ISOLATION AND IDENTIFICATION OF FLAVIN ADENINE DINUCLEOTIDE

II STUDIES ON PURIFICATION

Abstract

The mycelium obtained by the culture of Eremothecium ashbyii was found to be a suitable material for preparing FAD. Purification of FAD has so far been effected through its silver or barium salt by many workers. In the present study, however, it was found that the purification is effectively achieved by repeating the process of reducing crude FAD with sodium hydrosulfite and recovering by aeration, as in the purification of riboflavin. The yield was ca. 600mg from 6kg of pressed mycelium, and this is far superior to those hitherto reported to have obtained from animal organs or yeast. Purity of the product was estimated from its absorption spectrum. Since the extinction ratio (E_260mμ/E_450mμ) of the product was found to be 3.28, its purity was calculated from E450mμ to be 81.5%. The product, 10mg, was hydrolyzed by heating with 1.0ml of N/10 hydrochloric acid at 100° for one hour, and an examination by PPC of the product detected FAD, FMN, adenosine and AMP, but with the product of 4-hour hydrolysis, FAD, FMN, riboflavin and adenosine were detected. Paper ionophoresis of the latter product detected FAD, FMN, adenosine and AMP. In both cases adenine was not detected. The coenzymic activity of the product was found to be in accord with that given by Warburg et al.