Biochemical properties

DSN acquires its enzymatic activity in the presence of divalent cations (Mn²⁺, Co²⁺, or Mg²⁺). Mg²⁺ ion concentration for most applications should be at least 5 mM. DSN is inhibited by EDTA.

The temperature optimum for DSN activity is 60°C. However, already at 80°C DSN retains only 10% of activity. This sharp decrease in activity may be attributable, at least in part, to ds DNA substrate denaturation.

The optimal pH for DSN activity is 6.6. At pH values between 3 and 5, DSN displays only 10% of its maximal activity. The nuclease is stable at a wide range of pH (from 4 to 12) and temperatures below 65°C. About 60% of DSN activity remains after 30-min incubation at 70°C, and 40% – after incubation at 80°C.

Effects of various conditions on DSN activity. Activity of DNAse on ds DNA substrate was measured using modified Kunitz assay (Kunitz, 1950)

Incubation of DSN with aggressive chemicals like 1% SDS, 10 mM β-mercaptoethanol, and 0.3% hydrogen peroxide at 37°C results in only a moderate drop in activity, after 30 min incubation about 90% of activity is preserved. However, a sharp decrease in activity was observed upon chemical treatment at 60°C. SDS completely inhibits DSN activity, while β-mercaptoethanol and hydrogen peroxide induce approximately 70% and 80% loss in activity, respectively.

DSN is highly sensitive to ionic force (e.g., a 10 times decrease in catalytic activity is observed in the presence of 0.2 M NaCl). The addition of chaotropic agents or polyamines to the reaction mixture also results in suppression of enzyme activity.

DSN is tolerant to proteinase K treatment (incubation at 37°C for 30 min).