Deoxyribonuclease I Assay

Method: That developed by Kunitz (1950) based upon the increased absorbance at 260 nm observed during the depolymerization of DNA by DNase. A unit causes an increase in absorbance at 260 nm of 0.001 per minute per ml when acting upon highly polymerized DNA at 25deg.C and pH 5.0 under the specified conditions. A standard enzyme preparation should be run in parallel with an unknown because standardization of DNA preparations and their degree of polymerization in solution is not possible.

Reagents

• 1.0 M Acetate buffer, pH 5.0
• 6.25 mM Magnesium sulfate in reagent grade water
• Worthington Standard DNase Vial (Code:DSV) containing a defined activity of approximately 2000 DNase units per vial.
• Worthington Highly Polymerized DNA (Code: DNA). Dissolve 10 mg DNA in 200 ml of 6.25 mM magnesium sulfate. Let stand overnight at room temperature. Add 25 ml of 1.0 M acetate buffer, pH 5.0 and dilute to a final volume of 250 ml with reagent grade water. (Substrate solution may be prepared in larger batches and stored for 2-3 weeks at 0 - 4deg.C.)

Enzyme

Note: Pancreatic deoxyribonuclease is unusually sensitive to physical denaturation by shaking. Mixing should be done by gentle inversion. Dissolve the standard vial in 1.0 ml of reagent grade water. Care must be taken when opening the vial that no lyophilized material is lost. This solution will contain the number of units/ml as stated on the label. Dilute further to a concentration of 20-60 u/ml. All dilutions are made in reagent grade water.

Sample to be assayed: Dissolve at a concentration of 1 mg/ml. Dilute further to a concentration of 20-60 u/ml immediately before the assay.

Procedure

Adjust spectrophotometer at 260 nm and 25deg.C. Pipette 2.5 ml of substrate into cuvettes and incubate in spectrophotometer at 25deg.C for 3-4 minutes to establish blank rate if any, and to reach temperature equilibration. Add 0.5 ml of diluted standard and record A₂₆₀ for 8 - 10 minutes. Calculate ≥A₂₆₀/minute from linear portion of curve following a brief lag.

Note: The change in A₂₆₀ for this assay is not generally linear from the initial time and is linear for only short periods. The most linear portion should be used in determining the activity. A rate of 0.008 - 0.018 ≥[[Alpha]]/min. is recommended.

Calculate the "factor" for the standard vial.

Using the diluted sample to be tested, repeat the above procedure. Record the ≥A₂₆₀/minute from the linear portion of the curve.

Calculation:

Activity is compared to that of the standard vial.

Deoxyribonuclease activity can also be conveniently measured in a radial diffusion assay system.