Studies on bovine pancreatic deoxyribonuclease A: I. General properties and activation with different bivalent metals

Abstract

Bovine pancreatic deoxyribonuclease I (EC 3.1.4.5) was separated into five fractions by phosphocellulose chromatography. The main fraction (deoxyribonuclease A) was very stable both in the lyophilized form and in solution. Deoxyribonuclease A was activated by bivalent metals and inhibited by EDTA and high concentrations of NaCl and Tris-HCl buffer, pH 7.5. The rate of hydrolysis of DNA by the enzyme was a function of pH, ionic strength of the buffer, DNA concentration and bivalent metal concentration, all of which were interdependent. Activity was investigated at 0.05 and 0.5 mg/ml DNA. Mg 2+, Mn 2+ and Co 2+ were the most effective activators of the enzyme. The optimal rate of the Mn 2+-activated reaction was 50–70 % that of the Mg 2+-activated reaction with 0.5 mg/ml DNA as substrate but 2.2–2.5-fold higher with 0.05 mg/ml DNA in 5 mM Tris-HCl buffer, pH 7.6. Deoxyribonuclease A was also activated by Ca 2+, Ni 2+, Sr 2+, Zn 2+, Cd 2+, Cu 2+ and Ba 2+ but not by Sn 2+ and Fe 2+. Mg 2+ and Ca 2+ had a synergistic activating effect. The Mg 2+-activated reaction had a sharp pH optimum of 7 while the pH optima with the other bivalent metal-activated reactions varied between 6.5 and 8.5. The Mg 2+-, Sr 2+- and Zn 2+-activated reactions showed a lag in the rate of hydrolysis of native DNA supporting the concept of two different mechanisms of degradation by bovine pancreatic deoxyribonuclease I. The results presented define the optimal conditions for hydrolysis of DNA with various bivalent metals as activators and have provided an explanation of discrepancies in quantitative results, with bovine pancreatic deoxyribonuclease I, from various laboratories.