The Separation of the Phosphodiesterase and Deoxyribonuclease II Activities of Bovine Spleen

Deoxyribonuclease II (Deoxyribonucleate 3'-nucleotidohydrolase, EC 3.1.4.6) was purified 1250-fold from bovine spleen. The specific activity of the final preparation was 408. It was free of phosphatase and alkaline ribonuclease, and almost free of nonspecific phosphodiesterase, acid ribonuclease, and adenosine triphosphatase. Chromatography on carboxymethyl cellulose prior to heating caused the bulk of the deoxyribonuclease to be eluted early, in a symmetrical peak containing phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1). After the crude preparation was heated at 60°, the symmetrical peak disappeared and was replaced by separate phosphodiesterase and deoxyribonuclease peaks. Repeated chromatography of the unheated preparation or mild heating (37°, with or without 2-mercaptoethanol) of the symmetrical peak caused changes similar to those seen after heat treatment of the crude preparation. The chromatographic data, heat inactivation, pH optimum, and activity on a number of phosphodiesters support the contention that the phosphodiesterase and deoxyribonuclease activities of the unheated preparation are on separate proteins.