32] Purification of flavin-adenine dinucleotide and coenzyme A on p-acetoxymercurianiline-agarose

Abstract

Publisher Summary This chapter discusses the purification of flavin-adenine dinucleotide (FAD) and coenzyme A (CoA) on p -acetoxymercurianiline-agarose. Partially purified FAD and CoA can be obtained by the conventional methods, such as ion-exchange chromatography and extraction with organic solvents. However, these methods are not suitable for the isolation of highly pure FAD and reduced CoA in good yield, because the removal of contaminants, having absorption at 260 nm, from crude preparations is difficult. This chapter describes a method for immobilization of p -acetoxymercurianiline (PAMA) to agarose and the application of the immobilized PAMA for the purification of FAD and reduced CoA. In this method, PAMA is immobilized to CNBr-activated agarose, by the method of Sluyterman and Wijdenes, with some modifications. FAD is prepared from the culture broth of Sarcina lutea, according to the procedure of Watanabe. CoA is prepared from the culture broth of S. lutea, according to the method of Nishimura. With Assay method, FAD is determined by the fluorometric method. Reduced CoA is determined spectrophotometrically, using phosphate acetyltransferase from Clostridium kluyveri, according to the method of Michal and Bergmeyer. In this chapter, the purification of FAD and of the reduced CoA are also discussed in the chapter in detail.