The separation of flavins by paper electrophoresis and its application to the examination of the flavin contents of micro-organisms

Abstract

The separation of riboflavin, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD) by paper electrophoresis in 0.025 M ammonium formate-formic acid buffer, pH 4.6, is described. Recoveries of flavin from the paper are about 90%. The use of 10% trichloroacetic acid for 5 minutes at room temperature was selected as the most convenient procedure for extracting flavins from microbial cells. Flavins so extracted are obtained in a form suitable for electrophoresis by frac-tionation with a Florisil column giving 80% recovery. These methods were combined in an examination of the flavins present in a range of microorganisms. FMN and FAD were demonstrated in all organisms examined. Very little, if any, free riboflavin is present and no other flavin derivatives were detected. Flavin contents varied from 30 to 2930 m-moles/g dry weight with Rhodospirillum rubrum and Clostridium kluyveri respectively. High flavin contents are associated with the fermentative formation of butyrate and higher volatile fatty acids. With 3 hexanoate-oxidizing organisms isolated from soil, the oxidation of hexanoate is adaptive and is associated with a higher flavin content. The proportions of flavin present as FMN and FAD vary widely from 21% of FMN and 79% of FAD with Desulphovibrio to 68% of FMN and 30% of FAD with Lactobacillus helveticus. Those organisms oxidizing hexanoate or producing higher volatile fatty acids contain a high proportion of FAD.