins isolated from certain anaerobic bacteria bind FMN rapidly and tightly to form complexes that are nonfluorescent, but they do not react with FAD or riboflavin. The FMN content of a test solution, therefore, can be determined from the end point of a fluorescence titration with a standard solution of apoflavodoxin. FAD is determined from the difference between the end point of this titration and that of a second titration, following the treatment of the test sample, with phosphodiesterase to hydrolyze FAD to FMN. Commercial preparations of FMN contain up to 32% flavin impurity that is not bound by apoflavodoxin from Megasphaera elsdenii . The FMN and FAD in mixtures of the two flavins can be accurately determined by the method discussed in the chapter when the total flavin concentration is l μM and the ratio FMN:FAD is between 9:l and 1:9. Similar results are obtained when the total flavin concentration is about 0.1 μM, but only when the FMN in the mixture is greater than about 20 nM.
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