[12] Preparation of α-NADP

This chapter discusses the experimental study, focusing on the preparation of α- nicotinamide adenine dinucleotide phosphate (NADP+). The α-anomer of NADP+ can be readily prepared, by the direct phosphorylation of α-NAD+, and catalyzed, by Azotobacter NAD kinase. NAD kinase is prepared from Azotobacter vinelandii. The enzyme prepared has a specific activity of 9 μmol of NADP+ formed per hour per milligram protein. Into a 10-ml beaker, 77.6 mg of ATP, 1.25 ml of 20 mM α-NAD+, 1.25 ml of 0.1 M MnCl2, and 2.5 ml of water are placed. The beaker is transferred to a larger water-jacketed beaker maintained at 37° and equipped with magnetic stirring. The pH is adjusted from about 2.6 to pH 7.6, with 1 M NH4OH (approximately 0.3 ml), and the reaction is started by adding 1.5 ml of Azotobacter NAD kinase. The pH is monitored and kept near 7.6, with 10-μl aliquots of 1 M NH4OH. The results of a typical chromatographic analysis of the ultrafiltered reaction mixture are discussed in this chapter. In the procedure, the total recovery from 25 μmol of α-NAD was 15.8 μmol of α-NADP+.