Phosphodiesterase I (EC 3.1.4.1, Crotalus adamanteus venom)

Venom exonuclease (Phosphodiesterase I) successively hydrolyzes 5'-mononucleotides from 3'-hydroxy-terminated ribo-and deoxyribo-oligonucleotides. The enzyme has been widely utilized as a tool for structural and sequence studies of nucleic acids. The enzyme has been purified with endonuclease activity being eliminated as well as 5'-nucleotidase and nonspecific monophosphatase. It is nonspecific with respect to base or sugar moieties of nucleotides. A variety of synthetic substrates are hydrolyzed. The exonuclease will not recognize nucleoside units in the syn conformation. ADP-ribosylated proteins are cleaved at the pyrophosphate linkages by venom phosphodiesterase to yield phosphoribosyl-AMP. Activity: >20 units per mg dry weight, Absorbance (A280): As reported, Unit Definition: 1 unit hydrolyzes 1 umole of p-nitrophenyl thymidine-5-phosphate per minute at 25ºC, pH 8.9. Nucleotidase: <8%, Optimum pH: 9.8-10.4, Inhibitors: Reducing agents such as glutathione, cysteine and ascorbic acid. It is completely inhibited by 5mM EDTA while ATP, ADP and AMP are partial inhibitors. Activators: The enzyme has an absolute requirement for Mg2+ indicates an optimum concentration of 15mM. Storage and Stability: Lyophilized and reconstituted products are stable for 6 months after receipt at -20°C. Reconstitute with sterile dH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.