The NAD glycohydrolase (NADase) from Bungarus fasciatus venom was purified over 1000-fold to electrophoretic homogeneity through a 3-step procedure which included affinity chromatography on Cibacron Blue agarose. The enzyme exhibited a broad pH profile with the optimum range between 7-8. Studies on the substrate specificity of B. fasciatus venom NADase demonstrated that alterations in the purine ring were less pronounced then alterations in the pyridinium moiety of NAD. Product inhibition studies indicated nicotinamide to be a noncompetitive inhibitor with a K_i = 1.4 mM and ADP-ribose to be a competitive inhibitor with a K_i =0.4 mM. The purified enzyme was inactivated by both 2,4-pentane dione and Woodward's Reagent K suggesting the involvement of a lysine and carboxyl group in the catalytic process. In contrast to other known NADases, the snake venom enzyme did not self-inactivate.

The purified B. fasciatus venom NADase catalyzed a transglycosidation reaction (ADP-ribose transfer) with a number of acceptor molecules. The functioning of a variety of substituted pyridine bases as acceptor molecules was demonstrated through the formation of the corresponding NAD analogs. The enzyme also catalyzed the transfer of ADP-ribose to aliphatic alcohols (methanol to hexanol, inclusive) and a positive chainlength effect was observed in the functioning of these acceptors. Kinetic studies of transglycosidation reactions were consistent with the partitioning of an enzyme-ADP-ribose intermediate between water and nucleophilic acceptors as has been proposed in earlier studies of mammalian NADases. The partitioning of this intermediate between water and pyridine bases can be correlated with the basicity of the ring nitrogen of the pyridine derivative. The K_i of pyridine bases in the hydrolytic reaction did not equate to the K_m of these bases in the pyridine base exchange reaction suggesting two forms of the NADase with varying affinity for the pyridine bases. This implys the pyridine base exchange reaction to be more complicated than originally proposed.