Assay Procedure for Deoxyribonuclease
(Ref. No. FGAP008)
(Kunitz Method)
Based on that of Kunitz in which the rate of increase in
absorbance caused by the hydrolysis of deoxyribonucleic acid (DNA) is measured
spectrophotometrically at 260nm and 25°C.
Unit Definition
That amount of enzyme which causes an increase of absorbance at
260nm of 0.001 per minute at 25°C.
Reagents
| A |
1.0 M Acetate Buffer pH 5.0 1. Dissolve 13.6 g CH3 COONa . 3H2O [MM 136.1] in
distilled H2O and adjust volume to 100ml.
2. Dissolve 5.7 ml glacial acetic acid [MM60.01] in distilled H2O and dilute to
100ml.
Titrate solution 1 with solution 2 to yield buffer at pH5.0.
|
| B |
0.05 M Magnesium Sulphate Solution Dissolve 1.23g MgSO4 . 7 H2O [MM 246.48] in
distilled H2O and adjust volume to 100ml.
|
| C |
Substrate 4mg % DNA/0.005 M MgSO4/0.1 M Acetate
Buffer pH5.0 Dissolve 4mg DNA in 50ml distilled H2O.
Store overnight at 5°C. Add 10ml buffer [A] and 10ml MgSO4 [B] and
adjust volume to 100ml with distilled H2O. Store stock (bulk substrate) at 5°C
and equilibrate substrate at 25°C.
|
| D |
Sample Dissolve between
1mg and 5mg enzyme/ml ice-cold distilled H2O. Immediately before assay, dilute
solution to yield 45 - 75 u/ml ice-cold distilled H2O. (DA260/min 0.0075  0.0125) |
Procedure
l = 260nm
Temperature = 25°C
Light path = 10mm
Cuvette volume = 3.0ml
Into a 10mm quartz cuvette pipette:
| Substrate [C] |
2.5 ml |
| Equilibrate at 25°C and monitor DA 260/min |
|
| Enzyme at zero time |
0.5 ml |
|
3.00 ml |
Record rate of increase in absorbance at 260nm for 3-6 minutes
after initial time lag.
Calculation
|
DA 260/min
x 3 x dilution |
| Activity [u/mg] = |
 |
|
0.001 x 0.5 x mg enzyme / ml original solution |
[e = 0.001; cuvette
volume = 3.0ml; enzyme volume = 0.5ml]
Note
- Reference: Kunitz M.: (1950) J. Gen. Physiol 33 349.
- As the degree of polymerization of DNA in solution can not be
standardised, it is necessary to assay a standard DNase and to correct the unknown
activity accordingly.
- Prepare DNase standard at 1mg / ml ice-cold distilled H2O
and dilute appropriately immediately before assay.
|
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes