Assay Procedure for Ribonuclease (Ref.
No. FGAP011)
Based on the method of Kunitz, J. Biol. Chem. (1946), 164,
563.
Unit Definition
That amount of enzyme causing the hydrolysis of RNA at a rate
such that the velocity constant k equals units (Kunitz units) at 25°C and
pH5.0.
Reagents
| A |
0.1M Sodium Acetate Buffer pH5.0 i) 0.1M Sodium Acetate
Dissolve 4.1g of CH3COONa [MM82.03] in ± 300ml distilled H2O
and adjust volume to 500ml with same.
ii) 0.1M Acetic Acid
Dissolve 5.73ml of Glacial Acetic Acid [MM60.05] in ± 300ml distilled H2O
and adjust volume to 500ml with same.
Titrate (i) with (ii) to pH5.0. Store at 5°C. Stable
for NLT one month.
|
| B |
RNA Solution [Substrate] Accurately
weigh out Torula Yeast RNA [Sigma R6625] and dissolve in buffer [A] to a concentration of
1mg/ml. Store at 5°C. Prepare fresh daily.
|
| C |
Enzyme Solution [TO BE MADE UP IMMEDIATELY BEFORE USE] Dissolve to 2 mg/ml distilled H2O and
dilute immediately before assay to ± 2.0 u/ml in H2O. |
Procedure
l = 300nm
Temperature = 25°C
Path length = 10mm
Cuvette volume (Vt) = 3.0ml
Sample volume (Vs) = 0.1ml
Pipette into test tubes @ 25°C.
|
Sample |
Reference |
| Substrate [C] |
1.5 ml |
1.5 ml |
| Distilled H2O |
1.4 ml |
1.5 ml |
| Enzyme [C] at time zero |
0.1 ml |
- |
|
3.00 ml |
3.00 ml |
Transfer to 3 ml silica cuvettes to read A
340nm.
Read A340 at 15 second intervals for 4 minutes.
Incubate at 25°C and read absorbance after 3 hours or until steady state is
obtained.
Caclulation
Plot log (Et - Ef) vs time
(t).
where Et = E300 at time t
Ef = E300 at final reading.
|
-2,3 x S x Vt x dilution |
| Activity [u/ml] = |
 |
|
Vs |
where S = slope of the above graph.
| Activity [u/mg material] = |
u/ml of dissolved lyophilised material |
|
mg material/ml original solution |
 |
Determination |
Determine the E280nm of the dissolved lyophilised
material [2mg/ml] versus H2O, diluting if necessary in H2O.
|
= E280nm x dilution x 5 |
|
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes