Deoxyribonuclease from Salmon Testes

Abstract

A procedure is described for the purification of salmon testis deoxyribonuclease II b y means of acid extraction, fractional precipitation with ammonium sulfate, heat denaturation of extraneous proteins, and ethanol fractionation. This process separates the deoxyribonuclease activity from that of ribonuclease, phosphatase, phosphodiesterase, and protease. Over 50 per cent of the activity is retained with an over-all enrichment of 20,000-fold. The enzyme degrades both nadve and heat-denatured DNA, but the rate of degradation of the latter is only one-tenth that of the former. It does not hydrolyze apurinic acid.The enzyme is most stable in the pH range 4 to 5. Electrolytes are essential for the expression of its activity: monovalent ions satisfy the requirement, but divalent ones are much more effective. Above a certain optimum concentration, each electrolyte is inhibitory. The pH of maximal activity, under conditions of optimal ionic strength, is 4.8; the temperature optimum is near to 55°C.At least two types of deoxyribonuclease (DNase) have been identified in mammalian tissues. The best known is the by now "classical" crystalline DNase from beef pancreas (1, 2), representative of the DNase I (or neutral) class. This type is active in neutral solution and requires magnesium or other divalent cations (1-4) for expression of activity. The DNase II (or acid) class is most active at p H 4 to 5, and its electrolyte requirement can be satisfied with either mono-or divalent cations (5-8). DNase II predominates in most mammalian tissues (9).Procedures have been developed for the purification of DNase II from spleen and thymus (10-15). None of them appears to be operationally satisfactory for routine preparation of large amounts of enzyme. Furthermore, those purified DNases that have been tested still show measurable traces of ribonuclease and phosphodiesterase. Since the DNase II content of normal tissues appears to be intimately related to their capacity for synthesis of D N A 77

Comparative Assessment of Automated Nucleic Acid Sample Extraction Equipment for Biothreat Agents

“…After the discovery of DNases, RNases, and chaotropic salts, purification procedures were generated that yielded consistent, measurable nucleic acid product (2)(3)(4). Several current clinical diagnostic extraction procedures separate nucleic acid by magnetic beads, a process that harmonizes older methods with user-friendly and precise techniques (4,5).…”