Abstract
The incorporation of P^<32>-phosphate into FAD in the mycelium of Eremothecium ashbyii was successfully attempted. Eremothecium ashbyii was grown at 27℃ in shaking flasks, containing peptone-glucose medium. P^<32>-phosphate was added to the medium at a final concentration of 3 μc/ml. After 60 hours' cultivation, mycelium was harvested, from which FAD was extracted with a mixture of pyridine, methanol and water. The extract was purified by the following procedures : concentration in vacuo, extraction with liquid phenol, transfer to aqueous phase by adding ether, precipitation by Na_2S_2O_4 reduction, partition chromatography as well as electrophoresis on powdered cellulose column, and crystallisation by the addition of ethanol. The bright-orange powder of P^<32>-FAD thus obtained, was confirmed to be of such purity as 90% in spectrophotometric assay and entirely free from other flavins and other radioactive compounds. Its specific radioactivity, 6 μc/mg, will enable it to be useful in chemical and biochemical studies. In this method, the over-all yield of P^<32>-FAD from wet mycelium was nearly 30%.
