A New Method for the Determination of Deoxyribonuclease I

Abstract

Summary. A new tubidimetric method for the determination of deoxyribonuclease I (deoxyribonuclease-oligonucleotide hydrolase, E.C.3.1.4.5) with the basic antibiotic – Neomycin Sulfate – as DNA precipitant, is proposed. The method affords the determination in purified as well as in crude systems, of enzyme activities equivalent to as little as 25 mμg crystalline DNase I. The main factors affecting the reaction of DNA with Neomycin were found to be the following : the relative concentrations of the reactants, the molecular weight of the DNA and such medium features as: pH, temperature and the presence of micro- and macroions. With the present method the optimum pH was found to be at about 7.5. A broad temperature optimum between 26 and 33°C with a small peak at 28° C was observed. At pH 7.5, divalent cations activated the DNase I reaction in the order: Mn>Zn>Mg>Co>Cu>Cd>Ca>Ba. The rate of enzymic depolymerization appears to be related to the molecular weight and the biological origin of the DNA. At high substrate concentration (>1.2 mg/ml) or with high molecular weights DNAs (>2.10*) the rate of turbidity decrease was found not to parallel the actual degradation rate as determined by capillary viscometry. The relation between enzyme concentration and depolymerization rate was demonstrated by a modified Applequist plot to be approximately first order.