Properties of chromatographically purified bovine pancreatic deoxyribonuclease.

Preparations of bovine pancreatic deoxyribonuclease obtained by the ammonium sulfate precipitation procedure of Kunitz can be further purified by chromatography on sulfoethyl-Sephadex at pH 4.70. The instability of earlier preparations of the enzyme has usually been attributed to the sensitivity of the protein to proteolytic digestion. This hypothesis has been confirmed; treatment of the starting product in the present experiments with diisopropylphosphorofluoridate inactivates contaminating proteases and leads to a stable preparation of the enzyme. Chromatography on sulfoethyl-Sephadex shows the presence of two active components which have almost identical properties. The major product is homogeneous by chromatography on hydroxylapatite at pH 6.7, by gel electrophoresis at pH 8.4, and by gel filtration on Sephadex G-75. Its amino acid composition is consistent with the molecular weight of 31,000 determined by ultracentrifugation by Lindberg. The analyses also show that the enzyme contains glucosamine and mannose and establish DNase as a glycoprotein. Lindberg’s results, together with the present data, show the protein to be a single chain of about 270 residues with leucine at the NHz-terminal position and two disulfide bonds. The stabilized, purified product provides a suitable starting material for structural study. DNase can also be fully stabilized against proteolytic digestion by 5 mu Ca++; the concentration of Ca++ needed to reduce the rate of inactivation by one-half is 0.1 m&r. This property may permit the enzyme to remain active in the presence of the proteases concurrently excreted by the pancreas.