The preparation of diphosphopyridine nucleotide with ion exchange resin.

The older methods for the preparation of diphosphopyridine nucleotide (DPN) were reviewed in 1942 by Schlenk (1). In 1947 Sumner et al. (2) outlined the need for a greatly simplified method for the preparation of this coenzyme. These authors described a procedure which did away with heavy metal fractionation. Recently, Clark et al. (3) made several modifications in the above method. The LePage (4) preparation has been widely used, although it is stated by him to give a product only 63 per cent pure. Hogeboom and Barry (5) have applied counter-current distribution for the isolation of DPN. This technique is undoubtedly of value for the preparation of relatively small quantities of highly purified coenzyme. The seemingly constant evolution of new methods for the preparation of DPN and the high cost, of the coenzyme on the commercial market must be accepted as evidence that the above methods leave room for improvement. Jandorf (6) introduced the adsorption of DPN on charcoal, a process which has become an important step in nearly all subsequent methods. Very recently LePage and Mueller (7) published a method for the preparation of triphosphopyridine nucleotide (TPN) in which the dinucleotide is adsorbed directly on charcoal from a water extract of liver. Although they chose to ignore DPN, the authors st.ate that a large amount of it was also taken up by the charcoal. A new technique in nucleotide chemistry, chromatography on ion exchange resin, has yielded striking results for the isolation of mononucleotides (Cohn (8, 9); Cohn and Carter (10)). It was thought possible that a combination of the two steps, adsorption on charcoal and chromatography on ion exchange resin, might lead to good yields of highly purified DPN. A method based on these principles has been worked out in this laboratory and will be described below. It has been found to give DPN of purity 0.8 in yields of about 1.2 gm. from 10 kilos of fresh bakers’ yeast.